Symbiotic interactions between legumes and rhizobia lead to development of root nodules and nitrogen fixation by differentiated bacteroids within nodules. Differentiation of the endosymbionts is reversible or terminal, determined by plant effectors. In IRLC legumes, Nodule-Specific Cysteine-Rich Peptides (NCRs) control the terminal differentiation of bacteroids. Medicago truncatula contains ∼700 NCR-coding genes. However, the role of few NCRs has been demonstrated. Here, we report characterization of FN2106 (Fast Neutron 2106), a symbiotic nitrogen fixation defective (fix-) mutant of M. truncatula. Using a transcript-based approach, together with linkage and complementation tests, we showed that loss-of-function of NCR343 results in impaired bacteroid differentiation and/or maintenance and premature nodule senescence of the FN2106 mutant. NCR343 was specifically expressed in nodules. Subcellular localization studies showed that the functional NCR343-YFP fusion protein colocalizes with bacteroids in symbiosomes in infected nodule cells. Transcriptomic analyses identified senescence-, but not defense-related genes, as being significantly upregulated in ncr343 (FN2106) nodules. Taken together, results from our phenotypic and transcriptomic analyses of a loss-of-function ncr343 mutant demonstrate an essential role of NCR343 in bacteroid differentiation and/or maintenance required for symbiotic nitrogen fixation.

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA-sequencing of maize mesophyll and bundle sheath strands over a 24-h time-course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene co-expression analyses suggested members of the DNA binding with One Finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators as well as peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

The initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis for most life on Earth, is catalyzed by photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we used Arabidopsis (Arabidopsis thaliana) as a model to show that in plants, a eukaryote-exclusive assembly factor facilitates the early assembly step, during which the intrinsic antenna protein CP47 becomes associated with the PSII reaction center (RC) to form the RC47 intermediate. This factor, which we named DECREASED ELECTRON TRANSPORT AT PSII (DEAP2), works in concert with the conserved PHOTOSYNTHESIS AFFECTED MUTANT 68 (PAM68) assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins resulted in a loss of functional PSII and the inability of plants to grow photoautotrophically on soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulated at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.

Cell polarity results from the asymmetric distribution of cellular structures, molecules, and functions. Polarity is a fundamental cellular trait that can determine the orientation of cell division, the formation of particular cell shapes, and ultimately the development of a multicellular body. To maintain the distinct asymmetric distribution of proteins and lipids in cellular membranes, plant cells have developed complex trafficking and regulatory mechanisms. Major advances have been made in our understanding of how membrane microdomains influences the asymmetric distribution of proteins and lipids. In this review, we first give an overview of cell polarity. Next, we discuss current knowledge concerning membrane microdomains and their roles as structural and signaling platforms to establish and maintain membrane polarity, with a special focus on the asymmetric distribution of proteins and lipids, and advanced microscopy techniques to observe and characterize membrane microdomains. Finally, we review recent advances regarding membrane trafficking in cell polarity establishment, and how the balance between exocytosis and endocytosis affects membrane polarity.

Autophagy serves as an important recycling route for the growth and survival of eukaryotic organisms in nutrient-deficient conditions. Since starvation induces massive changes in metabolic flux that are coordinated by key metabolic enzymes, specific processing steps of autophagy may be linked with metabolic flux-monitoring enzymes. We attempted to identify carbon metabolic genes that modulate autophagy using VIGS screening of 45 glycolysis- and Calvin-Benson cycle-related genes in Arabidopsis (Arabidopsis thaliana). Here, we report that three consecutive triose-phosphate-processing enzymes involved in cytosolic glycolysis, TPI (triose-phosphate-isomerase), GAPC (glyceraldehyde-3-phosphate dehydrogenase), and PGK (phosphoglycerate kinase), designated TGP, negatively regulate autophagy. Depletion of TGP enzymes causes spontaneous autophagy induction and increases AUTOPHAGY-RELATED 1 (ATG1) kinase activity. TGP enzymes interact with ATG101, a regulatory component of the ATG1 kinase complex. Spontaneous autophagy induction and abnormal growth under insufficient sugar in the TGP mutants are suppressed by crossing with the atg101 mutant. Considering that triose-phosphates are photosynthates transported to the cytosol from active chloroplasts, the TGP enzymes would be strategically positioned to monitor the flow of photosynthetic sugars and modulate autophagy accordingly. Collectively, these results suggest that TGP enzymes negatively control autophagy acting upstream of the ATG1 complex, which is critical for seedling development.

Cis-regulatory elements (CREs) fine-tune gene transcription in eukaryotes. CREs with sequence variations play vital roles in driving plant or crop domestication. However, how global sequence and structural variations are responsible for multi-level changes between indica and japonica rice (Oryza sativa) is still not fully elucidated. To address this, we conducted multi-omics studies using MH-seq (MNase hypersensitivity sequencing) in combination with RNA-seq, ChIP-seq and BS-seq between the japonica rice variety Nipponbare (NIP) and indica rice variety 93-11. We found that differential MHSs exhibited some distinct intrinsic genomic sequence features between NIP and 93-11. Notably, through MHS-GWAS integration, we found that key sequence variations may be associated with differences of agronomic traits between NIP and 93-11, which is partly achieved by MHSs harboring CREs. In addition, SV-derived differential MHSs caused by TE insertion, especially by non-common TEs among rice varieties, were associated with genes with distinct functions, indicating that TE driven gene neo- or subfunctionalization is mediated by changes of chromatin openness. This study thus provides insights into how sequence and genomic structural variations control agronomic traits of NIP and 93-11; it also provides genome editing targets for molecular breeding aiming at improving favorable agronomic properties.

Organelles function as hubs of cellular metabolism and elements of cellular architecture. In addition to 3 spatial dimensions that describe the morphology and localization of each organelle, the time dimension describes complexity of the organelle life cycle, comprising formation, maturation, functioning, decay, and degradation. Thus, structurally identical organelles could be biochemically different. All organelles present in a biological system at a given moment of time constitute the organellome. The homeostasis of the organellome is maintained by complex feedback and feedforward interactions between cellular chemical reactions and by the energy demands. Synchronized changes of organelle structure, activity, and abundance in response to environmental cues generate the fourth dimension of plant polarity. Temporal variability of the organellome highlights the importance of organellomic parameters for understanding plant phenotypic plasticity and environmental resiliency. Organellomics involves experimental approaches for characterizing structural diversity and quantifying the abundance of organelles in individual cells, tissues, or organs. Expanding the arsenal of appropriate organellomics tools and determining parameters of the organellome complexity would complement existing -omics approaches in comprehending the phenomenon of plant polarity. To highlight the importance of the fourth dimension, this review provides examples of organellome plasticity during different developmental or environmental situations.

Distinct from animals, plants maintain organogenesis from specialized tissues termed meristems throughout life. In the shoot apex, the shoot apical meristem (SAM) produces all aerial organs, such as leaves, from its periphery. For this, the SAM needs to precisely balance stem cell renewal and differentiation, which is achieved through dynamic zonation of the SAM, and cell signaling within functional domains is key for SAM functions. The WUSCHEL-CLAVATA feedback loop plays a key role in SAM homeostasis, and recent studies have uncovered new components, expanding our understanding of the spatial expression and signaling mechanism. Advances in polar auxin transport and signaling have contributed to knowledge of the multifaceted roles of auxin in the SAM and organogenesis. Finally, single-cell techniques have expanded our understanding of the cellular functions within the shoot apex at single-cell resolution. In this review, we summarize the most up-to-date understanding of cell signaling in the SAM and focus on the multiple levels of regulation of SAM formation and maintenance.

Plant development is a complex task, and many processes involve changes in the asymmetric subcellular distribution of cell components that strongly depend on cell polarity. Cell polarity regulates anisotropic growth and polar localization of membrane proteins and helps to identify the cell's position relative to its neighbors within an organ. Cell polarity is critical in a variety of plant developmental processes, including embryogenesis, cell division, and response to external stimuli. The most conspicuous downstream effect of cell polarity is the polar transport of the phytohormone auxin, which is the only known hormone transported in a polar fashion in and out of cells by specialized exporters and importers. The biological processes behind the establishment of cell polarity are still unknown, and researchers have proposed several models that have been tested using computer simulations. The evolution of computer models has progressed in tandem with scientific discoveries, which have highlighted the importance of genetic, chemical, and mechanical input in determining cell polarity and regulating polarity-dependent processes such as anisotropic growth, protein subcellular localization, and the development of organ shapes. The purpose of this review is to provide a comprehensive overview of the current understanding of computer models of cell polarity establishment in plants, focusing on the molecular and cellular mechanisms, the proteins involved, and the current state of the field.

Cell polarity is intimately linked to numerous biological processes, such as oriented plant cell division, particular asymmetric division, cell differentiation, cell and tissue morphogenesis, and transport of hormones and nutrients. Cell polarity is typically initiated by a polarizing cue that regulates the spatiotemporal dynamic of polarity molecules, leading to the establishment and maintenance of polar domains at the plasma membrane. Despite considerable progress in identifying key polarity regulators in plants, the molecular and cellular mechanisms underlying cell polarity formation have yet to be fully elucidated. Recent work suggests a critical role for membrane protein/lipid nanodomains in polarized morphogenesis in plants. One outstanding question is how the spatiotemporal dynamics of signaling nanodomains are controlled to achieve robust cell polarization. In this review, we first summarize the current state of knowledge on potential regulatory mechanisms of nanodomain dynamics, with a special focus on Rho-like GTPases from plants. We then discuss the pavement cell system as an example of how cells may integrate multiple signals and nanodomain-involved feedback mechanisms to achieve robust polarity. A mechanistic understanding of nanodomains' roles in plant cell polarity is still in the early stages and will remain an exciting area for future investigations.

The unequal (asymmetric) distribution of cell structures and proteins within a cell is designated as cell polarity. Cell polarity is a crucial prerequisite for morphogenetic processes such as oriented cell division and directed cell expansion. Rho-related GTPase from plants (ROPs) are required for cellular morphogenesis through the reorganization of the cytoskeleton and vesicle transport in various tissues. Here, I review recent advances in ROP-dependent tip growth, vesicle transport, and tip architecture. I report on the regulatory mechanisms of ROP upstream regulators found in different cell types. It appears that these regulators assemble in nanodomains with specific lipid compositions and recruit ROPs for activation in a stimulus-dependent manner. Current models link mechanosensing/mechanotransduction to ROP polarity signaling involved in feedback mechanisms via the cytoskeleton. Finally, I discuss ROP signaling components that are upregulated by tissue-specific transcription factors and exhibit specific localization patterns during cell division, clearly suggesting ROP signaling in division plane alignment.

Polar callose deposition into the extracellular matrix is tightly controlled in time and space. Its presence in the cell wall modifies the properties of the surrounding area, which is fundamental for the correct execution of numerous processes such as cell division, male gametophyte development, intercellular transport, or responses to biotic and abiotic stresses. Previous studies have been invaluable in characterizing specific callose synthases (CalSs) during individual cellular processes. However, the complex view of the relationships between a particular CalS and a specific process is still lacking. Here we review the recent proceedings on the role of callose and individual CalSs in cell wall remodelling from an evolutionary perspective and with a particular focus on cytokinesis. We provide a robust phylogenetic analysis of CalS across the plant kingdom, which implies a 3-subfamily distribution of CalS. We also discuss the possible linkage between the evolution of CalSs and their function in specific cell types and processes.

Understanding mechanisms of nutrient allocation in organisms requires precise knowledge of the spatiotemporal dynamics of small molecules in vivo. Genetically encoded sensors are powerful tools for studying nutrient distribution and dynamics, as they enable minimally invasive monitoring of nutrient steady-state levels in situ. Numerous types of genetically encoded sensors for nutrients have been designed and applied in mammalian cells and fungi. However, to date, their application for visualizing changing nutrient levels in planta remains limited. Systematic sensor-based approaches could provide the quantitative, kinetic information on tissue-specific, cellular, and subcellular distributions and dynamics of nutrients in situ that is needed for the development of theoretical nutrient flux models that form the basis for future crop engineering. Here, we review various approaches that can be used to measure nutrients in planta with an overview over conventional techniques, as well as genetically encoded sensors currently available for nutrient monitoring, and discuss their strengths and limitations. We provide a list of currently available sensors and summarize approaches for their application at the level of cellular compartments and organelles. When used in combination with bioassays on intact organisms and precise, yet destructive analytical methods, the spatiotemporal resolution of sensors offers the prospect of a holistic understanding of nutrient flux in plants.

For multicellular organisms to develop, cells must grow, divide, and differentiate along preferential or exclusive orientations or directions. Moreover, those orientations, or axes, and directions, or polarities, must be coordinated between cells within and between tissues. Therefore, how axes and polarities are coordinated between cells is a key question in biology. In animals, such coordination mainly depends on cell migration and direct interaction between proteins protruding from the plasma membrane. Both cell movements and direct cell-cell interactions are prevented in plants by cell walls that surround plant cells and keep them apart and in place. Therefore, plants have evolved unique mechanisms to coordinate their cell axes and polarities. Here I will discuss evidence suggesting that understanding how leaf veins form may uncover those unique mechanisms. Indeed, unlike previously thought, the cell-to-cell polar transport of the plant hormone auxin along developing veins cannot account for many features of vein patterning. Instead, those features can be accounted for by models of vein patterning that combine polar auxin transport with auxin diffusion through plasmodesmata along the axis of developing veins. Though it remains unclear whether such a combination of polar transport and axial diffusion of auxin can account for the formation of the variety of vein patterns found in plant leaves, evidence suggests that such a combined mechanism may control plant developmental processes beyond vein patterning.

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

The visualization of photosynthesis-derived reactive oxygen species has been experimentally limited to pH-sensitive probes, unspecific redox dyes, and whole-plant phenotyping. Recent emergence of probes that circumvent these limitations permits advanced experimental approaches to investigate in situ plastid redox properties. Despite growing evidence of heterogeneity in photosynthetic plastids, investigations have not addressed the potential for spatial variation in redox and/or reactive oxygen dynamics. To study the dynamics of H2O2 in distinct plastid types, we targeted the pH-insensitive, highly specific probe HyPer7 to the plastid stroma in Arabidopsis (Arabidopsis thaliana). Using HyPer7 and glutathione redox potential (EGSH) probe for redox-active green fluorescent protein 2 genetically fused to the redox enzyme human glutaredoxin-1 with live cell imaging and optical dissection of cell types, we report heterogeneities in H2O2 accumulation and redox buffering within distinct epidermal plastids in response to excess light and hormone application. Our observations suggest that plastid types can be differentiated by their physiological redox features. These data underscore the variation in photosynthetic plastid redox dynamics and demonstrate the need for cell-type-specific observations in future plastid phenotyping.

Plant cells can reprogram their fate. The combinatorial actions of auxin and cytokinin dedifferentiate somatic cells to regenerate organs, which can develop into individual plants. As transgenic plants can be generated from genetically modified somatic cells through these processes, cell fate transition is an unavoidable step in crop genetic engineering. However, regeneration capacity closely depends on the genotype, and the molecular events underlying these variances remain elusive. In the present study, we demonstrated that WUSCHEL (WUS)-a homeodomain transcription factor-determines regeneration capacity in different potato (Solanum tuberosum) genotypes. Comparative analysis of shoot regeneration efficiency and expression of genes related to cell fate transition revealed that WUS expression coincided with regeneration rate in different potato genotypes. Moreover, in a high-efficiency genotype, WUS silencing suppressed shoot regeneration. Meanwhile, in a low-efficiency genotype, regeneration could be enhanced through the supplementation of a different type of cytokinin that promoted WUS expression. Computational modeling of cytokinin receptor-ligand interactions suggested that the docking pose of cytokinins mediated by hydrogen bonding with the core residues may be pivotal for WUS expression and shoot regeneration in potatoes. Furthermore, our whole-genome sequencing analysis revealed core sequence variations in the WUS promoters that differentiate low- and high-efficiency genotypes. The present study revealed that cytokinin responses, particularly WUS expression, determine shoot regeneration efficiency in different potato genotypes.

Posttranslationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small posttranslationally modified peptides are perceived by leucine-rich repeat receptor-like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibits anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is downregulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continues to act as negative regulators to limit pigment accumulation in the light.

Pollen germination is a process of polarity establishment, through which a single and unique growth axis is established. Although most of the intracellular activities associated with pollen germination are controlled by RHO OF PLANTs (ROPs) and increased ROP activation accompanies pollen germination, a critical role of ROPs in this process has not yet been demonstrated. Here, by genomic editing of all 4 Arabidopsis (Arabidopsis thaliana) ROPs that are preferentially expressed in pollen, we showed that ROPs are essential for polarity establishment during pollen germination. We further identified and characterized 2 ROP effectors in pollen germination (REGs) through genome-wide interactor screening, boundary of ROP domain (BDR) members BDR8 and BDR9, whose functional loss also resulted in no pollen germination. BDR8 and BDR9 were distributed in the cytosol and the vegetative nucleus of mature pollen grains but redistributed to the plasma membrane (PM) of the germination site and to the apical PM of growing pollen tubes. We demonstrated that the PM redistribution of BDR8 and BDR9 during pollen germination relies on ROPs but not vice versa. Furthermore, enhanced expression of BDR8 partially restored germination of rop1 pollen but had no effects on that of the quadruple rop pollen, supporting their genetic epistasis. Results presented here demonstrate an ROP signaling route essential for pollen germination, which supports evolutionarily conserved roles of Rho GTPases in polarity establishment.

Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development, including autophagy, which facilitates cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy affects nutrient remobilization from mother plants and functions in the embryo. However, it is impossible to distinguish between the contribution of autophagy in the source (i.e. the mother plant) and the sink tissue (i.e. the embryo) when using autophagy knockout (atg mutant) plants. To address this, we employed an approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between wild type and atg mutant Arabidopsis (Arabidopsis thaliana) plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from altered seed coat development. Our study emphasizes the importance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It also sheds light on the tissue-specific functions of autophagy, offering potential for research into the underlying mechanisms governing seed development and crop yield.

Soil salinity is progressively impacting agriculture, including viticulture. Identification of genetic factors rendering grapevine (Vitis vinifera L.) resilience that can be introgressed into commercial varieties is necessary for safeguarding viticulture against the consequences of global climate change. To gain insight into the physiological and metabolic responses enabling salt tolerance, we compared a salt-tolerant accession of Vitis sylvestris from Tunisia, "Tebaba", with "1103 Paulsen" rootstock widely used in the Mediterranean. Salt stress was slowly increased, simulating the situation of an irrigated vineyard. We determined that "Tebaba" does not sequester sodium in the root but can cope with salinity through robust redox homeostasis. This is linked with rechanneling of metabolic pathways toward antioxidants and compatible osmolytes, buffering photosynthesis, such that cell-wall breakdown can be avoided. We propose that salt tolerance of this wild grapevine cannot be attributed to a single genetic factor but emerges from favorable metabolic fluxes that are mutually supportive. We suggest that introgression of "Tebaba" into commercial varieties is preferred over the use of "Tebaba" as a rootstock for improving salt tolerance in grapevine.

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Oomycete pathogens can secrete hundreds of effectors into plant cells to interfere with the plant immune system during infection. Here, we identified a Arg-X-Leu-Arg (RXLR) effector protein from the most destructive pathogen of litchi (Litchi chinensis Sonn.), Peronophythora litchii, and named it P. litchii avirulence homolog 202 (PlAvh202). PlAvh202 could suppress cell death triggered by infestin 1 or avirulence protein 3a/resistance protein 3a in Nicotiana benthamiana and was essential for P. litchii virulence. In addition, PlAvh202 suppressed plant immune responses and promoted the susceptibility of N. benthamiana to Phytophthora capsici. Further research revealed that PlAvh202 could suppress ethylene (ET) production by targeting and destabilizing plant S-adenosyl-L-methionine synthetase (SAMS), a key enzyme in the ET biosynthesis pathway, in a 26S proteasome-dependent manner without affecting its expression. Transient expression of LcSAMS3 induced ET production and enhanced plant resistance, whereas inhibition of ET biosynthesis promoted P. litchii infection, supporting that litchi SAMS (LcSAMS) and ET positively regulate litchi immunity toward P. litchii. Overall, these findings highlight that SAMS can be targeted by the oomycete RXLR effector to manipulate ET-mediated plant immunity.

The apoplast of plant leaves, the intercellular space between mesophyll cells, is normally largely filled with air with a minimal amount of liquid water in it, which is essential for key physiological processes such as gas exchange to occur. Phytopathogens exploit virulence factors to induce a water-rich environment, or "water-soaked" area, in the apoplast of the infected leaf tissue to promote disease. We propose that plants evolved a "water soaking" pathway, which normally keeps a nonflooded leaf apoplast for plant growth but is disturbed by microbial pathogens to facilitate infection. Investigation of the "water soaking" pathway and leaf water control mechanisms is a fundamental, yet previously overlooked, aspect of plant physiology. To identify key components in the "water soaking" pathway, we performed a genetic screen to isolate Arabidopsis (Arabidopsis thaliana) severe water soaking (sws) mutants that show liquid water overaccumulation in the leaf under high air humidity, a condition required for visible water soaking. Here, we report the sws1 mutant, which displays rapid water soaking upon high humidity treatment due to a loss-of-function mutation in CURLY LEAF (CLF), encoding a histone methyltransferase in the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). We found that the sws1 (clf) mutant exhibits enhanced abscisic acid (ABA) levels and stomatal closure, which are indispensable for its water soaking phenotype and mediated by CLF's epigenetic regulation of a group of ABA-associated NAM, ATAF, and CUC (NAC) transcription factor genes, NAC019/055/072. The clf mutant showed weakened immunity, which likely also contributes to the water soaking phenotype. In addition, the clf plant supports a substantially higher level of Pseudomonas syringae pathogen-induced water soaking and bacterial multiplication, in an ABA pathway and NAC019/055/072-dependent manner. Collectively, our study sheds light on an important question in plant biology and demonstrates CLF as a key modulator of leaf liquid water status via epigenetic regulation of the ABA pathway and stomatal movement.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Banana (Musa spp.) fruits, as typical tropical fruits, are cold sensitive, and lower temperatures can disrupt cellular compartmentalization and lead to severe browning. How tropical fruits respond to low temperature compared to the cold response mechanisms of model plants remains unknown. Here, we systematically characterized the changes in chromatin accessibility, histone modifications, distal cis-regulatory elements, transcription factor binding, and gene expression levels in banana peels in response to low temperature. Dynamic patterns of cold-induced transcripts were generally accompanied by concordant chromatin accessibility and histone modification changes. These upregulated genes were enriched for WRKY binding sites in their promoters and/or active enhancers. Compared to banana peel at room temperature, large amounts of banana WRKYs were specifically induced by cold and mediated enhancer-promoter interactions regulating critical browning pathways, including phospholipid degradation, oxidation, and cold tolerance. This hypothesis was supported by DNA affinity purification sequencing, luciferase reporter assays, and transient expression assay. Together, our findings highlight widespread transcriptional reprogramming via WRKYs during banana peel browning at low temperature and provide an extensive resource for studying gene regulation in tropical plants in response to cold stress, as well as potential targets for improving cold tolerance and shelf life of tropical fruits.

Drought stress poses a serious threat to crop production worldwide. Genes encoding homocysteine methyltransferase (HMT) have been identified in some plant species in response to abiotic stress, but its molecular mechanism in plant drought tolerance remains unclear. Here, transcriptional profiling, evolutionary bioinformatics, and population genetics were conducted to obtain insight into the involvement of HvHMT2 from Tibetan wild barley (Hordeum vulgare ssp. agriocrithon) in drought tolerance. We then performed genetic transformation coupled with physio-biochemical dissection and comparative multiomics approaches to determine the function of this protein and the underlying mechanism of HvHMT2-mediated drought tolerance. HvHMT2 expression was strongly induced by drought stress in tolerant genotypes in a natural Tibetan wild barley population and contributed to drought tolerance through S-adenosylmethionine (SAM) metabolism. Overexpression of HvHMT2 promoted HMT synthesis and efficiency of the SAM cycle, leading to enhanced drought tolerance in barley through increased endogenous spermine and less oxidative damage and growth inhibition, thus improving water status and final yield. Disruption of HvHMT2 expression led to hypersensitivity under drought treatment. Application of exogenous spermine reduced accumulation of reactive oxygen species (ROS), which was increased by exogenous mitoguazone (inhibitor of spermine biosynthesis), consistent with the association of HvHMT2-mediated spermine metabolism and ROS scavenging in drought adaptation. Our findings reveal the positive role and key molecular mechanism of HvHMT2 in drought tolerance in plants, providing a valuable gene not only for breeding drought-tolerant barley cultivars but also for facilitating breeding schemes in other crops in a changing global climate.

Pentatricopeptide repeat (PPR) proteins form a large protein family and have diverse functions in plant development. Here, we identified an ALBINO EMBRYO AND SEEDLING (AES) gene that encodes a P-type PPR protein expressed in various tissues, especially the young leaves of Arabidopsis (Arabidopsis thaliana). Its null mutant aes exhibited a collapsed chloroplast membrane system, reduced pigment content and photosynthetic activity, decreased transcript levels of PEP (plastid-encoded polymerase)-dependent chloroplast genes, and defective RNA splicing. Further work revealed that AES could directly bind to psbB-psbT, psbH-petB, rps8-rpl36, clpP, ycf3, and ndhA in vivo and in vitro and that the splicing efficiencies of these genes and the expression levels of ycf3, ndhA, and cis-tron psbB-psbT-psbH-petB-petD decreased dramatically, leading to defective PSI, PSII, and Cyt b6f in aes. Moreover, AES could be transported into the chloroplast stroma via the TOC-TIC channel with the assistance of Tic110 and cpSRP54 and may recruit HCF244, SOT1, and CAF1 to participate in the target RNA process. These findings suggested that AES is an essential protein for the assembly of photosynthetic complexes, providing insights into the splicing of psbB operon (psbB-psbT-psbH-petB-petD), ycf3, and ndhA, as well as maintaining chloroplast homeostasis.

As a fundamental metabolic pathway, autophagy plays important roles in plant growth and development, particularly under stress conditions. A set of autophagy-related (ATG) proteins is recruited for the formation of a double-membrane autophagosome. Among them, the essential roles of ATG2, ATG18, and ATG9 have been well established in plant autophagy via genetic analysis; however, the underlying molecular mechanism for ATG2 in plant autophagosome formation remains poorly understood. In this study, we focused on the specific role of ATG2 in the trafficking of ATG18a and ATG9 during autophagy in Arabidopsis (Arabidopsis thaliana). Under normal conditions, YFP-ATG18a proteins are partially localized on late endosomes and translocated to ATG8e-labeled autophagosomes upon autophagic induction. Real-time imaging analysis revealed sequential recruitment of ATG18a on the phagophore membrane, showing that ATG18a specifically decorated the closing edges and finally disassociated from the completed autophagosome. However, in the absence of ATG2, most of the YFP-ATG18a proteins are arrested on autophagosomal membranes. Ultrastructural and 3D tomography analysis showed that unclosed autophagosome structures are accumulated in the atg2 mutant, displaying direct connections with the endoplasmic reticulum membrane and vesicular structures. Dynamic analysis of ATG9 vesicles suggested that ATG2 depletion also affects the association between ATG9 vesicles and the autophagosomal membrane. Furthermore, using interaction and recruitment analysis, we mapped the interaction relationship between ATG2 and ATG18a, implying a possible role of ATG18a in recruiting ATG2 and ATG9 to the membrane. Our findings unveil a specific role of ATG2 in coordinating ATG18a and ATG9 trafficking to mediate autophagosome closure in Arabidopsis.

Pollen tubes extend rapidly via tip growth. This process depends on a dynamic actin cytoskeleton, which has been implicated in controlling organelle movements, cytoplasmic streaming, vesicle trafficking, and cytoplasm organization in pollen tubes. In this update review, we describe the progress in understanding the organization and regulation of the actin cytoskeleton and the function of the actin cytoskeleton in controlling vesicle traffic and cytoplasmic organization in pollen tubes. We also discuss the interplay between ion gradients and the actin cytoskeleton that regulates the spatial arrangement and dynamics of actin filaments and the organization of the cytoplasm in pollen tubes. Finally, we describe several signaling components that regulate actin dynamics in pollen tubes.

Chromoplasts are plant organelles with a unique ability to sequester and store massive carotenoids. Chromoplasts have been hypothesized to enable high levels of carotenoid accumulation due to enhanced sequestration ability or sequestration substructure formation. However, the regulators that control the substructure component accumulation and substructure formation in chromoplasts remain unknown. In melon (Cucumis melo) fruit, β-carotene accumulation in chromoplasts is governed by ORANGE (OR), a key regulator for carotenoid accumulation in chromoplasts. By using comparative proteomic analysis of a high β-carotene melon variety and its isogenic line low-β mutant that is defective in CmOr with impaired chromoplast formation, we identified carotenoid sequestration protein FIBRILLIN1 (CmFBN1) as differentially expressed. CmFBN1 expresses highly in melon fruit tissue. Overexpression of CmFBN1 in transgenic Arabidopsis (Arabidopsis thaliana) containing ORHis that genetically mimics CmOr significantly enhances carotenoid accumulation, demonstrating its involvement in CmOR-induced carotenoid accumulation. Both in vitro and in vivo evidence showed that CmOR physically interacts with CmFBN1. Such an interaction occurs in plastoglobules and results in promoting CmFBN1 accumulation. CmOR greatly stabilizes CmFBN1, which stimulates plastoglobule proliferation and subsequently carotenoid accumulation in chromoplasts. Our findings show that CmOR directly regulates CmFBN1 protein levels and suggest a fundamental role of CmFBN1 in facilitating plastoglobule proliferation for carotenoid sequestration. This study also reveals an important genetic tool to further enhance OR-induced carotenoid accumulation in chromoplasts in crops.

Japanese apricot (Prunus mume Sieb. et Zucc.) is a traditional fruit tree with a long history. Multiple pistils (MP) lead to the formation of multiple fruits, decreasing fruit quality and yield. In this study, the morphology of flowers was observed at 4 stages of pistil development: undifferentiated stage (S1), predifferentiation stage (S2), differentiation stage (S3), and late differentiation stage (S4). In S2 and S3, the expression of PmWUSCHEL (PmWUS) in the MP cultivar was significantly higher than that in the single-pistil (SP) cultivar, and the gene expression of its inhibitor, PmAGAMOUS (PmAG), also showed the same trend, indicating that other regulators participate in the regulation of PmWUS during this period. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) showed that PmAG could bind to the promoter and the locus of PmWUS, and H3K27me3 repressive marks were also detected at these sites. The SP cultivar exhibited an elevated level of DNA methylation in the promoter region of PmWUS, which partially overlapped with the region of histone methylation. This suggests that the regulation of PmWUS involves both transcription factors and epigenetic modifications. Also, the gene expression of Japanese apricot LIKE HETEROCHROMATIN PROTEIN (PmLHP1), an epigenetic regulator, in MP was significantly lower than that in SP in S2 to 3, contrary to the trend in expression of PmWUS. Our results showed that PmAG recruited sufficient PmLHP1 to maintain the level of H3K27me3 on PmWUS during the S2 of pistil development. This recruitment of PmLHP1 by PmAG inhibits the expression of PmWUS at the precise time, leading to the formation of 1 normal pistil primordium.

Citrus, 1 of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and downregulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene β-cyclase (CsLCYb2), 2 key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

Bud dormancy is crucial for winter survival and is characterized by the inability of the bud meristem to respond to growth-promotive signals before the chilling requirement (CR) is met. However, our understanding of the genetic mechanism regulating CR and bud dormancy remains limited. This study identified PpDAM6 (DORMANCY-ASSOCIATED MADS-box) as a key gene for CR using a genome-wide association study analysis based on structural variations in 345 peach (Prunus persica (L.) Batsch) accessions. The function of PpDAM6 in CR regulation was demonstrated by transiently silencing the gene in peach buds and stably overexpressing the gene in transgenic apple (Malus × domestica) plants. The results showed an evolutionarily conserved function of PpDAM6 in regulating bud dormancy release, followed by vegetative growth and flowering, in peach and apple. The 30-bp deletion in the PpDAM6 promoter was substantially associated with reducing PpDAM6 expression in low-CR accessions. A PCR marker based on the 30-bp indel was developed to distinguish peach plants with non-low and low CR. Modification of the H3K27me3 marker at the PpDAM6 locus showed no apparent change across the dormancy process in low- and non-low- CR cultivars. Additionally, H3K27me3 modification occurred earlier in low-CR cultivars on a genome-wide scale. PpDAM6 could mediate cell-cell communication by inducing the expression of the downstream genes PpNCED1 (9-cis-epoxycarotenoid dioxygenase 1), encoding a key enzyme for ABA biosynthesis, and CALS (CALLOSE SYNTHASE), encoding callose synthase. We shed light on a gene regulatory network formed by PpDAM6-containing complexes that mediate CR underlying dormancy and bud break in peach. A better understanding of the genetic basis for natural variations of CR can help breeders develop cultivars with different CR for growing in different geographical regions.

Root-knot nematodes (Meloidogyne spp., RKN) are responsible for extensive crop losses worldwide. During infection, they penetrate plant roots, migrate between plant cells, and establish feeding sites, known as giant cells, near the root vasculature. Previously, we found that nematode perception and early responses in plants were similar to those of microbial pathogens and required the BRI1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 (BAK1/SERK3) coreceptor in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum). Here, we implemented a reverse genetic screen for resistance or sensitivity to RKN using Arabidopsis T-DNA alleles of genes encoding transmembrane receptor-like kinases to identify additional receptors involved in this process. This screen identified a pair of allelic mutations with enhanced resistance to RKN in a gene we named ENHANCED RESISTANCE TO NEMATODES1 (ERN1). ERN1 encodes a G-type lectin receptor kinase (G-LecRK) with a single-pass transmembrane domain. Further characterization showed that ern1 mutants displayed stronger activation of MAP kinases, elevated levels of the defense marker MYB51, and enhanced H2O2 accumulation in roots upon RKN elicitor treatments. Elevated MYB51 expression and ROS bursts were also observed in leaves of ern1 mutants upon flg22 treatment. Complementation of ern1.1 with 35S- or native promoter-driven ERN1 rescued the RKN infection and enhanced defense phenotypes. Our results indicate that ERN1 is an important negative regulator of immunity.

Drought stress limits woody species productivity and influences tree distribution. However, dissecting the molecular mechanisms that underpin drought responses in forest trees can be challenging due to trait complexity. Here, using a panel of 300 Chinese white poplar (Populus tomentosa) accessions collected from different geographical climatic regions in China, we performed a genome-wide association study (GWAS) on seven drought-related traits and identified PtoWRKY68 as a candidate gene involved in the response to drought stress. A 12-bp insertion and/or deletion and three nonsynonymous variants in the PtoWRKY68 coding sequence categorized natural populations of P. tomentosa into two haplotype groups, PtoWRKY68hap1 and PtoWRKY68hap2. The allelic variation in these two PtoWRKY68 haplotypes conferred differential transcriptional regulatory activities and binding to the promoters of downstream abscisic acid (ABA) efflux and signaling genes. Overexpression of PtoWRKY68hap1 and PtoWRKY68hap2 in Arabidopsis (Arabidopsis thaliana) ameliorated the drought tolerance of two transgenic lines and increased ABA content by 42.7% and 14.3% compared to wild-type plants, respectively. Notably, PtoWRKY68hap1 (associated with drought tolerance) is ubiquitous in accessions in water-deficient environments, whereas the drought-sensitive allele PtoWRKY68hap2 is widely distributed in well-watered regions, consistent with the trends in local precipitation, suggesting that these alleles correspond to geographical adaptation in Populus. Moreover, quantitative trait loci analysis and an electrophoretic mobility shift assay showed that SHORT VEGETATIVE PHASE (PtoSVP.3) positively regulates the expression of PtoWRKY68 under drought stress. We propose a drought tolerance regulatory module in which PtoWRKY68 modulates ABA signaling and accumulation, providing insight into the genetic basis of drought tolerance in trees. Our findings will facilitate molecular breeding to improve the drought tolerance of forest trees.

The identification of chemical compounds that affect intracellular processes has greatly contributed to our understanding of plant growth and development. In most cases, these compounds have been identified in germinated seedlings. However, chemical screening using mature plants would benefit and advance our understanding of environmental responses. In this study, we developed a high-throughput screening method using single leaves of mature plants to identify small molecules that affect cold-regulated gene expression. A single excised leaf of Arabidopsis (Arabidopsis thaliana) grown in submerged cultures responded to low temperatures in terms of COLD-REGULATED (COR) gene expression. We used transgenic Arabidopsis harboring a COLD-REGULATED 15A (COR15A) promoter::luciferase (COR15Apro::LUC) construct to screen natural compounds that affect the cold induction of COR15Apro::LUC. This approach allowed us to identify derivatives of 1,4-naphthoquinone as specific inhibitors of COR gene expression. Moreover, 1,4-naphthoquinones appeared to inhibit the rapid induction of upstream C-REPEAT BINDING FACTOR (CBF) transcription factors upon exposure to low temperature, suggesting that 1,4-naphthoquinones alter upstream signaling processes. Our study offers a chemical screening scheme for identifying compounds that affect environmental responses in mature plants. This type of analysis is likely to reveal an unprecedented link between certain compounds and plant environmental responses.

Cleavage and polyadenylation specificity factor (CPSF) is a protein complex that plays an essential biochemical role in mRNA 3'-end formation, including poly(A) signal recognition and cleavage at the poly(A) site. However, its biological functions at the organismal level are mostly unknown in multicellular eukaryotes. The study of plant CPSF73 has been hampered by the lethality of Arabidopsis (Arabidopsis thaliana) homozygous mutants of AtCPSF73-I and AtCPSF73-II. Here, we used poly(A) tag sequencing to investigate the roles of AtCPSF73-I and AtCPSF73-II in Arabidopsis treated with AN3661, an antimalarial drug with specificity for parasite CPSF73 that is homologous to plant CPSF73. Direct seed germination on an AN3661-containing medium was lethal; however, 7-d-old seedlings treated with AN3661 survived. AN3661 targeted AtCPSF73-I and AtCPSF73-II, inhibiting growth through coordinating gene expression and poly(A) site choice. Functional enrichment analysis revealed that the accumulation of ethylene and auxin jointly inhibited primary root growth. AN3661 affected poly(A) signal recognition, resulted in lower U-rich signal usage, caused transcriptional readthrough, and increased the distal poly(A) site usage. Many microRNA targets were found in the 3' untranslated region lengthened transcripts; these miRNAs may indirectly regulate the expression of these targets. Overall, this work demonstrates that AtCPSF73 plays important part in co-transcriptional regulation, affecting growth, and development in Arabidopsis.

Asymmetric cell division generates different cell types and is a feature of development in multicellular organisms. Prior to asymmetric cell division, cell polarity is established. Maize (Zea mays) stomatal development serves as an excellent plant model system for asymmetric cell division, especially the asymmetric division of the subsidiary mother cell (SMC). In SMCs, the nucleus migrates to a polar location after the accumulation of polarly localized proteins but before the appearance of the preprophase band. We examined a mutant of an outer nuclear membrane protein that is part of the LINC (linker of nucleoskeleton and cytoskeleton) complex that localizes to the nuclear envelope in interphase cells. Previously, maize linc kash sine-like2 (mlks2) was observed to have abnormal stomata. We confirmed and identified the precise defects that lead to abnormal asymmetric divisions. Proteins that are polarly localized in SMCs prior to division polarized normally in mlks2. However, polar localization of the nucleus was sometimes impaired, even in cells that have otherwise normal polarity. This led to a misplaced preprophase band and atypical division planes. MLKS2 localized to mitotic structures; however, the structure of the preprophase band, spindle, and phragmoplast appeared normal in mlks2. Time-lapse imaging revealed that mlks2 has defects in premitotic nuclear migration toward the polarized site and unstable position at the division site after formation of the preprophase band. Overall, our results show that nuclear envelope proteins promote premitotic nuclear migration and stable nuclear position and that the position of the nucleus influences division plane establishment in asymmetrically dividing cells.

Soil acidification in apple (Malus domestica) orchards results in the release of rhizotoxic aluminum ions (Al3+) into soil. Melatonin (MT) participates in plant responses to abiotic stress; however, its role in AlCl3 stress in apple remains unknown. In this study, root application of MT (1 μM) substantially alleviated AlCl3 stress (300 μM) in Pingyi Tiancha (Malus hupehensis), which was reflected by higher fresh and dry weight, increased photosynthetic capacity, and longer and more roots compared with plants that did not receive MT treatment. MT functioned mainly by regulating vacuolar H+/Al3+ exchange and maintaining H+ homeostasis in the cytoplasm under AlCl3 stress. Transcriptome deep sequencing analysis identified the transcription factor gene SENSITIVE TO PROTON RHIZOTOXICITY 1 (MdSTOP1) was induced by both AlCl3 and MT treatments. Overexpressing MdSTOP1 in apple increased AlCl3 tolerance by enhancing vacuolar H+/Al3+ exchange and H+ efflux to the apoplast. We identified 2 transporter genes, ALUMINUM SENSITIVE 3 (MdALS3) and SODIUM HYDROGEN EXCHANGER 2 (MdNHX2), as downstream targets of MdSTOP1. MdSTOP1 interacted with the transcription factor NAM ATAF and CUC 2 (MdNAC2) to induce MdALS3 expression, which reduced Al toxicity by transferring Al3+ from the cytoplasm to the vacuole. Furthermore, MdSTOP1 and MdNAC2 coregulated MdNHX2 expression to increase H+ efflux from the vacuole to the cytoplasm to promote Al3+ compartmentalization and maintain cation balance in the vacuole. Taken together, our findings reveal an MT-STOP1 + NAC2-NHX2/ALS3-vacuolar H+/Al3+ exchange model for the alleviation of AlCl3 stress in apple, laying a foundation for practical applications of MT in agriculture.

The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infects Arabidopsis (Arabidopsis thaliana) by forming structures called haustoria inside host cells. Previous transcriptome analyses have revealed that host genes are specifically induced during infection; however, RNA profiling from whole-infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells, where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis and H. arabidopsidis at the cellular level, we devised a translating ribosome affinity purification system using 2 high-affinity binding proteins, colicin E9 and Im9 (immunity protein of colicin E9), applicable to pathogen-responsive promoters, thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing insights into the Arabidopsis-downy mildew interaction. We propose that our protocol for profiling cell-specific transcripts will apply to several stimulus-specific contexts and other plant-pathogen interactions.

Movement proteins (MPs) encoded by plant viruses deliver viral genomes to plasmodesmata (PD) to ensure intracellular and intercellular transport. However, how the MPs encoded by monopartite geminiviruses are targeted to PD is obscure. Here, we demonstrate that the C5 protein of tomato yellow leaf curl virus (TYLCV) anchors to PD during the viral infection following trafficking from the nucleus along microfilaments in Nicotiana benthamiana. C5 could move between cells and partially complement the traffic of a movement-deficient turnip mosaic virus (TuMV) mutant (TuMV-GFP-P3N-PIPO-m1) into adjacent cells. The TYLCV-C5 null mutant (TYLCV-mC5) attenuates viral pathogenicity and decreases viral DNA and protein accumulation, and ectopic overexpression of C5 enhances viral DNA accumulation. Interaction assays between TYLCV-C5 and the other eight viral proteins described in TYLCV reveal that C5 associates with C2 in the nucleus and with V2 in the cytoplasm and at PD. The V2 protein is mainly localized in the nucleus and cytoplasmic granules when expressed alone; in contrast, V2 forms small punctate granules at PD when co-expressed with C5 or in TYLCV-infected cells. The interaction of V2 and C5 also facilitates their nuclear export. Furthermore, C5-mediated PD localization of V2 is conserved in two other geminiviruses. Therefore, this study solves a long-sought-after functional connection between PD and the geminivirus movement and improves our understanding of geminivirus-encoded MPs and their potential cellular and molecular mechanisms.

Viral RNAs can be uridylated in eukaryotic hosts. However, our knowledge of uridylation patterns and roles remains rudimentary for phytoviruses. Here, we report global 3' terminal RNA uridylation profiles for representatives of the main families of positive single-stranded RNA phytoviruses. We detected uridylation in all 47 viral RNAs investigated here, revealing its prevalence. Yet, uridylation levels of viral RNAs varied from 0.2% to 90%. Unexpectedly, most poly(A) tails of grapevine fanleaf virus (GFLV) RNAs, including encapsidated tails, were strictly monouridylated, which corresponds to an unidentified type of viral genomic RNA extremity. This monouridylation appears beneficial for GFLV because it became dominant when plants were infected with nonuridylated GFLV transcripts. We found that GFLV RNA monouridylation is independent of the known terminal uridylyltransferases (TUTases) HEN1 SUPPRESSOR 1 (HESO1) and UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) in Arabidopsis (Arabidopsis thaliana). By contrast, both TUTases can uridylate other viral RNAs like turnip crinkle virus (TCV) and turnip mosaic virus (TuMV) RNAs. Interestingly, TCV and TuMV degradation intermediates were differentially uridylated by HESO1 and URT1. Although the lack of both TUTases did not prevent viral infection, we detected degradation intermediates of TCV RNA at higher levels in an Arabidopsis heso1 urt1 mutant, suggesting that uridylation participates in clearing viral RNA. Collectively, our work unveils an extreme diversity of uridylation patterns across phytoviruses and constitutes a valuable resource to further decipher pro- and antiviral roles of uridylation.

Drought and flooding are the two most important environmental factors limiting maize (Zea mays L.) production globally. This study aimed to investigate the physiological mechanisms and accurate evaluation indicators and methods of maize germplasm involved in drought and flooding stresses. The twice replicated pot experiments with 60 varieties, combined with the field validation experiment with 3 varieties, were conducted under well-watered, drought, and flooding conditions. Most varieties exhibited stronger tolerance to drought than flooding due to higher antioxidant enzyme activities, osmotic adjustment substances, and lower reactive oxygen species. In contrast, flooding stress resulted in higher levels of reactive oxygen species (particularly O2-), ascorbate peroxidase, catalase, peroxidase, and soluble sugars but lower levels of superoxide dismutase, proline, and soluble protein compared with well-watered conditions. Superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, proline, soluble sugars, and protein contents, in addition to plant height, leaf area/plant, and stem diameter, were accurate and representative indicators for evaluating maize tolerance to drought and flooding stresses and could determine a relatively high mean forecast accuracy of 100.0% for the comprehensive evaluation value. A total of 4 principal components were extracted, in which different principal components played a vital role in resisting different water stresses. Finally, the accuracy of the 3 varieties screened by multivariate analysis was verified in the field. This study provides insights into the different physiological mechanisms and accurate evaluation methods of maize germplasm involved in drought and flooding stresses, which could be valuable for further research and breeding.

The circadian clock is responsible for the temporal regulation of various physiological processes in plants. Individual cells contain a circadian oscillator consisting of a clock gene circuit that coordinates physiological rhythms within the plant body in an orderly manner. The coordination of time information has been studied from the perspective of cell-cell local coupling and long-distance communication between tissues based on the view that the behavior of circadian oscillators represents physiological rhythms. Here, we report the cellular circadian rhythm of bioluminescence reporters that are not governed by the clock gene circuit in expressing cells. We detected cellular bioluminescence rhythms with different free-running periods in the same cells using a dual-color bioluminescence monitoring system in duckweed (Lemna minor) transfected with Arabidopsis CIRCADIAN CLOCK ASSOCIATED 1::luciferace+ (AtCCA1::LUC+) and Cauliflower mosaic virus 35S::modified click-beetle red-color luciferase (CaMV35S::PtRLUC) reporters. Co-transfection experiments with the two reporters and a clock gene-overexpressing effector revealed that the AtCCA1::LUC+ rhythm, but not the CaMV35S::PtRLUC rhythm, was altered in cells with a dysfunctional clock gene circuit. This indicated that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian oscillator, whereas the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared, whereas the AtCCA1::LUC+ rhythm persisted. This suggests that the CaMV35S::PtRLUC bioluminescence has a symplast/apoplast-mediated circadian rhythm generated at the organismal level. The CaMV35S::PtRLUC-type bioluminescence rhythm was also observed when other bioluminescence reporters were expressed. These results reveal that the plant circadian system consists of both cell-autonomous and noncell-autonomous rhythms that are unaffected by cellular oscillators.

Flowering time, maturity, and plant height are crucial agronomic traits controlled by photoperiod that affect soybean (Glycine max [L.] Merr.) yield and regional adaptability. It is important to cultivate soybean cultivars of earlier maturity that adapt to high latitudes. GAMYB-binding protein 1 (GmGBP1), a member of the SNW/SKIP family of transcriptional coregulators in soybean, is induced by short days and interacts with transcription factor GAMYB (GmGAMYB) during photoperiod control of flowering time and maturity. In the present study, GmGBP1:GmGBP1 soybean showed the phenotypes of earlier maturity and higher plant height. Chromatin immunoprecipitation sequencing (ChIP-seq) assays of GmGBP1-binding sites and RNA sequencing (RNA-seq) of differentially expressed transcripts in GmGBP1:GmGBP1 further identified potential targets of GmGBP1, including small auxin-up RNA (GmSAUR). GmSAUR:GmSAUR soybean also showed earlier maturity and higher plant height. GmGBP1 interacted with GmGAMYB, bound to the promoter of GmSAUR and promoted the expression of FLOWER LOCUS T homologs 2a (GmFT2a) and FLOWERING LOCUS D LIKE 19 (GmFDL19). Flowering repressors such as GmFT4 were negatively regulated, resulting in earlier flowering and maturity. Furthermore, the interaction of GmGBP1 with GmGAMYB increased the gibberellin (GA) signal to promote height and hypocotyl elongation by activating GmSAUR and GmSAUR bound to the promoter of the GA-positive activating regulator gibberellic acid-stimulated Arabidopsis 32 (GmGASA32). These results suggested a photoperiod regulatory pathway in which the interaction of GmGBP1 with GmGAMYB directly activated GmSAUR to promote earlier maturity and plant height in soybean.

Plant roots can absorb sugars from the rhizosphere, which reduces the consumption of carbon derived from photosynthesis. However, the underlying mechanisms that roots use to control sugar absorption from soil are poorly understood. Here, we identified an apple (Malus × domestica Borkh.) hexose transporter, MdHT1.2, that functions on the root epidermis to absorb glucose (Glc) from the rhizosphere. Based on RNA-seq data, MdHT1.2 showed the highest expression level among 29 MdHT genes in apple roots. Biochemical analyses demonstrated that MdHT1.2 was mainly expressed in the epidermal cells of fine roots, and its protein was located on the plasma membrane. The roots of transgenic apple and Solanum lycopersicum lines overexpressing MdHT1.2 had an increased capability to absorb Glc when fed with [13C]-labeled Glc or 2-NBDG, whereas silencing MdHT1.2 in apple showed the opposite results. Further studies established that MdHT1.2-mediated Glc absorption from the rhizosphere changed the carbon assimilate allocation between apple shoot and root, which regulated plant growth. Additionally, a grafting experiment in tomato confirmed that increasing the Glc uptake capacity in the root overexpressing MdHT1.2 could facilitate carbohydrate partitioning to the fruit. Collectively, our study demonstrated that MdHT1.2 functions on the root epidermis to absorb rhizospheric Glc, which regulates the carbohydrate allocation for plant growth and fruit sugar accumulation.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in 2 subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22), and chitin, including 1 feature from Candidatus Liberibacter species (csp22CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized 2 genetically linked FLS2 homologs from "Frost Lisbon" lemon (Citrus ×limon, responsive) and "Washington navel" orange (Citrus ×aurantium, nonresponsive). Surprisingly, FLS2 homologs from responsive and nonresponsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. "Washington navel" orange weakly responded to chitin, whereas "Tango" mandarin (C. ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the 2 genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.

Arabidopsis (Arabidopsis thaliana) seeds expressing the feedback-insensitive form of cystathionine γ-synthase (AtD-CGS), the key gene of methionine (Met) synthesis, under the control of a seed-specific phaseolin promoter (SSE plants) show a significant increase in Met content. This elevation is accompanied by increased levels of other amino acids (AAs), sugars, total protein, and starch, which are important from a nutritional aspect. Here, we investigated the mechanism behind this phenomenon. Gas chromatography-mass spectrometry (GC-MS) analysis of SSE leaves, siliques, and seeds collected at 3 different developmental stages showed high levels of Met, AAs, and sugars compared to the control plants. A feeding experiment with isotope-labeled AAs showed an increased flux of AAs from nonseed tissues toward the developing seeds of SSE. Transcriptome analysis of leaves and seeds displayed changes in the status of methylation-related genes in SSE plants that were further validated by methylation-sensitive enzymes and colorimetric assay. These results suggest that SSE leaves have higher DNA methylation rates than control plants. This occurrence apparently led to accelerated senescence, together with enhanced monomer synthesis, which further resulted in increased transport of monomers from the leaves toward the seeds. The developing seeds of SSE plants, however, show reduced Met levels and methylation rates. The results provide insights into the role of Met in DNA methylation and gene expression and how Met affects the metabolic profile of the plant.

Intracellular gene transfers (IGTs) between the nucleus and organelles, including plastids and mitochondria, constantly reshape the nuclear genome during evolution. Despite the substantial contribution of IGTs to genome variation, the dynamic trajectories of IGTs at the pangenomic level remain elusive. Here, we developed an approach, IGTminer, that maps the evolutionary trajectories of IGTs using collinearity and gene reannotation across multiple genome assemblies. We applied IGTminer to create a nuclear organellar gene (NOG) map across 67 genomes covering 15 Poaceae species, including important crops. The resulting NOGs were verified by experiments and sequencing data sets. Our analysis revealed that most NOGs were recently transferred and lineage specific and that Triticeae species tended to have more NOGs than other Poaceae species. Wheat (Triticum aestivum) had a higher retention rate of NOGs than maize (Zea mays) and rice (Oryza sativa), and the retained NOGs were likely involved in photosynthesis and translation pathways. Large numbers of NOG clusters were aggregated in hexaploid wheat during 2 rounds of polyploidization, contributing to the genetic diversity among modern wheat accessions. We implemented an interactive web server to facilitate the exploration of NOGs in Poaceae. In summary, this study provides resources and insights into the roles of IGTs in shaping interspecies and intraspecies genome variation and driving plant genome evolution.

Plants possess well-developed light sensing mechanisms and signal transduction systems for regulating photomorphogenesis. ELONGATED HYPOCOTYL5 (HY5), a basic leucine zipper (bZIP) transcription factor, has been extensively characterized in dicots. In this study, we show that OsbZIP1 is a functional homolog of Arabidopsis (Arabidopsis thaliana) HY5 (AtHY5) and is important for light-mediated regulation of seedling and mature plant development in rice (Oryza sativa). Ectopic expression of OsbZIP1 in rice reduced plant height and leaf length without affecting plant fertility, which contrasts with OsbZIP48, a previously characterized HY5 homolog. OsbZIP1 is alternatively spliced, and the OsbZIP1.2 isoform lacking the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-binding domain regulated seedling development in the dark. Rice seedlings overexpressing OsbZIP1 were shorter than the vector control under white and monochromatic light conditions, whereas RNAi knockdown seedlings displayed the opposite phenotype. While OsbZIP1.1 was light-regulated, OsbZIP1.2 showed a similar expression profile in both light and dark conditions. Due to its interaction with OsCOP1, OsbZIP1.1 undergoes 26S proteasome-mediated degradation under dark conditions. Also, OsbZIP1.1 interacted with and was phosphorylated by CASEIN KINASE2 (OsCK2α3). In contrast, OsbZIP1.2 did not show any interaction with OsCOP1 or OsCK2α3. We propose that OsbZIP1.1 likely regulates seedling development in the light, while OsbZIP1.2 is the dominant player under dark conditions. The data presented in this study reveal that AtHY5 homologs in rice have undergone neofunctionalization, and alternative splicing of OsbZIP1 has increased the repertoire of its functions.

Somatic embryogenesis (SE), like zygotic embryo development, is a progressive process. Early SE is the beginning of a switch from a somatic to an embryogenic state and is an important stage for initiating chromatin reprogramming of SE. Previous studies suggest that changes in chromatin accessibility occur during early SE, although information on the 3D structure of chromatin is not yet available. Here, we present a chromosome-level genome assembly of longan (Dimocarpus longan) using PacBio combined with high-through chromosome conformation capture scaffolding, which resulted in a 446 Mb genome assembly anchored onto 15 scaffolds. During early SE, chromatin was concentrated and then decondensed, and a large number of long terminal repeat retrotransposons (LTR-RTs) were enriched in the local chromatin interaction region, suggesting LTR-RTs were involved in chromatin reorganization. Early SE was accompanied by the transformation from A to B compartments, and the interactions between B compartments were enhanced. Results from chromatin accessibility, monomethylation of histone H3 at lysine 4 (H3K4me1) modification, and transcription analyses further revealed a gene regulatory network for cell wall thickening during SE. Particularly, we found that the H3K4me1 differential peak binding motif showed abnormal activation of ethylene response factor transcription factors and participation in SE. The chromosome-level genomic and multiomics analyses revealed the 3D conformation of chromatin during early SE, providing insight into the molecular mechanisms underlying cell wall thickening and the potential regulatory networks of TFs during early SE in D. longan. These results provide additional clues for revealing the molecular mechanisms of plant SE.

Flowers are critical for angiosperm reproduction and the production of food, fiber, and pharmaceuticals, yet for unknown reasons, they appear particularly sensitive to combined heat and drought stress. A possible explanation for this may be the co-occurrence of leaky cuticles in flower petals and a vascular system that has a low capacity to supply water and is prone to failure under water stress. These characteristics may render reproductive structures more susceptible than leaves to runaway cavitation-an uncontrolled feedback cycle between rising water stress and declining water transport efficiency that can rapidly lead to lethal tissue desiccation. We provide modeling and empirical evidence to demonstrate that flower damage in the perennial crop pyrethrum (Tanacetum cinerariifolium), in the form of irreversible desiccation, corresponds with runaway cavitation in the flowering stem after a combination of heat and water stress. We show that tissue damage is linked to greater evaporative demand during high temperatures rather than direct thermal stress. High floral transpiration dramatically reduced the soil water deficit at which runaway cavitation was triggered in pyrethrum flowering stems. Identifying runaway cavitation as a mechanism leading to heat damage and reproductive losses in pyrethrum provides different avenues for process-based modeling to understand the impact of climate change on cultivated and natural plant systems. This framework allows future investigation of the relative susceptibility of diverse plant species to reproductive failure under hot and dry conditions.

Grain size is important for yield in rice (Oryza sativa L.). Although many genes involved in grain size have been isolated, few can be used in breeding due to their interactions and phenotypic effects. Here, we describe natural variation in the granule-type quantitative trait locus GRAIN SIZE AND WEIGHT 3 (GSW3) located on chromosome 3 in wild rice (Oryza rufipogon Griff.) that encodes a GTPase-regulated protein and negatively regulates grain length, grain width, and 1,000-grain weight. The insertion of a 232-bp fragment of the genomic sequence in the wild rice, a natural allelic variant gene (GSW3), increased the expression levels and reduced the grain length and width and 1,000-grain weight. Knockout of GSW3 in the wild rice inbred line Huaye 3 increased the grain length and width and 1,000-grain weight. Introducing GSW3Huaye3 into cultivated rice line KJ01 and overexpressing GSW3Huaye3 in Huaye 3 resulted in reduced grain length and width and 1,000-grain weight, and grain size and 1,000-grain weight changes were closely related to GSW3 expression levels. GSW3 regulated the grain length and width simultaneously by promoting grain glume cell division and longitudinal and transverse cell growth. GSW3 was also involved in regulating the gibberellic acid signaling pathway and negatively regulated plant growth. Furthermore, a critical SNP in the GSW3 coding region was obviously correlated with grain size variation in a core collection of cultivated rice. This SNP resulted in an amino acid substitution from Gln to Arg at position 161 in GSW3, which reduced the grain size. Our study shows that GSW3 negatively regulates the grain shape, which could explain different grain shapes in modern cultivars and wild rice. GSW3 may also be used for breeding rice varieties with improved grain shapes and higher yield.

Late embryogenesis abundant (LEA) proteins are important stress-response proteins that participate in protecting plants against abiotic stresses. Here, we investigated LEA group 3 protein MsLEA1, containing the typically disordered and α-helix structure, via overexpression and RNA interference (RNAi) approaches in alfalfa (Medicago sativa L.) under drought and aluminum (Al) stresses. MsLEA1 highly expressed in leaves and localized in chloroplasts. Overexpressing MsLEA1 increased alfalfa tolerance to drought and Al stresses, but downregulating MsLEA1 decreased the tolerance. We observed a larger stomatal aperture and a lower water use efficiency in MsLEA1 RNAi lines compared to wild-type plants under drought stress. Photosynthetic rate, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, and superoxide dismutase (SOD) activity increased or decreased in MsLEA1-OE or -RNAi lines, respectively, under drought and Al stress. Cu/Zn-SOD, Fe-SOD and Rubisco large subunit proteins (Ms1770) were identified as binding partners of MsLEA1, which protected chloroplast structure and function under drought and Al stress. These results indicate that MsLEA1 recruits and protects its target proteins (SOD and Ms1770) and increases alfalfa tolerance against drought and Al stresses.

The plant cell wall (CW) is one of the most important physical barriers phytopathogens must conquer to invade their hosts. This barrier is a dynamic structure that responds to pathogen infection through a complex network of immune receptors, together with CW-synthesizing and CW-degrading enzymes. Callose deposition in the primary CW is a well-known physical response to pathogen infection. Notably, callose and cellulose biosynthesis share an initial substrate, UDP-glucose, which is the main load-bearing component of the CW. However, how these two critical biosynthetic processes are balanced during plant-pathogen interactions remains unclear. Here, using two different pathogen-derived molecules, bacterial flagellin (flg22) and the diffusible signal factor (DSF) produced by Xanthomonas campestris pv. campestris, we show a negative correlation between cellulose and callose biosynthesis in Arabidopsis (Arabidopsis thaliana). By quantifying the abundance of callose and cellulose under DSF or flg22 elicitation and characterizing the dynamics of the enzymes involved in the biosynthesis and degradation of these two polymers, we show that the balance of these two CW components is mediated by the activity of a β-1,3-glucanase (BG2). Our data demonstrate balanced cellulose and callose biosynthesis during plant immune responses.

Cold affects the growth and development of plants. MYB transcription factors and histone H3K4me3 transferases ARABIDOPSIS TRITHORAXs (ATXs) play important regulatory functions in the process of plant resistance to low temperature stress. In this study, DgMYB expression was responsive to low temperature, and overexpression of DgMYB led to increased tolerance, whereas the dgmyb mutant resulted in decreased tolerance of Chrysanthemum morifolium (Dendranthema grandiflorum var. Jinba) to cold stresses. Interestingly, we found that only peroxidase (POD) activity differed substantially between WT, overexpression lines, and the mutant line. A DgATX H3K4me3 methylase that interacts with DgMYB was isolated by further experiments. DgATX expression was also responsive to low temperature. Overexpression of DgATX led to increased tolerance, whereas the dgatx mutant resulted in decreased tolerance of chrysanthemum to cold stresses. Moreover, the dgmyb, dgatx, and dgmyb dgatx double mutants all led to reduced H3K4me3 levels at DgPOD, thus reducing DgPOD expression. Together, our results show that DgMYB interacts with DgATX, allowing DgATX to specifically target DgPOD, altering H3K4me3 levels, increasing DgPOD expression, and thereby reducing the accumulation of ROS in chrysanthemum.

The efficient infection of plants by the bacteria Xanthomonas campestris pv. campestris (Xcc) depends on its type III effectors (T3Es). Although the functions of AvrE-family T3Es have been reported in some bacteria, the member (XopAM) in Xcc has not been studied. As XopAM has low sequence similarity to reported AvrE-T3Es, and different reports have shown that these T3Es have different targets in hosts, we investigated the functions of XopAM in the Xcc-plant interaction. Deletion of xopAM from Xcc reduced its virulence in cruciferous crops but increased virulence in Arabidopsis (Arabidopsis thaliana) Col-0, indicating that XopAM may perform opposite functions depending on the host species. We further found that XopAM is a lipase that may target the cytomembrane and that this activity might be enhanced by its membrane-targeted protein XOPAM-ACTIVATED RESISTANCE 1 (AMAR1) in Arabidopsis Col-0. The binding of XopAM to AMAR1 induced an intense hypersensitive response that restricted Xcc proliferation. Our results showed that the roles of XopAM in Xcc infection are not the same as those of other AvrE-T3Es, indicating that the functions of this type of T3E have differentiated during long-term bacterium‒host interactions.

Plant flowering time is induced by environmental and endogenous signals perceived by the plant. The MCM1-AGAMOUSDEFICIENS-SRF-box (MADS-box) protein SHORT VEGETATIVE PHASE (SVP) is a pivotal repressor that negatively regulates the floral transition during the vegetative phase; however, the transcriptional regulatory mechanism remains poorly understood. Here, we report CmSVP, a chrysanthemum (Chrysanthemum morifolium Ramat.) homologue of SVP, can repress the expression of a key flowering gene, a chrysanthemum FLOWERING LOCUS T-like gene (CmFTL3), by binding its promoter CArG element to delay flowering in the ambient temperature pathway in chrysanthemum. Protein-protein interaction assays identified an interaction between CmSVP and CmTPL1-2, a chrysanthemum homologue of TOPLESS (TPL) that plays critical roles as transcriptional corepressor in many aspects of plant life. Genetic analyses revealed the CmSVP-CmTPL1-2 transcriptional complex is a prerequisite for CmSVP to act as a floral repressor. Furthermore, overexpression of CmSVP rescued the phenotype of the svp-31 mutant in Arabidopsis (Arabidopsis thaliana), overexpression of AtSVP or CmSVP in the Arabidopsis dominant negative mutation tpl-1 led to ineffective late flowering, and AtSVP interacted with AtTPL, confirming the conserved function of SVP in chrysanthemum and Arabidopsis. We have validated a conserved machinery wherein SVP partially relies on TPL to inhibit flowering via a thermosensory pathway.

Grafting can facilitate better scion performance and is widely used in plants. Numerous studies have studied the involvement of mRNAs, small RNAs, and epigenetic regulations in the grafting process. However, it remains unclear whether the mRNA N6-methyladenosine (m6A) modification participates in the apple (Malus x domestica Borkh.) grafting process. Here, we decoded the landscape of m6A modification profiles in 'Golden delicious' (a cultivar, Gd) and M. prunifolia 'Fupingqiuzi' (a unique rootstock with resistance to environmental stresses, Mp), as well as their heterografted and self-grafted plants. Interestingly, global hypermethylation of m6A occurred in both heterografted scion and rootstock compared with their self-grafting controls. Gene Ontology (GO) term enrichment analysis showed that grafting-induced differentially m6A-modified genes were mainly involved in RNA processing, epigenetic regulation, stress response, and development. Differentially m6A-modified genes harboring expression alterations were mainly involved in various stress responses and fatty acid metabolism. Furthermore, grafting-induced mobile mRNAs with m6A and gene expression alterations mainly participated in ABA synthesis and transport (e.g., carotenoid cleavage dioxygenase 1 (CCD1) and ATP-binding cassette G22 (ABCG22)) and abiotic and biotic stress responses, which might contribute to the better performance of heterografted plants. Additionally, the DNA methylome analysis also demonstrated the DNA methylation alterations during grafting. Down-regulated expression of m6A methyltransferase gene MdMTA (ortholog of METTL3) in apples induced the global m6A hypomethylation and distinctly activated the expression level of DNA demethylase gene MdROS1 (REPRESSOR OF SILENCING 1) showing the possible association between m6A and 5mC methylation in apples. Our results reveal the m6A modification profiles in the apple grafting process and enhance our understanding of the m6A regulatory mechanism in plant biological processes.

In thylakoid membranes, photosystem II (PSII) monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while PSII monomers (PSIIm) from granal regions lack these subunits. Here, we have isolated and characterized these 2 types of PSII complexes in tobacco (Nicotiana tabacum). PSIIm-S/27 showed enhanced fluorescence, the near absence of oxygen evolution, and limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•- couple, which controls the charge recombination route, and this limits chlorophyll triplet-mediated 1O2 formation. These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit using a bicarbonate-mediated switch and protective mechanism.

Light, temperature, water, and nutrient availability influence how plants grow to maximize access to resources. Axial growth, the linear extension of tissues by coordinated axial cell expansion, plays a central role in these adaptive morphological responses. Using Arabidopsis (Arabidopsis thaliana) hypocotyl cells to explore axial growth control mechanisms, we investigated WAVE-DAMPENED2-LIKE4 (WDL4), an auxin-induced, microtubule-associated protein and member of the larger WDL gene family shown to modulate hypocotyl growth under changing environmental conditions. Loss-of-function wdl4 seedlings exhibited a hyper-elongation phenotype under light conditions, continuing to elongate when wild-type Col-0 hypocotyls arrested and reaching 150% to 200% of wild-type length before shoot emergence. wdl4 seedling hypocotyls showed dramatic hyper-elongation (500%) in response to temperature elevation, indicating an important role in morphological adaptation to environmental cues. WDL4 was associated with microtubules under both light and dark growth conditions, and no evidence was found for altered microtubule array patterning in loss-of-function wdl4 mutants under various conditions. Examination of hormone responses showed altered sensitivity to ethylene and evidence for changes in the spatial distribution of an auxin-dependent transcriptional reporter. Our data provide evidence that WDL4 regulates hypocotyl cell elongation without substantial changes to microtubule array patterning, suggesting an unconventional role in axial growth control.

WUSCHEL (WUS) and WUSCHEL-RELATED HOMEOBOX (WOX) proteins determine stem cell maintenance for continual plant growth and development under changing environmental conditions. Nutrient availability is an environmental factor that substantially controls plant growth and development. However, how plant stem cell homeostasis is regulated under nutrient deficiency remains to be elucidated. Here, we showed that cytosolic isocitrate dehydrogenase (ICDH) plays an important role in nutrient sensing of stem cells in Arabidopsis (Arabidopsis thaliana). Nutrient deficiency induced the cytoplasmic-to-nuclear translocation of cytosolic ICDH protein. ICDH can interact with WUS/WOX protein as a complex that further promotes WUS/WOX expression by binding to its promoter. WUS/WOX expression in the icdh-2 mutant was lower than that of wild-type plants under nutrient deficiency. Consistently, loss of ICDH function caused a more serious growth repression under nutrient deficiency that was independent of ICDH's catalytic activity. Therefore, cytosolic ICDH regulates stem cell homeostasis of plants in response to nutrient deficiency.

Waterlogging stress (WLS) negatively impacts the growth and yield of crops resulting in heavy losses to agricultural production. Previous studies have revealed that WLS induces a systemic response in shoots that is partially dependent on the plant hormones ethylene and abscisic acid. However, the role of rapid cell-to-cell signaling pathways, such as the reactive oxygen species (ROS) and calcium waves, in systemic responses of plants to WLS is unknown at present. Here we reveal that an abrupt WLS treatment of Arabidopsis (Arabidopsis thaliana) plants growing in peat moss triggers systemic ROS and calcium wave responses and that the WLS-triggered ROS wave response of Arabidopsis is dependent on the ROS-generating RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), calcium-permeable channels GLUTAMATE-LIKE RECEPTOR 3.3 and 3.6 (GLR3.3 and GLR3.6), and aquaporin PLASMA MEMBRANE INTRINSIC PROTEIN 2; 1 (PIP2; 1) proteins. We further show that WLS is accompanied by a rapid systemic transcriptomic response that is evident as early as 10 min following waterlogging initiation, includes many hypoxia-response transcripts, and is partially dependent on RBOHD. Interestingly, the abrupt WLS of Arabidopsis resulted in the triggering of a rapid hydraulic wave response and the transient opening of stomata on leaves. In addition, it induced in plants a heightened state of tolerance to a subsequent submergence stress. Taken together, our findings reveal that the initiation of WLS in plants is accompanied by rapid systemic physiological and transcriptomic responses that involve the ROS, calcium, and hydraulic waves, as well as the induction of hypoxia acclimation mechanisms in systemic tissues.

Chloroplast-to-nucleus retrograde signaling (RS) pathways are critical in modulating plant development and stress adaptation. Among chloroplast proteins mediating RS pathways, GENOMES UNCOUPLED1 (GUN1) represses the transcription of the nuclear transcription factors GOLDEN2-LIKE1 (GLK1) and GLK2 that positively regulate chloroplast biogenesis. Given the extensive exploration of the function of GUN1 in biogenic RS carried out in previous years, our understanding of its role in plant stress responses remains scarce. Here, we revealed that GUN1 contributes to the expression of salicylic acid (SA)-responsive genes (SARGs) through transcriptional repression of GLK1/2 in Arabidopsis (Arabidopsis thaliana). Loss of GUN1 significantly compromised the SA responsiveness in plants, concomitant with the upregulation of GLK1/2 transcripts. In contrast, knockout of GLK1/2 potentiated the expression of SARGs and led to enhanced stress responses. Chromatin immunoprecipitation, coupled with quantitative PCR and related reverse genetic approaches, unveiled that in gun1, GLK1/2 might modulate SA-triggered stress responses by stimulating the expression of WRKY18 and WRKY40, transcriptional repressors of SARGs. In summary, we demonstrate that a hierarchical regulatory module, consisting of GUN1-GLK1/2-WRKY18/40, modulates SA signaling, opening a research avenue regarding a latent GUN1 function in plant-environment interactions.

Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomic approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole-genome duplicates were typically enriched for CG-only gene body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was a characteristic of more recent single-gene duplicates. Core angiosperm gene families were differentiated into those which preferentially retain paralogs and "duplication-resistant" families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence-absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

Cassava (Manihot esculenta Crantz) is an important staple crop for food security in Africa and South America. The present study describes an integrated genomic and metabolomic approach to the characterization of Latin American cassava germplasm. Classification based on genotyping correlated with the leaf metabolome and indicated a key finding of adaption to specific eco-geographical environments. In contrast, the root metabolome did not relate to genotypic clustering, suggesting the different spatial regulation of this tissue's metabolome. The data were used to generate pan-metabolomes for specific tissues, and the inclusion of phenotypic data enabled the identification of metabolic sectors underlying traits of interest. For example, tolerance to whiteflies (Aleurotrachelus socialis) was not linked directly to cyanide content but to cell wall-related phenylpropanoid or apocarotenoid content. Collectively, these data advance the community resources and provide valuable insight into new candidate parental breeding materials with traits of interest directly related to combating food security.

Fruit crops cultivated in almost all countries and regions around the world serve as important agricultural commodities of significant economic value because they contribute to overall food security by providing a diverse food and nutrient supply to sustain human life and human health. Recent advances in high-throughput sequencing technologies offer unprecedented opportunities for pursuing genomic and genetic studies of fruit crops. Here, we will review major advances in fruit crop genome sequencing efforts undertaken over the past 15 years that have contributed to significant accumulation of publicly available genomic resources. We will highlight the expanding pool of genomic data that offer unprecedented opportunities to better unravel the genetic origin and domestication of fruit trees, as well as in deciphering the genetics of important horticultural traits of these fruit trees. Furthermore, we will explore how utilization of these genetic features of fruit trees along with new genomic-assisted tools, including genomic selection and gene editing, are informing and guiding plant geneticists and breeders in moving forward in their fruit crop breeding efforts. Finally, we will outline future prospects and unresolved questions that remain in both genomic research and genetic improvement of fruit crops.

NLP7 (NIN-LIKE-PROTEIN 7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its homolog, NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared with single mutants, the nlp6nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of the PNR showed that NLP6 and NLP7 govern ∼50% of nitrate-induced genes, with cluster analysis highlighting 2 distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high- and low-nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in high ammonium conditions. Growth phenotypes and transcriptome data revealed that NLP6 and NLP7 are completely functionally redundant and may act as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that differ depending on nitrogen sources and gene clusters.

Alternative splicing (AS) is a gene regulatory mechanism that generates multiple transcripts of the same gene precursor by the spliceosome complex, promoting messenger RNA complexity, and proteome diversity. Although AS is extensively studied in response to environmental stresses, whether it mediates age-dependent development and how it is adjusted by growth transitions are largely unknown. Here, we comprehensively explored the AS landscape at different developmental stages in the grass model plant Brachypodium (Brachypodium distachyon). We identified abundant coding genes and noncoding transcripts subject to dynamic AS regulation during juvenile, adult, and reproductive transitions. Moreover, we revealed that SC35-LIKE SPLICING FACTOR 33 (SCL33), a serine/arginine-rich splicing factor in spliceosomes, plays a redundant and antagonistic role with its putative paralog, SCL33L, in regulating intron assembly across distinct developmental stages. In addition, we determined global AS variations in microRNA156 (miR156)-overproducing plants, in which growth transitions are delayed, and found that SPLs were regulated by miR156 in intron retention alteration in addition to mRNA clearance and translation inhibition manners. Finally, we demonstrated a complex regulatory process of age-dependent AS events in B. distachyon that was coincidently or separately regulated by miR156 and SCL33/SCL33L. These results illustrate a substantial machinery of AS that mediates phase transitions in plants.

Light signals promote photomorphogenesis and photosynthesis, allowing plants to establish photoautotrophic growth. Chloroplasts are organelles responsible for photosynthesis in which light energy is converted into chemical energy and stored as organic matter. However, how light regulates chloroplast photomorphogenesis remains unclear. Here, we isolated a cucumber (Cucumis sativus L.) mutant albino seedling (as) from an ethyl methane sulfonate mutagenesis library with an albino phenotype. Map-based cloning revealed that the mutation occurred in a component of cucumber translocon at the inner membrane of chloroplasts (CsTIC21). Subsequently, virus-induced gene silencing and CRISPR/Cas9 analyses confirmed the association between the mutant gene and the as phenotype. Loss-of-function of CsTIC21 induces malformation of chloroplast formation, leading to albinism and death in cucumber. Notably, CsTIC21 transcription was very low in etiolated seedlings grown in the dark and was upregulated by light, with expression patterns similar to those of Nuclear factor-YC (NF-YC) genes. Here, 7 cucumber NF-YC family genes (CsNF-YC) were identified, among which the expression of 4 genes (CsNF-YC1, -YC2, -YC9, and -YC13) responded to light. Gene silencing of all CsNF-YC genes in cucumber indicated that CsNF-YC2, -YC9, -YC11-1, and -YC11-2 induced distinct etiolated growth and decreased chlorophyll content. Interaction studies verified that CsNF-YC2 and CsNF-YC9 target the CsTIC21 promoter directly and promote gene transcription. These findings provide mechanistic insights on the role of the NF-YCs-TIC21 module in chloroplast photomorphogenesis promoted by light in cucumber.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of 5 variants of xanthone 4-prenyltransferase (HpPT4px) comprising 4 long variants (HpPT4px-v1 to HpPT4px-v4) and 1 short variant (HpPT4px-sh). The full-length sequences of all 5 variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone; 1,3,5-trihydroxyxanthone; and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.

Bacterial blight seriously affects the growth and production of cassava (Manihot esculenta Crantz), but disease resistance genes and the underlying molecular mechanism remain unknown. In this study, we found that LESION SIMULATING DISEASE 3 (MeLSD3) is essential for disease resistance in cassava. MeLSD3 physically interacts with SIRTUIN 1 (MeSRT1), inhibiting MeSRT1-mediated deacetylation modification at the acetylation of histone 3 at K9 (H3K9Ac). This leads to increased H3K9Ac levels and transcriptional activation of SUPPRESSOR OF BIR1 (SOBIR1) and FLAGELLIN-SENSITIVE2 (FLS2) in pattern-triggered immunity, resulting in immune responses in cassava. When MeLSD3 was silenced, the release of MeSRT1 directly decreased H3K9Ac levels and inhibited the transcription of SOBIR1 and FLS2, leading to decreased disease resistance. Notably, DELLA protein GIBBERELLIC ACID INSENSITIVE 1 (MeGAI1) also interacted with MeLSD3, which enhanced the interaction between MeLSD3 and MeSRT1 and further strengthened the inhibition of MeSRT1-mediated deacetylation modification at H3K9Ac of defense genes. In summary, this study illustrates the mechanism by which MeLSD3 interacts with MeSRT1 and MeGAI1, thereby mediating the level of H3K9Ac and the transcription of defense genes and immune responses in cassava.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

Glandular trichomes (GTs) are outgrowths of plant epidermal cells that secrete and store specialized secondary metabolites that protect plants against biotic and abiotic stresses and have economic importance for human use. While extensive work has been done to understand the molecular mechanisms of trichome organogenesis in Arabidopsis (Arabidopsis thaliana), which forms unicellular, nonglandular trichomes (NGTs), little is known about the mechanisms of GT development or regulation of secondary metabolites in plants with multicellular GTs. Here, we identified and functionally characterized genes associated with GT organogenesis and secondary metabolism in GTs of cucumber (Cucumis sativus). We developed a method for effective separation and isolation of cucumber GTs and NGTs. Transcriptomic and metabolomic analyses showed that flavonoid accumulation in cucumber GTs is positively associated with increased expression of related biosynthesis genes. We identified 67 GT development-related genes, the functions of 7 of which were validated by virus-induced gene silencing. We further validated the role of cucumber ECERIFERUM1 (CsCER1) in GT organogenesis by overexpression and RNA interference transgenic approaches. We further show that the transcription factor TINY BRANCHED HAIR (CsTBH) serves as a central regulator of flavonoid biosynthesis in cucumber GTs. Work from this study provides insight into the development of secondary metabolite biosynthesis in multicellular GTs.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here, we identified a knockout mutant of oxysterol binding protein-related protein 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar, and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of 2 ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2, and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gβ in the regulation of G protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the endoplasmic reticulum (ER), plasma membrane (PM), and ER-PM contact sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their two phenylalanines in an acidic track-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the pleckstrin homology domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

Somatic embryogenesis (SE) is a key regeneration pathway in various biotechnology approaches to crop improvement, especially for economically important perennial woody crops like citrus. However, maintenance of SE capability has long been a challenge and becomes a bottleneck in biotechnology-facilitated plant improvement. In the embryogenic callus (EC) of citrus, we identified 2 csi-miR171c-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 (CsSCL2/3), which exert positive feedback regulation on csi-miR171c expression. Suppression of CsSCL2 expression by RNA interference (RNAi) enhanced SE in citrus callus. A thioredoxin superfamily protein CsClot was identified as an interactive protein of CsSCL2/3. Overexpression of CsClot disturbed reactive oxygen species (ROS) homeostasis in EC and enhanced SE. Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq identified 660 genes directly suppressed by CsSCL2 that were enriched in biological processes including development-related processes, auxin signaling pathway, and cell wall organization. CsSCL2/3 bound to the promoters of regeneration-related genes, such as WUSCHEL-RELATED HOMEOBOX 2 (CsWOX2), CsWOX13, and Lateral Organ Boundaries Domain 40 (LBD40), and repressed their expression. Overall, CsSCL2/3 modulate ROS homeostasis through the interactive protein CsClot and directly suppress the expression of regeneration-related genes, thus regulating SE in citrus. We uncovered a regulatory pathway of miR171c-targeted CsSCL2/3 in SE, which shed light on the mechanism of SE and regeneration capability maintenance in citrus.

β-1,3-Glucanases are considered key regulators responsible for the degradation of callose in plants, yet little is known about the role and mode of action of their encoding genes in tomato (Solanum lycopersicum). In the present study, we identified the β-1,3-glucanase encoding gene β-1,3-GLUCANASE10 (SlBG10) and revealed its regulation in tomato pollen and fruit development, seed production, and disease resistance by modulating callose deposition. Compared with wild-type (WT) or SlBG10 overexpressing (SlBG10-OE) lines, knockout of SlBG10 caused pollen arrest and failure to set fruit with reduced male rather than female fecundity. Further analyses showed that SlBG10-knockout promoted callose deposition in anther at the tetrad-to-microspore stages, resulting in pollen abortion and male sterility. Moreover, loss-of-function SlBG10 delayed degradation of endosperm cell wall calloses during cellularization and impeded early seed development. We also uncovered that Botrytis cinerea infection induces SlBG10 expression in WT tomato, and the knockout lines showed increased callose accumulation in fruit pericarps, reduced susceptibility to B. cinerea, and enhanced antioxidant capacity to maintain tomato fruit quality. However, the expression of genes encoding cell wall hydrolases decreased in SlBG10-knockout tomatoes and thus led to an increase in pericarp epidermal thickness, enhancement in fruit firmness, reduction of fruit water loss, and extension of tomato shelf life. These findings not only expand our understanding of the involvement of β-1,3-glucanases as callose regulators in multiple developmental processes and pathogen resistance but also provide additional insight into the manipulation of multiagronomic traits for targeted tomato breeding.

As a climbing organ, the tendril undergoes rapid elongation to increase its length to locate support within a short growth time. However, the molecular mechanism underlying this observation is poorly understood. Here, tendril development was divided into 4 stages in cucumber (Cucumis sativus L.) along with its growth. Phenotypic observations and section analyses showed that the rapid elongation of tendril primarily happened during stage 3 and was mainly due to cell expansion. RNA-seq analysis showed that PACLOBUTRAZOL-RESISTANCE4 (CsPRE4) was highly expressed in the tendril. Our RNAi studies in cucumber and transgenic overexpression in Arabidopsis (Arabidopsis thaliana) suggested that CsPRE4 functions as a conserved activator of cell expansion to promote cell expansion and tendril elongation. Through a triantagonistic HLH (helix-loop-helix)-HLH-bHLH (basic helix-loop-helix) cascade, CsPRE4-CsPAR1 (PHYTOCHROME RAPIDLY REGULATED1)-CsBEE1 (BR-ENHANCED EXPRESSION 1), CsPRE4 released the transcription factor CsBEE1, which activated expansin A12 (CsEXPA12) to loosen the cell wall structure in tendrils. Gibberellin (GA) promoted tendril elongation by modulating cell expansion, and CsPRE4 expression was induced by exogenous GA treatment, suggesting that CsPRE4 acts downstream of GA in regulating tendril elongation. In summary, our work suggested a CsPRE4-CsPAR1-CsBEE1-CsEXPA12 pathway in regulating cell expansion in cucumber tendrils, which might enable rapid tendril elongation to quickly locate support.

Transcriptional regulation mediated by combinatorial interaction of transcription factors (TFs) is a key molecular mechanism modulating plant development and metabolism. Basic leucine zipper (bZIP) TFs play important roles in various plant developmental and physiological processes. However, their involvement in fatty acid biosynthesis is largely unknown. Arabidopsis (Arabidopsis thaliana) WRINKLED1 (WRI1) is a pivotal TF in regulation of plant oil biosynthesis and interacts with other positive and negative regulators. In this study, we identified two bZIP TFs, bZIP21 and bZIP52, as interacting partners of AtWRI1 by yeast-two-hybrid (Y2H)-based screening of an Arabidopsis TF library. We found that coexpression of bZIP52, but not bZIP21, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves. The AtWRI1-bZIP52 interaction was further verified by Y2H, in vitro pull-down, and bimolecular fluorescence complementation assays. Transgenic Arabidopsis plants overexpressing bZIP52 showed reduced seed oil accumulation, while the CRISPR/Cas9-edited bzip52 knockout mutant exhibited increased seed oil accumulation. Further analysis revealed that bZIP52 represses the transcriptional activity of AtWRI1 on the fatty acid biosynthetic gene promoters. Together, our findings suggest that bZIP52 represses fatty acid biosynthesis genes through interaction with AtWRI1, resulting in a reduction of oil production. Our work reports a previously uncharacterized regulatory mechanism that enables fine-tuning of seed oil biosynthesis.

Gummosis is 1 of the most common and destructive diseases threatening global peach (Prunus persica) production. Our previous studies have revealed that ethylene and methyl jasmonate enhance peach susceptibility to Lasiodiplodia theobromae, a virulent pathogen inducing gummosis; however, the underlying molecular mechanisms remain obscure. Here, 2 ethylene response factors (ERFs), PpERF98 and PpERF1, were identified as negative regulators in peach response to L. theobromae infection. Expression of 2 putative paralogs, PpERF98-1/2, was dramatically induced by ethylene and L. theobromae treatments and accumulated highly in the gummosis-sensitive cultivar. Silencing of PpERF98-1/2 increased salicylic acid (SA) content and pathogenesis-related genes PpPR1 and PpPR2 transcripts, conferring peach resistance to L. theobromae, whereas peach and tomato (Solanum lycopersicum) plants overexpressing either of PpERF98-1/2 showed opposite changes. Also, jasmonic acid markedly accumulated in PpERF98-1/2-silenced plants, but reduction in PpPR3, PpPR4, and PpCHI (Chitinase) transcripts indicated a blocked signaling pathway. PpERF98-1 and 2 were further demonstrated to directly bind the promoters of 2 putative paralogous PpERF1 genes and to activate the ERF branch of the jasmonate/ethylene signaling pathway, thus attenuating SA-dependent defenses. The lesion phenotypes of peach seedlings overexpressing PpERF1-1/2 and PpERF98-1/2 were similar. Furthermore, PpERF98-1/2 formed homodimers/heterodimers and interacted with the 2 PpERF1 proteins to amplify the jasmonate/ethylene signaling pathway, as larger lesions were observed in peach plants cooverexpressing PpERF98 with PpERF1 relative to individual PpERF98 overexpression. Overall, our work deciphers an important regulatory network of ethylene-mediated peach susceptibility to L. theobromae based on a PpERF98-PpERF1 transcriptional cascade, which could be utilized as a potential target for genetic engineering to augment protection against L. theobromae-mediated diseases in crops and trees.

Orange carotenoid protein (OCP) is a photoactive protein that participates in the photoprotection of cyanobacteria. There are 2 full-length OCP proteins, 4 N-terminal paralogs (helical carotenoid protein [HCP]), and 1 C-terminal domain-like carotenoid protein (CCP) found in Nostoc flagelliforme, a desert cyanobacterium. All HCPs (HCP1 to 3 and HCP6) from N. flagelliforme demonstrated their excellent singlet oxygen quenching activities, in which HCP2 was the strongest singlet oxygen quencher compared with others. Two OCPs, OCPx1 and OCPx2, were not involved in singlet oxygen scavenging; instead, they functioned as phycobilisome fluorescence quenchers. The fast-acting OCPx1 showed more effective photoactivation and stronger phycobilisome fluorescence quenching compared with OCPx2, which behaved differently from all reported OCP paralogs. The resolved crystal structure and mutant analysis revealed that Trp111 and Met125 play essential roles in OCPx2, which is dominant and long acting. The resolved crystal structure of OCPx2 is maintained in a monomer state and showed more flexible regulation in energy quenching activities compared with the packed oligomer of OCPx1. The recombinant apo-CCP obtained the carotenoid pigment from holo-HCPs and holo-OCPx1 of N. flagelliforme. No such carotenoid transferring processes were observed between apo-CCP and holo-OCPx2. The close phylogenetic relationship of OCP paralogs from subaerial Nostoc species indicates an adaptive evolution toward development of photoprotection: protecting cellular metabolism against singlet oxygen damage using HCPs and against excess energy captured by active phycobilisomes using 2 different working modes of OCPx.

As photosynthetic organisms, plants have a potential role in the sustainable production of high-value products such as medicines, biofuels, and chemical feedstocks. With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the costs and waste of production for materials that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. Information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed to achieve these complex goals. A genetic Boolean AND logic gate is a device that computes the presence or absence of 2 inputs (signals and stimuli) and produces an output (response) only when both inputs are present. We optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate 2 hormonal inputs in transgenic Arabidopsis thaliana plants. These AND gates produce an output only in the presence of both abscisic acid and auxin but not when either or neither hormone is present. The AND logic gate can also integrate signals resulting from 2 plant stresses, cold temperature and bacterial infection, to produce a response. The design principles used here are generalizable, and, therefore, multiple orthogonal AND gates could be assembled and rationally layered to process complex genetic information in plants. These layered logic gates may be used in genetic circuits to probe fundamental questions in plant biology, such as hormonal crosstalk, in addition to plant engineering for bioproduction.

Lysine malonylation (Kmal) is a recently discovered posttranslational modification, and its role in the response to abiotic stress has not been reported in plants. In this study, we isolated a nonspecific lipid transfer protein, DgnsLTP1, from chrysanthemum (Dendranthema grandiflorum var. Jinba). Overexpression and CRISPR-Cas9-mediated gene editing of DgnsLTP1 demonstrated that the protein endows chrysanthemum with cold tolerance. Yeast 2-hybrid, bimolecular fluorescence complementation, luciferase complementation imaging, and coimmunoprecipitation experimental results showed that DgnsLTP1 interacts with a plasma membrane intrinsic protein (PIP) DgPIP. Overexpressing DgPIP boosted the expression of DgGPX (glutathione peroxidase), increased the activity of GPX, and decreased the accumulation of reactive oxygen species (ROS), thereby enhancing the low-temperature stress tolerance of chrysanthemum, while the CRISPR-Cas9-mediated mutant dgpip inhibited this process. Transgenic analyses in chrysanthemum showed that DgnsLTP1 improves the cold resistance of chrysanthemum in a DgPIP-dependent manner. Moreover, Kmal of DgnsLTP1 at the K81 site prevented the degradation of DgPIP in Nicotiana benthamiana and chrysanthemum, further promoted DgGPX expression, enhanced GPX activity, and scavenged excess ROS produced by cold stress, thereby further enhancing the cold resistance of chrysanthemum.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA-containing intron. Consequently, the suppressed ccr1-6 allele was named epiccr1-6. Long-read sequencing revealed that epiccr1-6, not ccr1-6, carries dense cytosine methylation over the full length of the T-DNA. We showed that the SAIL T-DNA in the UGT72E3 locus could trigger the trans T-DNA suppression of the GABI-Kat T-DNA in the CCR1 locus. Furthermore, we scanned the literature for other potential cases of trans T-DNA suppression in Arabidopsis and found that 22% of the publications matching our query report on double or higher-order T-DNA mutants that meet the minimal requirements for trans T-DNA suppression. These combined observations indicate that intronic T-DNA mutants need to be used with caution since methylation of intronic T-DNA might derepress gene expression and can thereby confound results.

Viral synergism occurs when mixed infection of a susceptible plant by 2 or more viruses leads to increased susceptibility to at least 1 of the viruses. However, the ability of 1 virus to suppress R gene-controlled resistance against another virus has never been reported. In soybean (Glycine max), extreme resistance (ER) against soybean mosaic virus (SMV), governed by the Rsv3 R-protein, manifests a swift asymptomatic resistance against the avirulent strain SMV-G5H. Still, the mechanism by which Rsv3 confers ER is not fully understood. Here, we show that viral synergism broke this resistance by impairing downstream defense mechanisms triggered by Rsv3 activation. We found that activation of the antiviral RNA-silencing pathway and the proimmune mitogen-activated protein kinase 3 (MAPK3), along with the suppression of the proviral MAPK6, are hallmarks of Rsv3-mediated ER against SMV-G5H. Surprisingly, infection with bean pod mottle virus (BPMV) disrupted this ER, allowing SMV-G5H to accumulate in Rsv3-containing plants. BPMV subverted downstream defenses by impairing the RNA-silencing pathway and activating MAPK6. Further, BPMV reduced the accumulation of virus-related siRNAs and increased the virus-activated siRNA that targeted several defense-related nucleotide-binding leucine-rich repeat receptor (NLR) genes through the action of the suppression of RNA-silencing activities encoded in its large and small coat protein subunits. These results illustrate that viral synergism can result from abolishing highly specific R gene resistance by impairing active mechanisms downstream of the R gene.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) reversibly converts 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate coupled with the reduction of NADPH to NADP+. The GAPDH enzyme that functions in the Calvin-Benson cycle is assembled either from 4 glyceraldehyde-3-phosphate dehydrogenase A (GAPA) subunit proteins forming a homotetramer (A4) or from 2 GAPA and 2 glyceraldehyde-3-phosphate dehydrogenase B (GAPB) subunit proteins forming a heterotetramer (A2B2). The relative importance of these 2 forms of GAPDH in determining the rate of photosynthesis is unknown. To address this question, we measured the photosynthetic rates of Arabidopsis (Arabidopsis thaliana) plants containing reduced amounts of the GAPDH A and B subunits individually and jointly, using T-DNA insertion lines of GAPA and GAPB and transgenic GAPA and GAPB plants with reduced levels of these proteins. Here, we show that decreasing the levels of either the A or B subunits decreased the maximum efficiency of CO2 fixation, plant growth, and final biomass. Finally, these data showed that the reduction in GAPA protein to 9% wild-type levels resulted in a 73% decrease in carbon assimilation rates. In contrast, eliminating GAPB protein resulted in a 40% reduction in assimilation rates. This work demonstrates that the GAPA homotetramer can compensate for the loss of GAPB, whereas GAPB alone cannot compensate fully for the loss of the GAPA subunit.

In eukaryotes, a target of rapamycin (TOR) is a well-conserved kinase that controls cell metabolism and growth in response to nutrients and environmental factors. Nitrogen (N) is an essential element for plants, and TOR functions as a crucial N and amino acid sensor in animals and yeast. However, knowledge of the connections between TOR and the overall N metabolism and assimilation in plants is still limited. In this study, we investigated the regulation of TOR in Arabidopsis (Arabidopsis thaliana) by the N source as well as the impact of TOR deficiency on N metabolism. Inhibition of TOR globally decreased ammonium uptake while triggering a massive accumulation of amino acids, such as Gln, but also of polyamines. Consistently, TOR complex mutants were hypersensitive to Gln. We also showed that the glutamine synthetase inhibitor glufosinate abolishes Gln accumulation resulting from TOR inhibition and improves the growth of TOR complex mutants. These results suggest that a high level of Gln contributes to the reduction in plant growth resulting from TOR inhibition. Glutamine synthetase activity was reduced by TOR inhibition while the enzyme amount increased. In conclusion, our findings show that the TOR pathway is intimately connected to N metabolism and that a decrease in TOR activity results in glutamine synthetase-dependent Gln and amino acid accumulation.

Heat stress is a major factor limiting the production and geographic distribution of rice (Oryza sativa), and breeding rice varieties with tolerance to heat stress is of immense importance. Although extensive studies have revealed that reactive oxygen species (ROS) play a critical role in rice acclimation to heat stress, the molecular basis of rice controlling ROS homeostasis remains largely unclear. In this study, we discovered a novel heat-stress-responsive strategy that orchestrates ROS homeostasis centering on an immune activator, rice ENHANCED DISEASE SUSCEPTIBILITY 1 (OsEDS1). OsEDS1, which confers heat stress tolerance, promotes hydrogen peroxide (H2O2) scavenging by stimulating catalase activity through the OsEDS1-catalase association. The loss-of-function mutation in OsEDS1 causes increased sensitivity to heat stress, whereas the overexpression of OsEDS1 enhances thermotolerance. Furthermore, overexpression lines greatly improved rice tolerance to heat stress during the reproductive stage, which was associated with substantially increased seed setting, grain weight, and plant yield. Rice CATALASE C (OsCATC), whose activity is promoted by OsEDS1, degrades H2O2 to activate rice heat stress tolerance. Our findings greatly expand our understanding of heat stress responses in rice. We reveal a molecular framework that promotes heat tolerance through ROS homeostasis regulation, suggesting a theoretical basis and providing genetic resources for breeding heat-tolerant rice varieties.

Plants have evolved multiple mechanisms to cope with diverse types of light stress, particularly the regulation of the electron transport chain (ETC). Under high light (HL) conditions, the balance of electron flux in the ETC is disturbed, which leads to the overaccumulation of reactive oxygen species (ROS) and results in photodamage and photoinhibition. The cytochrome (Cyt) b6/f complex, which coordinates electron transfer between photosystems I and II (PSI and PSII), plays an essential role in regulating the ETC and initiating photoprotection. However, how the Cyt b6/f complex is maintained under HL conditions remains unclear. Here, we report that the activity of the Cyt b6/f complex is sustained by thylakoid-localized cyclophilin 37 (CYP37) in Arabidopsis (Arabidopsis thaliana). Compared with wild-type plants, cyp37 mutants displayed an imbalance in electron transport from Cyt b6/f to PSI under HL stress, which led to increased ROS accumulation, decreased anthocyanin biosynthesis, and increased chlorophyll degradation. Surprisingly, CYP37's role in regulating ETC balance was independent of photosynthesis control, which was indicated by a higher Y (ND), an indicator of P700 oxidation in PSI. Furthermore, the interaction between CYP37 and photosynthetic electron transfer A (PetA), a subunit of the Cyt b6/f complex, suggests that the central function of CYP37 is to maintain Cyt b6/f complex activity rather than to serve as an assembly factor. Our study provides insights into how plants balance electron flow between PSII and PSI via Cyt b6/f complex under HL.

In flowering plants, hundreds of RNA editing events occur in the chloroplasts and mitochondria during posttranscriptional processes. Although several pentatricopeptide repeat (PPR) proteins have been shown to form the editosome core, the precise interactions between the different editing factors are still obscure. Here, we isolated an Arabidopsis (Arabidopsis thaliana) PPR protein, designated DELAYED GREENING 409 (DG409), that was dually targeted to chloroplasts and mitochondria. This protein consists of 409 amino acids with 7 PPR motifs but lacks a C-terminal E, E+, or DYW domain. A mild dg409 knockdown mutant displays a sickly phenotype. In this mutant, the young leaves are pale green and turn green at maturity, and the development of chloroplasts and mitochondria is severely disrupted. Complete loss of DG409 function results in defective embryos. Transcriptomic analysis of the dg409 knockdown plants showed some editing defects in genes from both organelles, including CASEINOLYTIC PROTEASE P (clpP)-559, RNA POLYMERASE SUBUNIT ALPHA (rpoA)-200, ACETYL-COA CARBOXYLASE CARBOXYL TRANSFERASE SUBUNIT BETA (accD)-1568, NADH DEHYDROGENASE SUBUNIT 7 (nad7)-1505, and RIBOSOMAL PROTEIN S3 (rps3)-1344. RNA immunoprecipitation showed that DG409 was associated with the targeted transcripts in vivo. Interaction assays revealed that DG409 directly interacted with 2 DYW-type PPR proteins (EARLY CHLOROPLAST BIOGENESIS2 [AtECB2] and DYW DOMAIN PROTEIN2 [DYW2]) and 3 multiple organellar RNA editing factors (MORF2, MORF8, and MORF9). These results indicate that DG409 is involved in RNA editing via protein complexes and is therefore essential for chloroplast and mitochondrial development.

Magnesium chelatase (MgCh) catalyzes the insertion of magnesium into protoporphyrin IX, a vital step in chlorophyll (Chl) biogenesis. The enzyme consists of 3 subunits, MgCh I subunit (CHLI), MgCh D subunit (CHLD), and MgCh H subunit (CHLH). The CHLI subunit is an ATPase that mediates catalysis. Previous studies on CHLI have mainly focused on model plant species, and its functions in other species have not been well described, especially with regard to leaf coloration and metabolism. In this study, we identified and characterized a CHLI mutant in strawberry species Fragaria pentaphylla. The mutant, noted as p240, exhibits yellow-green leaves and a low Chl level. RNA-Seq identified a mutation in the 186th amino acid of the CHLI subunit, a base conserved in most photosynthetic organisms. Transient transformation of wild-type CHLI into p240 leaves complemented the mutant phenotype. Further mutants generated from RNA-interference (RNAi) and CRISPR/Cas9 gene editing recapitulated the mutant phenotype. Notably, heterozygous chli mutants accumulated more Chl under low light conditions compared with high light conditions. Metabolite analysis of null mutants under high light conditions revealed substantial changes in both nitrogen and carbon metabolism. Further analysis indicated that mutation in Glu186 of CHLI does not affect its subcellular localization nor the interaction between CHLI and CHLD. However, intramolecular interactions were impaired, leading to reduced ATPase and MgCh activity. These findings demonstrate that Glu186 plays a key role in enzyme function, affecting leaf coloration via the formation of the hexameric ring itself, and that manipulation of CHLI may be a means to improve strawberry plant fitness and photosynthetic efficiency under low light conditions.

Mitogen-activated protein kinase (MAPK/MPK) cascades are key signaling modules that regulate plant immunity. ENHANCED DISEASE RESISTANCE1 (EDR1) encodes a Raf-like MAPK kinase kinase (MAPKKK) that negatively regulates plant defense in Arabidopsis (Arabidopsis thaliana). The enhanced resistance of edr1 requires MAPK KINASE4 (MKK4), MKK5, and MPK3. Although the edr1 mutant displays higher MPK3/6 activation, the mechanism by which plants increase MAPK cascade activation remains elusive. Our previous study showed that MAPKKK5 is phosphorylated at the Ser-90 residue in edr1 mutants. In this study, we demonstrated that the enhanced disease resistance of edr1 required MAPKKK5. Phospho-dead MAPKKK5S90A partially impaired the resistance of edr1, and the expression of phospho-mimetic MAPKKK5S90D in mapkkk5-2 resulted in enhanced resistance to the powdery mildew Golovinomyces cichoracearum strain UCSC1 and the bacterial pathogen Pseudomonas syringae pv. tomato (Pto) strain DC3000. Thus, Ser-90 phosphorylation in MAPKKK5 appears to play a crucial role in disease resistance. However, MAPKKK5-triggered cell death was not suppressed by EDR1. Furthermore, activated MPK3 phosphorylated the N terminus of MAPKKK5, and Ser-90 was one of the phosphorylated sites. Ser-90 phosphorylation increased MAPKKK5 stability, and EDR1 might negatively regulate MAPK cascade activation by suppressing the MPK3-mediated feedback regulation of MAPKKK5. Taken together, these results indicate that MPK3 phosphorylates MAPKKK5 to enhance MAPK cascade activation and disease resistance in edr1 mutants.

Marine diatoms are responsible for up to 20% of annual global primary production by performing photosynthesis in seawater where CO2 availability is limited whilst HCO3- is abundant. Our previous studies have demonstrated that solute carrier 4 proteins at the plasma membrane of the diatom Phaeodactylum tricornutum facilitate the use of the abundant seawater HCO3-. There has been an unconcluded debate as to whether such HCO3- use capacity may itself supply enough dissolved inorganic carbon (DIC) to saturate the enzyme Rubisco. Here, we show that the θ-type carbonic anhydrase, Ptθ-CA1, a luminal factor of the pyrenoid-penetrating thylakoid membranes, plays an essential role in saturating photosynthesis of P. tricornutum. We isolated and analyzed genome-edited mutants of P. tricornutum defective in Ptθ-CA1. The mutants showed impaired growth in seawater aerated with a broad range of CO2 levels, from atmospheric to 1%. Independently of growth CO2 conditions, the photosynthetic affinity measured as K0.5 for DIC in mutants reached around 2 mM, which is about 10 times higher than K0.5[DIC] of high-CO2-grown wild-type cells that have repressed CCM levels. The results clearly indicate that diatom photosynthesis is not saturated with either seawater level DIC or even under a highly elevated CO2 environment unless the CO2-evolving machinery is at the core of the pyrenoid.

Although the sources of molecular hydrogen (H2) synthesis in plants remain to be fully elucidated, ample evidence shows that plant-based H2 can regulate development and stress responses. Here, we present genetic and molecular evidence indicating that nitrate reductase (NR) might be a target of H2 sensing that positively regulates nitrogen use efficiency (NUE) and seed size in Arabidopsis (Arabidopsis thaliana). The expression level of NR and changes of NUE under control and, in particular, low nitrogen supply, were positively associated with H2 addition supplied exogenously or through genetic manipulation. The improvement in nitrate assimilation achieved by H2 was also mediated via NR dephosphorylation. H2 control of seed size was impaired by NR mutation. Further genetic evidence revealed that H2, NR, and nitric oxide can synergistically regulate nitrate assimilation in response to N starvation conditions. Collectively, our data indicate that NR might be a target for H2 sensing, ultimately positively regulating nitrate uptake and seed size. These results provide insights into H2 signaling and its functions in plant metabolism.

Lipid droplets (LDs) of seed tissues are storage organelles for triacylglycerols (TAGs) that provide the energy and carbon for seedling establishment. In the major route of LD degradation (lipolysis), TAGs are mobilized by lipases. However, LDs may also be degraded via lipophagy, a type of selective autophagy, which mediates LD delivery to vacuoles or lysosomes. The exact mechanisms of LD degradation and the mobilization of their content in plants remain unresolved. Here, we provide evidence that LDs are degraded via a process morphologically resembling microlipophagy in Arabidopsis (Arabidopsis thaliana) seedlings. We observed the entry and presence of LDs in the central vacuole as well as their breakdown. Moreover, we show co-localization of AUTOPHAGY-RELATED PROTEIN 8b (ATG8b) and LDs during seed germination and localization of lipidated ATG8 (ATG8-PE) to the LD fraction. We further demonstrate that structural LD proteins from the caleosin family, CALEOSIN 1 (CLO1), CALEOSIN 2 (CLO2), and CALEOSIN 3 (CLO3), interact with ATG8 proteins and possess putative ATG8-interacting motifs (AIMs). Deletion of the AIM localized directly before the proline knot disrupts the interaction of CLO1 with ATG8b, suggesting a possible role of this region in the interaction between the two proteins. Collectively, we provide insights into LD degradation by microlipophagy in germinating seeds with a particular focus on the role of structural LD proteins in this process.

Fusarium head blight (FHB) of barley (Hordeum vulgare) causes yield losses and accumulation of trichothecene mycotoxins (e.g., deoxynivalenol (DON)) in grains. Glucosylation of DON to the nontoxic DON-3-O-glucoside (D3G) is catalyzed by UDP-glucosyltransferases (UGTs), such as barley UGT13248. We explored the natural diversity of UGT13248 in 496 barley accessions and showed that all carried potential functional alleles of UGT13248, as no genotypes showed strongly increased seedling sensitivity to DON. From a TILLING population, we identified two mutant alleles (T368I and H369Y) that, based on protein modeling, likely affect the UDP-glucose binding of UGT13248. In DON feeding experiments, DON-to-D3G conversion was strongly reduced in spikes of these mutants compared to controls, and plants overexpressing UGT13248 showed increased resistance to DON and increased DON-to-D3G conversion. Moreover, field-grown plants carrying the T368I or H369Y mutations inoculated with F. graminearum showed increased FHB disease severity and reduced D3G production. Barley is generally considered to have type II resistance that limits the spread of F. graminearum from the infected spikelet to adjacent spikelets. Point inoculation experiments with F. graminearum showed increased infection spread in T368I and H369Y across the spike compared to wild type, while overexpression plants showed decreased spread of FHB symptoms. Confocal microscopy revealed that F. graminearum spread to distant rachis nodes in T368I and H369Y mutants but was arrested at the rachis node of the inoculated spikelet in wild-type plants. Taken together, our data reveal that UGT13248 confers type II resistance to FHB in barley via conjugation of DON to D3G.

The lipid transport protein (LTP) product of the AsE246 gene of Chinese milk vetch (Astragalus sinicus) contributes to the transport of plant-synthesized lipids to the symbiosome membranes (SMs) that are required for nodule organogenesis in this legume. However, the mechanisms used by nodule-specific LTPs remain unknown. In this study, a functional protein in the DnaJ-like family, designated AsDJL1, was identified and shown to interact with AsE246. Immunofluorescence showed that AsDJL1 was expressed in infection threads (ITs) and in nodule cells and that it co-localized with rhizobium, and an immunoelectron microscopy assay localized the protein to SMs. Via co-transformation into Nicotiana benthamiana cells, AsDJL1 and AsE246 displayed subcellular co-localization in the cells of this heterologous host. Co-immunoprecipitation assays confirmed that AsDJL1 interacted with AsE246 in nodules. The essential interacting region of AsDJL1 was determined to be the zinc finger domain at its C-terminus. Chinese milk vetch plants transfected with AsDJL1-RNAi had significantly decreased numbers of ITs, nodule primordia and nodules as well as reduced (by 83%) nodule nitrogenase activity compared with the controls. By contrast, AsDJL1 overexpression led to increased nodule fresh weight and nitrogenase activity. RNAi-AsDJL1 also significantly affected the abundance of lipids, especially digalactosyldiacylglycerol, in early-infected roots and transgenic nodules. Taken together, the results of this study provide insights into the symbiotic functions of AsDJL1, which may participate in lipid transport to SMs and play an essential role in rhizobial infection and nodule organogenesis.

Understanding the molecular regulation of plant response to drought is the basis of drought resistance improvement through molecular strategies. Here, we characterized apple (Malus × domestica) histone deacetylase 6 (MdHDA6), which negatively regulates apple drought tolerance by catalyzing deacetylation on histones associated with drought-responsive genes. Transgenic apple plants over-expressing MdHDA6 were less drought-tolerant, while those with down-regulated MdHDA6 expression were more drought-resistant than non-transgenic apple plants. Transcriptomic and histone 3 acetylation (H3ac) ChIP-seq analyses indicated that MdHDA6 could facilitate histone deacetylation on the drought-responsive genes, repressing gene expression. Moreover, MdHDA6 interacted with the abscisic acid (ABA) signaling transcriptional factor, ABSCISIC ACID-INSENSITIVE 5 (MdABI5), forming the MdHDA6-MdABI5 complex. Interestingly, MdHDA6 facilitated histone deacetylation on the drought-responsive genes regulated by MdABI5, resulting in gene repression. Furthermore, a dual-Luc experiment showed that MdHDA6 could repress the regulation of a drought-responsive gene, RESPONSIVE TO DESICCATION 29A (MdRD29A) activated by MdABI5. On the one hand, MdHDA6 can facilitate histone deacetylation and gene repression on the positive drought-responsive genes to negatively regulate drought tolerance in apple. On the other hand, MdHDA6 directly interacts with MdABI5 and represses the expression of genes downstream of MdABI5 via histone deacetylation around these genes to reduce drought tolerance. Our study uncovers a different drought response regulatory mechanism in apple based on the MdHDA6-MdABI5 complex function and provides the molecular basis for drought resistance improvement in apple.

Arabidopsis (Arabidopsis thaliana) root development is regulated by multiple dynamic growth cues that require central metabolism pathways such as β-oxidation and auxin. Loss of the pectin biosynthesizing enzyme GALACTURONOSYLTRANSFERASE 10 (GAUT10) leads to a short root phenotype under sucrose-limited conditions. The present study focused on determining the specific contributions of GAUT10 to pectin composition in primary roots and the underlying defects associated with gaut10 roots. Using live-cell microscopy, we determined reduced root growth in gaut10 is due to a reduction in both root apical meristem size and epidermal cell elongation. In addition, GAUT10 was required for normal pectin and hemicellulose composition in primary Arabidopsis roots. Specifically, loss of GAUT10 led to a reduction in galacturonic acid and xylose in root cell walls and altered the presence of rhamnogalacturonan I (RG-I) and homogalacturonan (HG) polymers in the root. Transcriptomic analysis of gaut10 roots compared to wild-type uncovered hundreds of genes differentially expressed in the mutant, including genes related to auxin metabolism and peroxisome function. Consistent with these results, both auxin signaling and metabolism were modified in gaut10 roots. The sucrose-dependent short root phenotype in gaut10 was linked to β-oxidation based on hypersensitivity to indole-3-butyric acid (IBA) and an epistatic interaction with TRANSPORTER OF IBA1 (TOB1). Altogether, these data support a growing body of evidence suggesting that pectin composition may influence auxin pathways and peroxisome activity.

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behaviour, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas-exchange signalling in S. viridis. We identified two accessions, SHA and ZHA, with contrasting behaviour at the leaf, root, and whole-plant level. Our results indicated a role for root aquaporin plasma membrane intrinsic proteins in the differential behaviour of SHA and ZHA. Moreover, a different root hydraulics response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root aquaporins (AQPs). Using cell imaging, biochemical and reverse-genetic approaches, we identified plasma membrane intrinsic protein 1; 6 (PIP1; 6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signalling of gas exchange in S. viridis. In heterologous systems, PIP1; 6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, re-localization to the plasma membrane was observed. PIP1; 6 was predominantly expressed at the root endodermis. Generation of knockout PIP1; 6 plants (KO-PIP1; 6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.

Meiotic recombination drives genetic diversity and crop genome optimization. In plant breeding, parents with favorable traits are crossed to create elite varieties. Different hybridizations produce diverse types of segment reshuffling between homologous chromosomes. However, little is known about the factors that cause hybrid-specific changes in crossovers (COs). Here, we constructed two F2 populations from crosses between a semi-wild and two domesticated cucumber (Cucumis sativus) accessions and examined CO events. COs mainly occurred around genes and differed unevenly along chromosomes between the two hybrids. Fine-scale CO distributions were suppressed in regions of heterozygous structural variations (SVs) and were accelerated by high sequence polymorphism. C. sativus RADiation sensitive 51A (CsRAD51A) binding, histone H3 lysine 4 tri-methylation (H3K4me3) modification, chromatin accessibility, and hypomethylation were positively associated with global CO landscapes and in local DNA double-strand break (DSB) hotspots and genes. The frequency and suppression of COs could be roughly predicted based on multi-omic information. Differences in CO events between hybrids could be partially traced to distinct genetic and epigenetic features and were significantly associated with specific DSB hotspots and heterozygous SVs. Our findings identify the genomic and epigenetic features that contribute to CO formation and hybrid-specific divergence in cucumber and provide theoretical support for selecting parental combinations and manipulating recombination events at target genomic regions during plant breeding.

Source and sink interactions play a critical but mechanistically poorly understood role in the regulation of senescence. To disentangle the genetic and molecular mechanisms underlying source-sink regulated senescence (SSRS), we performed a phenotypic, transcriptomic, and systems genetics analysis of senescence induced by the lack of a strong sink in maize (Zea mays). Comparative analysis of genotypes with contrasting SSRS phenotypes revealed that feedback inhibition of photosynthesis, a surge in reactive oxygen species, and the resulting endoplasmic reticulum (ER) stress were the earliest outcomes of weakened sink demand. Multi-environmental evaluation of a biparental population and a diversity panel identified 12 quantitative trait loci and 24 candidate genes, respectively, underlying SSRS. Combining the natural diversity and coexpression networks analyses identified seven high-confidence candidate genes involved in proteolysis, photosynthesis, stress response, and protein folding. The role of a cathepsin B like protease 4 (ccp4), a candidate gene supported by systems genetic analysis, was validated by analysis of natural alleles in maize and heterologous analyses in Arabidopsis (Arabidopsis thaliana). Analysis of natural alleles suggested that a 700-bp polymorphic promoter region harboring multiple ABA-responsive elements is responsible for differential transcriptional regulation of ccp4 by ABA and the resulting variation in SSRS phenotype. We propose a model for SSRS wherein feedback inhibition of photosynthesis, ABA signaling, and oxidative stress converge to induce ER stress manifested as programmed cell death and senescence. These findings provide a deeper understanding of signals emerging from loss of sink strength and offer opportunities to modify these signals to alter senescence program and enhance crop productivity.

Arabidopsis (Arabidopsis thaliana) high-affinity NITRATE TRANSPORTER2.1 (NRT2.1) plays a dominant role in the uptake of nitrate, the most important nitrogen (N) source for most terrestrial plants. The nitrate-inducible expression of NRT2.1 is regulated by NIN-LIKE PROTEIN (NLP) family transcriptional activators and NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) family transcriptional repressors. Phosphorus (P) availability also affects NRT2.1 expression because the PHOSPHATE STARVATION RESPONSE1 (PHR1) transcriptional activator activates NIGT1 genes in P-deficient environments. Here, we show a biology-based mathematical understanding of the complex regulation of NRT2.1 expression by multiple transcription factors using two different approaches: a microplate-based assay for the real-time measurement of temporal changes in NRT2.1 promoter activity under different nutritional conditions, and an ordinary differential equation (ODE)-based mathematical modeling of the NLP- and NIGT1-regulated expression pattern of NRT2.1. Both approaches consistently revealed that NIGT1 stabilizes the amplitude of NRT2.1 expression under a wide range of nitrate concentrations. Furthermore, the ODE model suggested that parameters such as the synthesis rate of NIGT1 mRNA and NIGT1 proteins and the affinity of NIGT1 proteins for the NRT2.1 promoter substantially influence the temporal expression patterns of NRT2.1 in response to nitrate. These results suggest that the NIGT1-NRT2.1 incoherent type I feedforward loop allows precise control of nitrate uptake. Hence, this study paves the way for understanding the complex regulation of nutrient acquisition in plants, thus facilitating engineered nutrient uptake and plant response patterns using synthetic biology approaches.

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Common purslane (Portulaca oleracea) integrates both C4 and crassulacean acid metabolism (CAM) photosynthesis pathways, and is a promising model plant to explore C4-CAM plasticity. Here, we report a high-quality chromosome-level genome of NAD-ME subtype common purslane that provides evidence for two rounds of whole-genome duplication (WGD) with an ancient WGD (P-β) in the common ancestor to Portulacaceae and Cactaceae around 66.30 million years ago (Mya) and another (Po-α) specific to common purslane lineage around 7.74 Mya. A larger number of gene copies encoding key enzymes/transporters involved in C4 and CAM pathways were detected in common purslane than in related species. Phylogeny, conserved functional site and collinearity analyses revealed that the Po-α WGD produced the phosphoenolpyruvate carboxylase (PEPC) encoded gene copies used for photosynthesis in common purslane, while the P-β WGD event produced two ancestral genes of functionally differentiated (C4- and CAM-specific) beta carbonic anhydrases (β-CA) involved in the C4+CAM pathways. Additionally, cis-element enrichment analysis in the promoters showed CAM-specific genes have recruited both evening and midnight circadian elements as well as the ABA independent regulatory module mediated by ethylene-response factor (ERF) cis-elements. Overall, this study provides insights into the origin and evolutionary process of C4 and CAM pathways in common purslane, as well as potential targets for engineering crops by integrating C4 or CAM metabolism.

Endosperm cell number is critical in determining grain size in maize (Zea mays). Here, zma-miR159 overexpression led to enlarged grains in independent transgenic lines, suggesting that zma-miR159 positively to maize grain size. Targeting of ZmMYB74 and ZmMYB138 transcription factor genes by zma-miR159 was validated using 5'RACE and dual luciferase assay. Lines in which ZmMYB74 was knocked out using CRISPR/Cas9 presented a similar enlarged grain phenotype as those with zma-miR159 overexpression. Downstream genes regulating cell division were identified through DNA affinity purification sequencing using ZmMYB74 and ZmMYB138. Our results suggest that zma-miR159-ZmMYB modules act as an endosperm development hub, participating in the division and proliferation of endosperm cells.

Plants have evolved various resistance mechanisms to cope with biotic stresses that threaten their survival. The BBE23 member (At5g44360/BBE23) of the Arabidopsis berberine bridge enzyme-like (BBE-l) protein family (Arabidopsis thaliana) has been characterized in this paper in parallel with the closely related and previously described CELLOX (At4g20860/BBE22). Both enzymes, renamed here as CELLOX2 and CELLOX1, respectively, oxidize, besides cellodextrins, the mixed-linked β-1➝3/β-1➝4-glucans (MLGs), recently described as capable of activating plant immunity, reinforcing the view that the BBE-l family includes members that are devoted to the control of the homeostasis of potential cell wall-derived damage-associated molecular patterns (DAMPs). The two putatively paralogous genes display different expression profiles. Unlike CELLOX1, CELLOX2 is not expressed in seedlings or adult plants and is not involved in immunity against Botrytis cinerea. Both are instead expressed in a concerted manner in the seed coat during development. Whereas CELLOX2 is expressed mainly during the heart stage, CELLOX1 is expressed at the immediately later stage, when the expression of CELLOX2 decreases. Analysis of seeds of cellox1 and cellox2 knock-out mutants show alterations in the coat structure: the columella area is smaller in cellox1, radial cell walls are thicker in both cellox1 and cellox2, and the mucilage halo is reduced in cellox2. However, the coat monosaccharide composition is not significantly altered, suggesting an alteration of the organization of the cell wall, thus reinforcing the notion that the architecture of the cell wall in specific organs is determined not only by the dynamics of the synthesis/degradation of the main polysaccharides but also by its enzymatic oxidation.

Volatile esters in apple (Malus domestica) fruit are the critical aroma components determining apple flavor quality. While the exact molecular regulatory mechanism remains unknown, jasmonic acid (JA) plays a crucial role in stimulating the synthesis of ester aromas in apples. In our study, we investigated the effects of methyl jasmonate (MeJA) on the production of ester aroma in apples. MeJA treatment significantly increased ester aroma synthesis, accompanied by the upregulation of several genes involved in the jasmonate pathway transduction. Specifically, expression of the gene MdMYC2, which encodes a transcription factor associated with the jasmonate pathway, and the R2R3-MYB transcription factor gene MdMYB85 increased upon MeJA treatment. Furthermore, the essential gene ALCOHOL ACYLTRANSFERASE 1 (MdAAT1), encoding an enzyme responsible for ester aroma synthesis, showed increased expression levels as well. Our investigation revealed that MdMYC2 and MdMYB85 directly interacted with the promoter region of MdAAT1, thereby enhancing its transcriptional activity. In addition, MdMYC2 and MdMYB85 directly bind their promoters and activate transcription. Notably, the interaction between MdMYC2 and MdMYB85 proteins further amplified the regulatory effect of MdMYB85 on MdMYC2 and MdAAT1, as well as that of MdMYC2 on MdMYB85 and MdAAT1. Collectively, our findings elucidate the role of the gene module consisting of MdMYC2, MdMYB85, and MdAAT1 in mediating the effects of JA and promoting ester aroma synthesis in apples.

Plant development continues post-embryonically with a lifelong ability to form new tissues and organs. Asymmetric cell division, coupled with fate segregation, is essential to create cellular diversity during tissue and organ formation. Arabidopsis (Arabidopsis thaliana) plants harboring mutations in the SCHIZORIZA (SCZ) gene display fate segregation defects in their roots, resulting in the presence of an additional layer of endodermis, production of root hairs from sub-epidermal tissue, and misexpression of several tissue identity markers. Some of these defects are observed in tissues where SCZ is not expressed, indicating that part of the SCZ function is non-autonomous. As a class B HEAT-SHOCK TRANSCRIPTION FACTOR (HSFB), the SCZ protein contains several conserved domains and motifs. However, which domain(s) discriminates SCZ from its family members to obtain a role in development remains unknown. Here, we investigate how each domain contributes to SCZ function in Arabidopsis root patterning by generating altered versions of SCZ by domain swapping and mutation. We show that the SCZ DNA-Binding Domain is the main factor for its developmental function and that SCZ likely acts as a non-motile transcriptional repressor. Our results demonstrate how members of the HSF family can evolve toward functions beyond stress response.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that two cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert phenylacetaldoxime (PAOx) to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least three days after insect feeding. This, together with the fact that gut protein extracts of three insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in three other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

The lateral organs of watermelon (Citrullus lanatus), including lobed leaves, branches, flowers, and tendrils, together determine plant architecture and yield. However, the genetic controls underlying lateral organ initiation and morphogenesis remain unclear. Here, we found that knocking out the homologous gene of shoot branching regulator LATERAL SUPPRESSOR in watermelon (ClLs) repressed the initiation of branches, flowers, and tendrils and led to developing round leaves, indicating that ClLs undergoes functional expansion compared with its homologs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum). Using ClLs as the bait to screen against the cDNA library of watermelon, we identified several ClLs-interacting candidate proteins, including TENDRIL (ClTEN), PINOID (ClPID), and APETALA1 (ClAP1). Protein-protein interaction assays further demonstrated that ClLs could directly interact with ClTEN, ClPID, and ClAP1. The mRNA in situ hybridization assay revealed that the transcriptional patterns of ClLs overlapped with those of ClTEN, ClPID, and ClAP1 in the axillary meristems and leaf primordia. Mutants of ClTEN, ClPID, and ClAP1 generated by the CRISPR/Cas9 gene editing system lacked tendrils, developed round leaves, and displayed floral diapause, respectively, and all these phenotypes could be observed in ClLs knockout lines. Our findings indicate that ClLs acts as lateral organ identity protein by forming complexes with ClTEN, ClPID, and ClAP1, providing several gene targets for transforming the architecture of watermelon.

Chilling temperatures induce an increase in cytoplasmic calcium (Ca2+) ions to transmit cold signals, but the precise role of Calmodulins (CaMs), a type of Ca2+ sensor, in plant tolerance to cold stress remains elusive. In this study, we characterized a tomato (Solanum lycopersicum) CaM gene, CALMODULIN6 (CaM6), which responds to cold stimulus. Overexpressing CaM6 increased tomato sensitivity to cold stress whereas silencing CaM6 resulted in a cold-insensitive phenotype. We showed that CaM6 interacts with Inducer of CBF expression 1 (ICE1) in a Ca2+-independent process and ICE1 contributes to cold tolerance in tomato plants. By integrating RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) assays, we revealed that ICE1 directly altered the expression of 76 downstream cold-responsive (COR) genes that potentially confer cold tolerance to tomato plants. Moreover, the physical interaction of CaM6 with ICE1 attenuated ICE1 transcriptional activity during cold stress. These findings reveal that CaM6 attenuates the cold tolerance of tomato plants by suppressing ICE1-dependent COR gene expression. We propose a CaM6/ICE1 module in which ICE1 is epistatic to CaM6 under cold stress. Our study sheds light on the mechanism of plant response to cold stress and reveals CaM6 is involved in the regulation of ICE1.

Jasmonate (JA) and gibberellins (GAs) exert antagonistic effects on plant growth and development in response to environmental and endogenous stimuli. Although the crosstalk between JA and GA has been elucidated, the role of JA in GA biosynthesis remains unclear. Therefore, in this study, we investigated the mechanism underlying JA-mediated regulation of endogenous GA levels in Arabidopsis (Arabidopsis thaliana). Transient and electrophoretic mobility shift assays showed that transcription factor MYC2 regulates GA inactivation genes. Using transgenic plants, we further evaluated the contribution of MYC2 in regulating GA inactivation genes. JA treatment increased DELLA accumulation but did not inhibit DELLA protein degradation. Additionally, JA treatment decreased bioactive GA content, including GA4, significantly decreased the expression of GA biosynthesis genes, including ent-kaurene synthase (AtKS), GA 3β-hydroxylase (AtGA3ox1), and AtGA3ox2, and increased the expression of GA inactivation genes, including GA 2 oxidase (AtGA2ox4), AtGA2ox7, and AtGA2ox9. Conversely, JA treatment did not significantly affect gene expression in the myc2 myc3 myc4 triple mutant, demonstrating the MYC2-4-dependent effects of JA in GA biosynthesis. Additionally, JA post-transcriptionally regulated AtGA3ox1 expression. We identified microRNA miR5998 as an AtGA3ox1-associated miRNA; its overexpression inhibited plant growth by suppressing AtGA3ox1 expression. Overall, our findings indicate that JA treatment inhibits endogenous GA levels and plant growth by decreasing the expression of GA biosynthesis genes and increasing the expression of GA inactivation genes via miR5998 and MYC2 activities.

Cell elongation is a fundamental process for plant growth and development. Studies have shown lipid metabolism plays important roles in cell elongation; however, the related functional mechanisms remain largely unknown. Here, we report that cotton (Gossypium hirsutum) LIPID TRANSFER PROTEIN4 (GhLTP4) promotes fiber cell elongation via elevating ceramides (Cers) content and activating auxin-responsive pathways. GhLTP4 was preferentially expressed in elongating fibers. Over-expression and down-regulation of GhLTP4 led to longer and shorter fiber cells, respectively. Cers were greatly enriched in GhLTP4-overexpressing lines and decreased dramatically in GhLTP4-down-regulating lines. Moreover, auxin content and transcript levels of IAA-responsive genes were significantly increased in GhLTP4-overexpressing cotton fibers. Exogenous application of Cers promoted fiber elongation, while NPA (N-1-naphthalic acid, a polar auxin transport inhibitor) counteracted the promoting effect, suggesting that IAA functions downstream of Cers in regulating fiber elongation. Furthermore, we identified a bHLH transcription factor, GhbHLH105, that binds to the E-box element in the GhLTP4 promoter region and promotes the expression of GhLTP4. Suppression of GhbHLH105 in cotton reduced the transcripts level of GhLTP4, resulting in smaller cotton bolls and decreased fiber length. These results provide insights into the complex interactions between lipids and auxin signaling pathways to promote plant cell elongation.

High-affinity potassium (K+) transporter (HAK)/K+ uptake permease (KUP)/K+ transporter (KT) have been identified in all genome-sequenced terrestrial plants. They play an important role in K+ acquisition and translocation and in enhancing salt tolerance. Here, we report that plasma-membrane-located OsHAK18 functions in K+ and sodium (Na+) circulation and sugar translocation in rice (Oryza sativa). OsHAK18 was expressed mainly, though not exclusively, in vascular tissues and particularly in the phloem. Knockout (KO) of OsHAK18 reduced K+ concentration in phloem sap and roots but increased K+ accumulation in the shoot of both Nipponbare and Zhonghua11 cultivars, while overexpression (OX) of OsHAK18 driven by its endogenous promoter increased K+ concentration in phloem sap and roots and promoted Na+ retrieval from shoot to root under salt stress. Split-root experimental analysis of rubidium (Rb+) uptake and circulation indicated that OsHAK18-OX promoted Rb+ translocation from the shoot to the root. In addition, OsHAK18-KO increased while OsHAK18-OX reduced soluble sugar content in the shoot and oppositely affected the sugar concentration in the phloem and its content in the root. Moreover, OsHAK18-OX dramatically increased grain yield and physiological K+ utilization efficiency. Our results suggest that - unlike other OsHAKs analyzed heretofore - OsHAK18 is critical for K+ and Na+ re-circulation from shoot to root and enhances the source-to-sink translocation of photoassimilates.

The vacuole is an important organelle for nitrate storage, and reuse of vacuolar nitrate under nitrate starvation helps plants adapt to low-nitrate environments. CHLORIDE CHANNEL-b (CLC-b) in the vacuolar membrane is a nitrate transporter; however, its regulation and effects on nitrate efflux have not been established. Here, we evaluated CLC-b expression and its effects on physiological parameters under low nitrate conditions. CLC-b expression increased significantly in the roots of wild-type Arabidopsis (Arabidopsis thaliana) Col-0 under nitrate starvation. Under low nitrate, clcb mutants showed reductions in chlorophyll content and xylem sap nitrate concentration, shoot/root nitrate ratios, shoot/root total N ratios, and biomass. CLC-b overexpression yielded opposite phenotypes and increased nitrogen use efficiency. CLC-b mutants showed elevated chlorate tolerance and increased proportion of vacuolar nitrate relative to the total protoplast nitrate content as compared to the wild type. Yeast one-hybrid, EMSA, and ChIP experiments showed that HRS1 HOMOLOG2 (HHO2), the expression of which is downregulated under low nitrate, binds directly to the promoter of CLC-b. clcb/hho2 double mutants and HHO2-overexpressing clcb plants had similar phenotypes under low nitrate to those of clcb single mutants. Thus, CLC-b mediates vacuolar nitrate efflux and is negatively regulated by HHO2, providing a theoretical basis for improving plant adaptability to low nitrate.