FUSCA 3 (FUS3), a seed master regulator, plays critical roles in seed dormancy and oil accumulation. However, its downstream regulation mechanisms remain poorly understood. Here, we explored the roles of AINTEGUMENTA-like 6 (AIL6), a seed transcription factor, in these processes. The activation of AIL6 by FUS3 was demonstrated by dual-LUC assay. Seeds of ail6 mutants showed alterations of fatty acid compositions, and both AtAIL6 (AIL6 from Arabidopsis thaliana) and BnaAIL6 (AIL6 from Brassica napus) rescued the phenotype. Over-expression (OE) of AIL6s reversed changes in seed fatty acid composition. Notably, OE lines showed low seed germination rates down to 12% compared to 100% of wild-type Col-0. Transcriptome analysis of the mutant and an OE line indicated widespread expression changes of genes involved in lipid metabolism and phytohormone pathways. In OE mature seeds, GA4 content decreased more than 15-fold, while ABA and IAA contents clearly increased. Exogenous GA3 treatments did not effectively rescue the low germination rate. Nicking seed coats increased germination rates from 25% to nearly 80% while the wild-type rdr6-11 is 100% and 98% respectively, and elongation of storage time also improved seed germination. Furthermore, dormancy imposed by AIL6 was fully released in the della quintuple mutant. Together, our results indicate AIL6 acts as a manager downstream of FUS3 in seed dormancy and lipid metabolism.

Plant metaxylem vessels provide physical support to promote upright growth and the transport of water and nutrients. A detailed characterization of the molecular network controlling metaxylem development is lacking. However, knowledge of the events that regulate metaxylem development could contribute to the development of germplasm with improved yield. In this paper, we screened an ethyl methane sulfonate (EMS)-induced B73 mutant library, which covers 92% of maize (Zea mays) genes, to identify drought-sensitive phenotypes. Three mutants were identified, named iqd27-1, iqd27-2, and iqd27-3, and genetic crosses showed that they were allelic to each other. The causal gene in these three mutants encodes the IQ domain-containing protein ZmIQD27. Our study showed that defective metaxylem vessel development likely causes the drought sensitivity and abnormal water transport phenotypes in the iqd27 mutants. ZmIQD27 was expressed in the root meristematic zone where secondary cell wall deposition is initiated, and loss-of-function iqd27 mutants exhibited a microtubular arrangement disorder. We propose that association of functional ZmIQD27 with microtubules is essential for correct targeted deposition of the building blocks for secondary cell wall development in maize.

Sex chromosomes have evolved independently in many different plant lineages. Here, we describe reference genomes for spinach (Spinacia oleracea) X and Y haplotypes by sequencing homozygous XX females and YY males. The long arm of the 185 Mb chromosome 4 carries a 13 Mb X-linked region (XLR) and 24.1 Mb Y-linked region (YLR), of which 10 Mb is Y-specific. We describe evidence that this reflects insertions of autosomal sequences creating a "Y duplication region" or "YDR" whose presence probably directly reduces genetic recombination in the immediately flanking regions, although both the X and Y Sex-Linked Regions are within a large pericentromeric region of chromosome 4 that recombines rarely in meiosis of both sexes. Sequence divergence estimates using synonymous sites indicate that YDR genes started diverging from their likely autosomal progenitors about 3 MYA, around the time when the flanking YLR stopped recombining with the XLR. These flanking regions have a higher density of repetitive sequences in the YY than the XX assembly and include slightly more pseudogenes compared with the XLR, and the YLR has lost about 11% of the ancestral genes, suggesting some degeneration. Insertion of a male-determining factor would have caused Y-linkage across the entire pericentromeric region, creating physically small, highly recombining, terminal pseudo-autosomal regions. These findings provide a broader understanding of the origin of sex chromosomes in spinach.

Thermal dissipation of excess excitation energy, called non-photochemical quenching (NPQ), is one of the main photoprotective mechanisms in oxygenic photosynthetic organisms. Here, we investigated the function of the monomeric photosystem II antenna protein CP26 in photoprotection and light harvesting in Chlamydomonas reinhardtii, a model organism for green algae. We used CRISPR/Cas9 genome editing and complementation to generate cp26 knock-out mutants (named k6#) that did not negatively affect CP29 accumulation, which differed from previous cp26 mutants, allowing us to compare mutants specifically deprived of CP26, CP29 or both. The absence of CP26 partially affected photosystem II activity causing reduced growth at low or medium light but not at high irradiances. However, the main phenotype observed in k6# mutants was a more than 70% reduction of NPQ compared to the wild type. This phenotype was fully rescued by genetic complementation and complemented strains accumulating different levels of CP26, demonstrating that ∼50% of CP26 content, compared to the wild type, was sufficient to restore the NPQ capacity. Our findings demonstrate a pivotal role for CP26 in NPQ induction, while CP29 is crucial for photosystem II activity. The genetic engineering of these two proteins could be a promising strategy to regulate the photosynthetic efficiency of microalgae under different light regimes.

Environmental influences and differential growth subject plants to mechanical forces. Forces on the whole plant resolve into tensile forces on its primary cell walls and both tensile and compression forces on the secondary cell-wall layers of woody tissues. Forces on cell walls are further resolved into forces on cellulose microfibrils and on the non-cellulosic polymers between them. Many external forces on plants oscillate, with time constants that vary from seconds to milliseconds. Sound waves are a high-frequency example. Forces on the cell wall lead to responses that direct the oriented deposition of cellulose microfibrils and the patterned expansion of the cell wall, leading to complex cell and tissue morphology. Recent experiments have established many of the details of which cell-wall polymers associate with one another in both primary and secondary cell walls, but questions remain about which of the interconnections are load-bearing, especially in primary cell walls. Direct cellulose-cellulose interactions appear to have a more important mechanical role than was previously thought, and some of the non-cellulosic polymers may have a role in keeping microfibrils apart rather than cross-linking them as formerly envisaged.

Plant immune responses involve transcriptional reprogramming of defense response genes, and chromatin remodeling is important for transcriptional regulation. However, nucleosome dynamics induced by pathogen infection and its association with gene transcription is largely unexplored in plants. Here, we investigated the role of the rice (Oryza sativa) gene CHROMATIN REMODELING 11 (OsCHR11) in nucleosome dynamics and disease resistance. Nucleosome profiling revealed that OsCHR11 is required for the maintaining of genome-wide nucleosome occupancy in rice. Nucleosome occupancy of 14% of the genome was regulated by OsCHR11. Infection of bacterial leaf blight Xoo (Xanthomonas oryzae pv. oryzae) repressed genome-wide nucleosome occupancy, and this process depended on OsCHR11 function. Furthermore, OsCHR11/Xoo-dependent chromatin accessibility correlated with gene transcript induction by Xoo. In addition, accompanied by increased resistance to Xoo, several defense response genes were differentially expressed in oschr11 after Xoo infection. Overall, this study reports the genome-wide effects upon pathogen infection on nucleosome occupancy, its regulation, and its contribution to disease resistance in rice.

Flower senescence is genetically regulated and developmentally controlled. The phytohormone ethylene induces flower senescence in rose (Rosa hybrida), but the underlying signaling network is not well understood. Given that calcium regulates senescence in animals and plants, we explored the role of calcium in petal senescence. Here, we report that the expression of calcineurin B-like protein 4 (RhCBL4), which encodes a calcium receptor, is induced by senescence and ethylene signaling in rose petals. RhCBL4 interacts with CBL-interacting protein kinase 3 (RhCIPK3), and both positively regulate petal senescence. Furthermore, we determined that RhCIPK3 interacts with the jasmonic acid response repressor jasmonate ZIM-domain 5 (RhJAZ5). RhCIPK3 phosphorylates RhJAZ5 and promotes its degradation in the presence of ethylene. Our results reveal that the RhCBL4-RhCIPK3-RhJAZ5 module mediates ethylene-regulated petal senescence. These findings provide insights into flower senescence, which may facilitate innovations in postharvest technology for extending rose flower longevity.

Subgenome expression dominance plays a crucial role in the environmental adaptation of polyploids. However, the epigenetic molecular mechanism underlying this process has not been thoroughly investigated, particularly in perennial woody plants. Persian walnut (Juglans regia) and its wild relative, Manchurian walnut (J. mandshurica), are woody plants of great economic importance and are both paleopolyploids that have undergone whole-genome duplication events. In this study, we explored the characteristics of subgenome expression dominance in these two Juglans species and examined its epigenetic basis. We divided their genomes into dominant subgenome (DS) and submissive subgenome (SS) and found that the DS-specific genes might play critical roles in biotic stress response or pathogen defense. We comprehensively elucidated the characteristics of biased gene expression, asymmetric DNA methylation, transposable elements (TEs), and alternative splicing (AS) events of homoeologous gene pairs between subgenomes. The results showed that biased expression genes (BEGs) in two Juglans species were mainly related to external stimuli response, while non-BEGs were related to complexes that might be involved in signal transduction.DS genes had higher expression and more AS events while having less DNA methylation and TEs than homoeologous genes from the submissive subgenome in the two Juglans species. Further studies showed that DNA methylation might contribute to the biased expression of gene pairs by modifying LTR/TIR/nonTIR TEs and improving the AS efficiency of corresponding precursor mRNAs in a particular context. Our study contributes to understanding the epigenetic basis of subgenome expression dominance and the environmental adaptation of perennial woody plants.

Grapevine (Vitis ssp.) is a deciduous perennial fruit crop, and the canes and buds of grapevine should withstand low temperatures (LTs) annually during winter. However, the widely cultivated Vitis vinifera is cold-sensitive and cannot survive the severe winter in regions with extremely LTs, such as viticulture regions in northern China. By contrast, a few wild Vitis species like V. amurensis and V. riparia exhibit excellent freezing tolerance. However, the mechanisms underlying grapevine cold tolerance remain largely unknown. In recent years, much progress has been made in elucidating the mechanisms, owing to the advances in sequencing and molecular biotechnology. Assembly of grapevine genomes together with resequencing and transcriptome data enable researchers to conduct genomic and transcriptomic analyses in various grapevine genotypes and populations to explore genetic variations involved in cold tolerance. In addition, a number of pivotal genes have been identified and functionally characterized. In this review, we summarize recent major advances in physiological and molecular analyses of cold tolerance in grapevine and put forward questions in this field. We also discuss the strategies for improving the tolerance of grapevine to cold stress. Understanding grapevine cold tolerance will facilitate the development of grapevines for adaption to global climate change.

Green algae display a wide range of extracellular matrix (ECM) components that include various types of cell walls, scales, crystalline glycoprotein coverings, hydrophobic compounds and complex gels or mucilage. Recently, new information derived from genomic/transcriptomic screening, advanced biochemical analyses, immunocytochemical studies and ecophysiology has significantly enhanced and refined our understanding of the green algal ECM. In later diverging charophyte group of green algae, the cell wall and other ECM components provide insight into the evolution of plants and the ways the ECM modulates during environmental stress. Chlorophytes produce diverse ECM components, many of which have been exploited for various uses in medicine, food and the production of biofuels. This review highlights major advances in ECM studies of green algae.

Drought is a common stress in agricultural production. Thus, it is imperative to understand how fruit crops respond to drought and to develop drought-tolerant varieties. This paper provides an overview of the effects of drought on the vegetative and reproductive growth of fruits. We summarize the empirical studies that have assessed the physiological and molecular mechanisms of the drought response in fruit crops. This review focuses on the roles of calcium (Ca2+) signaling, abscisic acid (ABA), reactive oxygen species signaling, and protein phosphorylation underlying the early drought response in plants. We review the resulting downstream ABA-dependent and ABA-independent transcriptional regulation in fruit crops under drought stress. Moreover, we highlight the positive and negative regulatory mechanisms of microRNAs in the drought response of fruit crops. Lastly, strategies (including breeding and agricultural practices) to improve the drought resistance of fruit crops are outlined.

During ripening, fleshy fruits undergo irreversible changes in color, texture, sugar content, aroma, and flavor to appeal to seed-dispersal vectors. The onset of climacteric fruit ripening is accompanied by an ethylene burst. Understanding the factors triggering this ethylene burst is important for manipulating climacteric fruit ripening. Here, we review the current understanding and recent insights into the possible factors triggering climacteric fruit ripening: DNA methylation and histone modification, including methylation and acetylation. Understanding the initiation factors of fruit ripening is important for exploring and accurately regulating the mechanisms of fruit ripening. Lastly, we discuss the potential mechanisms responsible for climacteric fruit ripening.

Excessive softening during fleshy fruit ripening leads to physical damage and infection that reduce quality and cause massive supply chain losses. Changes in cell wall (CW) metabolism, involving loosening and disassembly of the constituent macromolecules, are the main cause of softening. Several genes encoding CW metabolizing enzymes have been targeted for genetic modification to attenuate softening. At least 9 genes encoding CW-modifying proteins have increased expression during ripening. Any alteration of these genes could modify CW structure and properties and contribute to softening, but evidence for their relative importance is sparse. The results of studies with transgenic tomato (Solanum lycopersicum), the model for fleshy fruit ripening, investigations with strawberry (Fragaria spp.) and apple (Malus domestica), and results from naturally occurring textural mutants provide direct evidence of gene function and the contribution of CW biochemical modifications to fruit softening. Here we review the revised CW structure model and biochemical and structural changes in CW components during fruit softening and then focus on and integrate the results of changes in CW characteristics derived from studies on transgenic fruits and mutants. Potential strategies and future research directions to understand and control the rate of fruit softening are also discussed.

Climate change and rapid adaption of invasive pathogens pose a constant pressure on fruit industry to develop improved varieties. Aiming to accelerate the development of better adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis and CRISPR/Cas genome editing hold significant potential for crop trait improvement, which have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance, tolerance to abiotic stress, and to improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. Regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.

L-Ascorbic acid (AsA) is more commonly known as vitamin C and is an indispensable compound for human health. As a major antioxidant, AsA not only maintains redox balance and resists biological and abiotic stress but also regulates plant growth, induces flowering, and delays senescence through complex signal transduction networks. However, AsA content varies greatly in horticultural crops, especially in fruit crops. The AsA content of the highest species is approximately 1,800 times higher than that of the lowest species. There have been significant advancements in the understanding of AsA accumulation in the past 20 years. The most noteworthy accomplishment was the identification of the critical rate-limiting genes for the 2 major AsA synthesis pathways (L-galactose pathway and D-galacturonic acid pathway) in fruit crops. The rate-limiting genes of the former are GMP, GME, GGP, and GPP, and the rate-limiting gene of the latter is GalUR. Moreover, APX, MDHAR, and DHAR are also regarded as key genes in degradation and regeneration pathways. Interestingly, some of these key genes are sensitive to environmental factors, such as GGP being induced by light. The efficiency of enhancing AsA content is high by editing upstream open reading frames (uORF) of the key genes and constructing multi-gene expression vectors. In summary, the AsA metabolism has been well understood in fruit crops, but the transport mechanism of AsA and the synergistic improvement of AsA and other traits is less known, which will be the focus of AsA research in fruit crops.

Dwarfism is an agronomic trait that has substantial effects on crop yield, lodging resistance, planting density, and a high harvest index. Ethylene plays an important role in plant growth and development, including the determination of plant height. However, the mechanism by which ethylene regulates plant height, especially in woody plants, remains unclear. In this study, a 1-aminocyclopropane-1-carboxylic acid synthase (ACC) gene (ACS), which is involved in ethylene biosynthesis, was isolated from lemon (Citrus limon L. Burm) and named CiACS4. Overexpression of CiACS4 resulted in a dwarf phenotype in Nicotiana tabacum and lemon and increased ethylene release and decreased gibberellin (GA) content in transgenic plants. Inhibition of CiACS4 expression in transgenic citrus significantly increased plant height compared with the controls. Yeast two-hybrid assays revealed that CiACS4 interacted with an ethylene response factor (ERF), CiERF3. Further experiments revealed that the CiACS4-CiERF3 complex can bind to the promoters of 2 citrus GA20-oxidase genes, CiGA20ox1 and CiGA20ox2, and suppress their expression. In addition, another ERF transcription factor, CiERF023, identified using yeast one-hybrid assays, promoted CiACS4 expression by binding to its promoter. Overexpression of CiERF023 in N. tabacum caused a dwarfing phenotype. CiACS4, CiERF3, and CiERF023 expression was inhibited and induced by GA3 and ACC treatments, respectively. These results suggest that the CiACS4-CiERF3 complex may be involved in the regulation of plant height by regulating CiGA20ox1 and CiGA20ox2 expression levels in citrus.

Plants have evolved sophisticated mechanisms to detect various forms of danger. Damage-associated molecular patterns (DAMPs) are endogenous danger molecules that are released from damaged cells and activate the innate immunity. Recent evidence suggests that plant extracellular self-DNA (esDNA) can serve as a DAMP molecule. However, the mechanisms by which esDNA functions are largely unknown. In this study, we confirmed that esDNA inhibits root growth and triggers reactive oxygen species (ROS) production in a concentration- and species-specific manner in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum L.). Furthermore, by combining RNA sequencing, hormone measurement, and genetic analysis, we found that esDNA-mediated growth inhibition and ROS production are achieved through the jasmonic acid (JA) signaling pathway. Specifically, esDNA induces JA production and the expression of JA-responsive genes. The esDNA-mediated growth inhibition, ROS production, and gene expression are impaired in the JA-related mutants. Finally, we found that the JA signaling pathway is required for the esDNA-elicited resistance against the pathogens Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000. This finding highlights the importance of JA signaling in esDNA-mediated biological effects, thereby providing insight into how esDNA functions as a DAMP.

Anthocyanin is a vital indicator for both fruit nutritional and commercial value. Anthocyanin accumulation is a surprisingly complicated process mediated by multiple networks associated with genetic, developmental, hormonal, and environmental factors. Transcriptional regulation along with epigenetic regulation constitutes the dominant molecular framework for anthocyanin biosynthesis. Here, we focus on current knowledge on regulatory mechanisms of anthocyanin accumulation, with emphasis on the latest progress in transcriptional and epigenetic regulation and the crosstalk between various signaling pathways. We present an emerging picture of how various internal and external stimuli control anthocyanin biosynthesis. Additionally, we discuss the synergistic or antagonistic effect of developmental, hormonal and environmental cues on anthocyanin accumulation in fruit.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

Malate impacts fruit acidity and plays a vital role in stress tolerance. Malate accumulation is induced by salinity in various plants as a metabolite in coping with this stress. However, the exact molecular mechanism responsible for salinity-induced malate accumulation remains unclear. Here, we determined that salinity treatment induces malate accumulation in pear (Pyrus spp.) fruit, calli, and plantlets compared to the control. Genetic and biochemical analyses established the key roles of PpWRKY44 and ABRE-BINDING FACTOR3 (PpABF3) transcription factors in promoting malate accumulation in response to salinity. We found that PpWRKY44 is involved in salinity-induced malate accumulation by directly binding to a W-box on the promoter of the malate-associated gene aluminum-activated malate transporter 9 (PpALMT9) to activate its expression. A series of in-vivo and in-vitro assays revealed that the G-box cis-element in the promoter of PpWRKY44 was targeted by PpABF3, which further enhanced salinity-induced malate accumulation. Taken together, these findings suggest that PpWRKY44 and PpABF3 play positive roles in salinity-induced malate accumulation in pears. This research provides insights into the molecular mechanism by which salinity affects malate accumulation and fruit quality.

The strawberry is one of the world's most popular fruits, providing humans with vitamins, fibers, and antioxidants. Cultivated strawberry (Fragaria × ananassa) is an allo-octoploid and highly heterozygous, making it a challenge for breeding, QTL mapping, and gene discovery. Some wild strawberry relatives, such as Fragaria vesca, have diploid genomes and are becoming laboratory models for the cultivated strawberry. Recent advances in genome sequencing and CRISPR-mediated genome-editing have greatly improved the understanding of various aspects of strawberry growth and development in both cultivated and wild strawberries. This review focuses on fruit quality traits that are most relevant to the consumers, including fruit aroma, sweetness, color, firmness, and shape. Recently available phased-haplotype genomes, SNP arrays, extensive fruit transcriptomes, and other big data have made it possible to locate key genomic regions or pinpoint specific genes that underlie volatile synthesis, anthocyanin accumulation for fruit color, and sweetness intensity or perception. These new advances will greatly facilitate marker-assisted breeding, the introgression of missing genes into modern varieties, and precise genome editing of selected genes and pathways. Strawberries are poised to benefit from these recent advances, providing consumers with fruit that is tastier, longer-lasting, healthier, and more beautiful.

Cucumber (Cucumis sativus L.) flesh is typically colorless or pale green. Flesh with yellow or orange pigment, determined mainly by carotenoid content and composition, is mostly found in semi-wild Xishuangbanna cucumber, which has a very narrow genetic background. Here, we identified a spontaneous cucumber mutant with yellow flesh (yf-343), which accumulated more β-cryptoxanthin and less lutein than regular cultivated European glasshouse-type cucumbers. Genetic analysis revealed that the yellow flesh phenotype was controlled by a single recessive gene. Through fine mapping and gene sequencing, we identified the candidate gene Cucumis sativus yellow flesh 2 (Csyf2), encoding an abscisic acid (ABA) 8'-hydroxylase. Overexpression and RNAi-silencing of Csyf2 in cucumber hairy roots produced lower and higher ABA contents than in non-transgenic controls, respectively. Further, RNA-seq analysis suggested that genes related to ABA signal transduction were differentially expressed in fruit flesh between yf-343 and its wild type, BY, with white flesh. The carotenoid biosynthesis pathway was specifically enriched in fruit flesh at 30 days after pollination, when yf-343 fruit flesh turns yellow. Our findings highlight a promising target for gene editing to increase carotenoid content, expanding our genetic resources for pigmented cucumber flesh breeding for improving the nutritional quality of cucumber.

Plants integrate environmental information into their developmental program throughout their lifetime. Light and temperature are particularly critical cues for plants to correctly time developmental transitions. Here, we investigated the role of photo-thermal cues in the regulation of the end-of-flowering developmental transition in the model plant Arabidopsis (Arabidopsis thaliana). We found that increased day length and higher temperature during flowering promote earlier inflorescence arrest by accelerating the rate at which the inflorescence meristem (IM) initiates floral primordia. Specifically, we show that plants arrest at a photo-thermal threshold and demonstrate that this photo-thermally mediated arrest is mediated by the floral integrator FLOWERING LOCUS T (FT), a known activator of flowering. FT expression increased over the duration of flowering, peaking during IM arrest, and we show that this is necessary and sufficient for photo-thermally induced arrest. Our data demonstrate the role of light and temperature, through FT, as key regulators of end-of-flowering. Overall, our results have important implications for understanding and modulating the flowering duration of crop species in changing light and temperature conditions in a warming global climate.

Banana (Musa acuminata) fruit ripening under high temperatures (>24 °C) undergoes green ripening due to failure of chlorophyll degradation, which greatly reduces marketability. However, the mechanism underlying high temperature-repressed chlorophyll catabolism in banana fruit is not yet well understood. Here, using quantitative proteomic analysis, 375 differentially expressed proteins were identified in normal yellow and green ripening in banana. Among these, one of the key enzymes involved in chlorophyll degradation, NON-YELLOW COLORING 1 (MaNYC1), exhibited reduced protein levels when banana fruit ripened under high temperature. Transient overexpression of MaNYC1 in banana peels resulted in chlorophyll degradation under high temperature, which weakens the green ripening phenotype. Importantly, high temperature induced MaNYC1 protein degradation via the proteasome pathway. A banana RING E3 ligase, NYC1-interacting protein 1 (MaNIP1), was found to interact with and ubiquitinate MaNYC1, leading to its proteasomal degradation. Furthermore, transient overexpression of MaNIP1 attenuated MaNYC1-induced chlorophyll degradation in banana fruits, indicating that MaNIP1 negatively regulates chlorophyll catabolism by affecting MaNYC1 degradation. Taken together, the findings establish a post-translational regulatory module of MaNIP1-MaNYC1 that mediates high temperature-induced green ripening in bananas.

Sugars are fundamental to plant developmental processes. For fruits, the accumulation and proportion of sugars play crucial roles in the development of quality and attractiveness. In citrus (Citrus reticulata Blanco.), we found that the difference in sweetness between mature fruits of "Gongchuan" and its bud sport "Youliang" is related to hexose contents. Expression of a SuS (sucrose synthase) gene CitSUS5 and a SWEET (sugars will eventually be exported transporter) gene CitSWEET6, characterized by transcriptome analysis at different developmental stages of these 2 varieties, revealed higher expression levels in "Youliang" fruit. The roles of CitSUS5 and CitSWEET6 were investigated by enzyme activity and transient assays. CitSUS5 promoted the cleavage of sucrose to hexoses, and CitSWEET6 was identified as a fructose transporter. Further investigation identified the transcription factor CitZAT5 (ZINC FINGER OF ARABIDOPSIS THALIANA) that contributes to sucrose metabolism and fructose transportation by positively regulating CitSUS5 and CitSWEET6. The role of CitZAT5 in fruit sugar accumulation and hexose proportion was investigated by homologous transient CitZAT5 overexpression, -VIGS, and -RNAi. CitZAT5 modulates the hexose proportion in citrus by mediating CitSUS5 and CitSWEET6 expression, and the molecular mechanism explained the differences in sugar composition of "Youliang" and "Gongchuan" fruit.

The relationships between aerial organ morpho-anatomy of woody polyploid plants with their functional hydraulics under water stress remain largely understudied. We evaluated growth-associated traits, aerial organ xylem anatomy, and physiological parameters of diploid, triploid, and tetraploid genotypes of atemoyas (Annona cherimola × Annona squamosa), which belong to the woody perennial genus Annona (Annonaceae), testing their performance under long-term soil water reduction. The contrasting phenotypes of vigorous triploids and dwarf tetraploids consistently showed stomatal size-density tradeoff. The vessel elements in aerial organs were ∼1.5 times wider in polyploids compared with diploids, and triploids displayed the lowest vessel density. Plant hydraulic conductance was higher in well-irrigated diploids while their tolerance to drought was lower. The phenotypic disparity of atemoya polyploids associated with contrasting leaf and stem xylem porosity traits that coordinate to regulate water balances between the trees and the belowground and aboveground environments. Polyploid trees displayed better performance under soil water scarcity, and consequently, could present more sustainable agricultural and forestry genotypes to cope with water stress.

Drought stress substantially reduces the productivity of apple plants and severely restricts the development of apple industry. Malus sieversii, wild apples with excellent drought resistance, is a valuable wild resource for a rootstock improvement of cultivated apple (Malus domestica). miRNAs and their targets play essential roles in plant growth and stress responses, but their roles in drought stress responses in apple are unknown. Here, we demonstrate that microRNA156ab is upregulated in M. sieversii in response to drought stress. Overexpressing msi-miR156ab promoted auxin accumulation, maintained the growth of apple plants, and increased plant resistance to osmotic stress. Antioxidant enzyme activities and proline contents were also increased in miR156ab-OE transgenic apple lines, which improved drought resistance. The squamosa promoter binding protein-like transcription factor MsSPL13 is the target of msi-miR156ab, as demonstrated by 5'-RACE and dual luciferase assays. Heterologous expression of MsSPL13 decreased auxin contents and inhibited growth in Arabidopsis (Arabidopsis thaliana) under normal and stress conditions. The activities of antioxidant enzymes were also suppressed in MsSPL13-OE transgenic Arabidopsis, reducing drought resistance. We showed that MsSPL13 regulates the expression of the auxin-related genes MsYUCCA5, PIN-FORMED7 (MsPIN7), and Gretchen Hagen3-5 (MsGH3-5) by binding to the GTAC cis-elements in their promoters, thereby regulating auxin metabolism. Finally, we demonstrated that the miR156ab-SPL13 module is involved in mediating the difference in auxin metabolism and stress responses between M. sieversii and M26 (M. domestica) rootstocks. Overall, these findings reveal that the miR156ab-SPL13 module enhances drought stress tolerance in apples by regulating auxin metabolism and antioxidant enzyme activities.

Soybean (Glycine max L. Merrill) is one of the most important economical crops. A large number of whole-genome resequencing datasets have been generated and are increasingly expanded for exploring genetic diversity and mining important quantitative trait loci. Most genome-wide association studies have focused on single-nucleotide polymorphisms, short insertions and deletions. Nevertheless, structure variants mainly caused by transposon element (TE) mobilization are not fully considered. To fill this gap, we uniformly processed the publicly available whole-genome resequencing data from 5,521 soybean germplasm accessions and built an online soybean transposon insertion polymorphisms database named SoyTIPdb (https://biotec.njau.edu.cn/soytipdb). The collected germplasm accessions derived from more than 45 countries and 160 regions representing the most comprehensive genetic diversity of soybean. SoyTIPdb implements easy-to-use query, analysis and browse functions to help understand and find meaningful structural variations from TE insertions. In conclusion, SoyTIPdb is a valuable data resource and will help soybean breeders/researchers take advantage of the whole-genome sequencing datasets available in the public depositories.

Phytosulfokine (PSK) is a danger-associated molecular pattern recognized by PHYTOSULFOKINE RECEPTOR 1 (PSKR1) and initiates intercellular signaling to coordinate different physiological processes, especially in the defense response to the necrotrophic fungus Botrytis cinerea. The activity of peptide receptors is largely influenced by different posttranslational modifications, which determine intercellular peptide signal outputs. To date, the posttranslational modification to PHYTOSULFOKINE RECEPTOR 1 (PSKR1) remains largely unknown. Here, we show that tomato (Solanum lycopersicum) PSKR1 is regulated by the ubiquitin/proteasome degradation pathway. Using multiple protein-protein interactions and ubiquitylation analyses, we identified that plant U-box E3 ligases PUB12 and PUB13 interacted with PSKR1, among which PUB13 caused PSKR1 ubiquitylation at Lys-748 and Lys-905 sites to control PSKR1 abundance. However, this posttranslational modification was attenuated upon addition of PSK. Moreover, the disease symptoms observed in PUB13 knock-down and overexpression lines demonstrated that PUB13 significantly suppressed the PSK-initiated defense response. This highlights an important regulatory function for the turnover of a peptide receptor by E3 ligase-mediated ubiquitylation in the plant defense response.

Chlorophyll degradation and anthocyanin biosynthesis, which often occur almost synchronously during fruit ripening, are crucial for vibrant coloration of fruits. However, the interlink point between their regulatory pathways remains largely unknown. Here, 2 litchi (Litchi chinensis Sonn.) cultivars with distinctively different coloration patterns during ripening, i.e. slow-reddening/stay-green "Feizixiao" (FZX) vs rapid-reddening/degreening "Nuomici" (NMC), were selected as the materials to study the key factors determining coloration. Litchi chinensis STAY-GREEN (LcSGR) was confirmed as the critical gene in pericarp chlorophyll loss and chloroplast breakdown during fruit ripening, as LcSGR directly interacted with pheophorbide a oxygenase (PAO), a key enzyme in chlorophyll degradation via the PAO pathway. Litchi chinensis no apical meristem (NAM), Arabidopsis transcription activation factor 1/2, and cup-shaped cotyledon 2 (LcNAC002) was identified as a positive regulator in the coloration of litchi pericarp. The expression of LcNAC002 was significantly higher in NMC than in FZX. Virus-induced gene silencing of LcNAC002 significantly decreased the expression of LcSGR as well as L. chinensis MYELOBLASTOSIS1 (LcMYB1), and inhibited chlorophyll loss and anthocyanin accumulation. A dual-luciferase reporter assay revealed that LcNAC002 significantly activates the expression of both LcSGR and LcMYB1. Furthermore, yeast-one-hybrid and electrophoretic mobility shift assay results showed that LcNAC002 directly binds to the promoters of LcSGR and LcMYB1. These findings suggest that LcNAC002 is an important ripening-related transcription factor that interlinks chlorophyll degradation and anthocyanin biosynthesis by coactivating the expression of both LcSGR and LcMYB1.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears (Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and the expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation by a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 and PyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provided a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. These subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.

Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short-form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.

Time to flowering (vegetative to reproductive phase) is tightly regulated by endogenous factors and environmental cues to ensure proper and successful reproduction. How endogenous factors coordinate with environmental signals to regulate flowering time in plants is unclear. Transcription factors ETHYLENE INSENSITIVE 3 (EIN3) and its homolog EIN3 LIKE 1 (EIL1) are the core downstream regulators in ethylene signal transduction, and their null mutants exhibit late flowering in Arabidopsis (Arabidopsis thaliana); however, the precise mechanism of floral transition remains unknown. Here, we reveal that FLOWERING LOCUS D (FLD), encoding a histone demethylase acting in the autonomous pathway of floral transition, physically associates with EIN3 and EIL1. Loss of EIN3 and EIL1 upregulated transcriptional expression of the floral repressor FLOWERING LOCUS C (FLC) and its homologs in Arabidopsis, and ethylene-insensitive mutants displayed inhibited flowering in an FLC-dependent manner. We further demonstrated that EIN3 and EIL1 directly bind to FLC loci, modulating their expression by recruiting FLD and thereafter removing di-methylation of lysine 4 on histone H3 (H3K4me2). In plants treated with 1-aminocyclopropane-1-carboxylic acid, decreased expression of FLD resulted in increased enrichment of H3K4me2 at FLC loci and transcriptional activation of FLC, leading to floral repression. Our study reveals the role of EIN3 and EIL1 in FLC-dependent and ethylene-induced floral repression and elucidates how phytohormone signals are transduced into chromatin-based transcriptional regulation.

Rice (Oryza sativa L.) microRNA156/529-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7/14/17 (miR156/529-SPL7/14/17) modules have pleiotropic effects on many biological pathways. OsSPL7/14 can interact with DELLA protein SLENDER RICE1 (SLR1) to modulate gibberellin acid (GA) signal transduction against the bacterial pathogen Xanthomonas oryzae pv. oryzae. However, whether the miR156/529-OsSPL7/14/17 modules also regulate resistance against other pathogens is unclear. Notably, OsSPL7/14/17 functioning as transcriptional activators, their target genes, and the corresponding downstream signaling pathways remain largely unexplored. Here, we demonstrate that miR156/529 play negative roles in plant immunity and that miR156/529-regulated OsSPL7/14/17 confer broad-spectrum resistance against 2 devastating bacterial pathogens. Three OsSPL7/14/17 proteins directly bind to the promoters of rice Allene Oxide Synthase 2 (OsAOS2) and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (OsNPR1) and activate their transcription, regulating jasmonic acid (JA) accumulation and the salicylic acid (SA) signaling pathway, respectively. Overexpression of OsAOS2 or OsNPR1 impairs the susceptibility of the osspl7/14/17 triple mutant. Exogenous application of JA enhances resistance of the osspl7/14/17 triple mutant and the miR156 overexpressing plants. In addition, genetic evidence confirms that bacterial pathogen-activated miR156/529 negatively regulate pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, such as pattern recognition receptor Xa3/Xa26-initiated PTI. Our findings demonstrate that bacterial pathogens modulate miR156/529-OsSPL7/14/17 modules to suppress OsAOS2-catalyzed JA accumulation and the OsNPR1-promoted SA signaling pathway, facilitating pathogen infection. The uncovered miR156/529-OsSPL7/14/17-OsAOS2/OsNPR1 regulatory network provides a potential strategy to genetically improve rice disease resistance.

Anthocyanin production in bicolored sweet cherry (Prunus avium cv. Rainier) fruit is induced by light exposure, leading to red coloration. The phytohormone abscisic acid (ABA) is essential for this process, but the regulatory relationships that link light and ABA with anthocyanin-associated coloration are currently unclear. In this study, we determined that light treatment of bicolored sweet cherry fruit increased anthocyanin accumulation and induced ABA production and that ABA participates in light-modulated anthocyanin accumulation in bicolored sweet cherry. Two B-box (BBX) genes, PavBBX6/9, were highly induced by light and ABA treatments, as was anthocyanin accumulation. The ectopic expression of PavBBX6 or PavBBX9 in Arabidopsis (Arabidopsis thaliana) increased anthocyanin biosynthesis and ABA accumulation. Overexpressing PavBBX6 or PavBBX9 in sweet cherry calli also enhanced light-induced anthocyanin biosynthesis and ABA accumulation. Additionally, transient overexpression of PavBBX6 or PavBBX9 in sweet cherry peel increased anthocyanin and ABA contents, whereas silencing either gene had the opposite effects. PavBBX6 and PavBBX9 directly bound to the G-box elements in the promoter of UDP glucose-flavonoid-3-O-glycosyltransferase (PavUFGT), a key gene for anthocyanin biosynthesis, and 9-cis-epoxycarotenoid dioxygenase 1 (PavNCED1), a key gene for ABA biosynthesis, and enhanced their activities. These results suggest that PavBBX6 and PavBBX9 positively regulate light-induced anthocyanin and ABA biosynthesis by promoting PavUFGT and PavNCED1 expression, respectively. Our study provides insights into the relationship between the light-induced ABA biosynthetic pathway and anthocyanin accumulation in bicolored sweet cherry fruit.

Introns are non-coding sequences spliced out of pre-mRNAs by the spliceosome to produce mature mRNAs. The 5' ends of introns mostly begin with GU and have a conserved sequence motif of AG/GUAAGU that could base-pair with the core sequence of U1 snRNA of the spliceosome. Intriguingly, approximately 1% of introns in various eukaryotic species begin with GC. This occurrence could cause mis-annotation of genes; however, the underlying splicing mechanism is unclear. We analyzed the sequences around the intron 5' splice site (ss) in Arabidopsis (Arabidopsis thaliana) and found sequences at the GC intron ss are much more stringent than those of GT introns. Mutational analysis at various positions of the intron 5' ss revealed that although mutations impair base pairing, different mutations at the same site can have different effects, suggesting that steric hindrance also affects splicing. Moreover, mutations of 5' ss often activate a hidden ss nearby. Our data suggest that the 5' ss is selected via a competition between the major ss and the nearby minor ss. This work not only provides insights into the splicing mechanism of intron 5' ss but also improves the accuracy of gene annotation and the study of the evolution of intron 5' ss.

S-RNase-mediated self-incompatibility (SI) prevents self-fertilization and promotes outbreeding to ensure genetic diversity in many flowering plants, including pear (Pyrus sp.). Brassinosteroids (BRs) have well-documented functions in cell elongation, but their molecular mechanisms in pollen tube growth, especially in the SI response, remain elusive. Here, exogenously applied brassinolide (BL), an active BR, countered incompatible pollen tube growth inhibition during the SI response in pear. Antisense repression of BRASSINAZOLE-RESISTANT1 (PbrBZR1), a critical component of BR signaling, blocked the positive effect of BL on pollen tube elongation. Further analyses revealed that PbrBZR1 binds to the promoter of EXPANSIN-LIKE A3 (PbrEXLA3) to activate its expression. PbrEXLA3 encodes an expansin that promotes pollen tube elongation in pear. The stability of dephosphorylated PbrBZR1 was substantially reduced in incompatible pollen tubes, where it is targeted by ARIADNE2.3 (PbrARI2.3), an E3 ubiquitin ligase that is strongly expressed in pollen. Our results show that during the SI response, PbrARI2.3 accumulates and negatively regulates pollen tube growth by accelerating the degradation of PbrBZR1 via the 26S proteasome pathway. Together, our results show that an ubiquitin-mediated modification participates in BR signaling in pollen and reveal the molecular mechanism by which BRs regulate S-RNase-based SI.

Programed cell death (PCD) plays fundamental roles in plant development and responses to environmental stresses. Here, we report a protein, SICKLE (SIC), which represses PCD. In Arabidopsis (Arabidopsis thaliana), the loss-of-function mutant of SIC, sic-4, hyperaccumulated lariat intronic RNAs (lariRNAs) and exhibited PCD. The gene encoding an RNA debranching enzyme 1 (DBR1), a rate-limiting enzyme for lariRNAs decay, was overexpressed to reduce the level of lariRNAs in the sic-4 mutant, which led to suppression of PCD. Meanwhile, another lariRNAs hyper-accumulating mutant, dbr1-2, also exhibited PCD, further indicating that sic-4 PCD is caused by hyper-accumulation of lariRNAs. Transcriptional profiling analyses revealed that the sic-4 mutation disturbed alternative splicing and decay of mRNAs associated with salicylic acid (SA) homeostasis, a well-known molecule functioning in PCD regulation. Moreover, SA is dramatically increased in sic-4 and the disruption of SA biosynthesis and signaling suppressed PCD in the mutant, demonstrating that SA functions downstream of sic-4. Taken together, our results demonstrate that SIC is involved in regulating SA-triggered PCD.

The genus Vaccinium L. (Ericaceae) contains premium berryfruit crops, including blueberry, cranberry, bilberry, and lingonberry. Consumption of Vaccinium berries is strongly associated with various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids, including the anthocyanins that provide the attractive red and blue berry colors. Because these phytochemicals are increasingly appealing to consumers, they have become a crop breeding target. There has been substantial recent progress in Vaccinium genomics and genetics together with new functional data on the transcriptional regulation of flavonoids. This is helping to unravel the developmental control of flavonoids and identify genetic regions and genes that can be selected for to further improve Vaccinium crops and advance our understanding of flavonoid regulation and biosynthesis across a broader range of fruit crops. In this update we consider the recent progress in understanding flavonoid regulation in fruit crops, using Vaccinium as an example and highlighting the significant gains in both genomic tools and functional analysis.

Plant somatic embryogenesis (SE) is an in vitro biological process wherein bipolar structures are induced to form somatic cells and regenerate into whole plants. MicroRNA (miRNA) is an essential player in plant SE. However, the mechanism of microRNA408 (miR408) in SE remains elusive. Here, we used stable transgenic technology in longan (Dimocarpus longan) embryogenic calli to verify the mechanism by which miR408 promotes cell division and differentiation of longan early SE. dlo-miR408-3p regulated riboflavin biosynthesis by targeting nudix hydrolase 23 (DlNUDT23), a previously unidentified gene mediating N6-methyladenosine (m6A) modification and influencing RNA homeostasis and cell cycle gene expression during longan early SE. We showed that DlMIR408 overexpression (DlMIR408-OE) promoted 21-nt miRNA biosynthesis. In DlMIR408-OE cell lines, dlo-miR408-3p targeted and downregulated DlNUDT23, promoted riboflavin biosynthesis, decreased flavin mononucleotide (FMN) accumulation, promoted m6A level, and influenced miRNA homeostasis. DNA replication, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, the pentose phosphate pathway, and taurine and hypotaurine metabolism were also closely associated with riboflavin metabolism. In a riboflavin feeding assay, dlo-miR408-3p and pre-miR408 were upregulated and DlNUDT23 was downregulated, increasing the m6A level and cell division and differentiation in longan globular embryos. When riboflavin biosynthesis was inhibited, dlo-miR408-3p was downregulated and DlNUDT23 was upregulated, which decreased m6A modification and inhibited cell division but did not inhibit cell differentiation. FMN artificial demethylated m6A modification affected the homeostasis of precursor miRNA and miRNA. Our results revealed a mechanism underlying dlo-miR408-3p-activated riboflavin biosynthesis in which DlNUDT23 is targeted, m6A modification is dynamically mediated, and cell division is affected, promoting early SE in plants.

Terpene synthases (TPSs) play pivotal roles in conferring the structural diversity of terpenoids, which are mainly emitted from flowers, whereas the genetic basis of the release of floral volatile terpenes remains largely elusive. Though quite similar in sequence, TPS allelic variants still function divergently, and how they drive floral terpene diversity in closely related species remains unknown. Here, TPSs responsible for the floral scent of wild Freesia species were characterized, and the functions of their natural allelic variants, as well as the causal amino acid residues, were investigated in depth. Besides the 8 TPSs previously reported in modern cultivars, 7 additional TPSs were functionally evaluated to contribute to the major volatiles emitted from wild Freesia species. Functional characterization of allelic natural variants demonstrated that allelic TPS2 and TPS10 variants changed the enzymatic capacity while allelic TPS6 variants drove the diversity of floral terpene products. Further residue substitution analysis revealed the minor residues determining the enzyme catalytic activity and product specificity. The clarification of TPSs in wild Freesia species reveals that allelic TPS variants evolved differently to determine the interspecific floral volatile terpenes in the genus and might be used for modern cultivar improvement.

ETHYLENE-INSENSITIVE 3/ETHYLENE-INSENSITIVE 3-LIKEs (EIN3/EILs) are important ethylene response factors during fruit ripening. Here, we discovered that EIL2 controls carotenoid metabolism and ascorbic acid (AsA) biosynthesis in tomato (Solanum lycopersicum). In contrast to the red fruits presented in the wild type (WT) 45 d after pollination, the fruits of CRISPR/Cas9 eil2 mutants and SlEIL2 RNA interference lines (ERIs) showed yellow or orange fruits. Correlation analysis of transcriptome and metabolome data for the ERI and WT ripe fruits revealed that SlEIL2 is involved in β-carotene and AsA accumulation. ETHYLENE RESPONSE FACTORs (ERFs) are the typical components downstream of EIN3 in the ethylene response pathway. Through a comprehensive screening of ERF family members, we determined that SlEIL2 directly regulates the expression of 4 SlERFs. Two of these, SlERF.H30 and SlERF.G6, encode proteins that participate in the regulation of LYCOPENE-β-CYCLASE 2 (SlLCYB2), encoding an enzyme that mediates the conversion of lycopene to carotene in fruits. In addition, SlEIL2 transcriptionally repressed L-GALACTOSE 1-PHOSPHATE PHOSPHATASE 3 (SlGPP3) and MYO-INOSITOL OXYGENASE 1 (SlMIOX1) expression, which resulted in a 1.62-fold increase of AsA via both the L-galactose and myoinositol pathways. Overall, we demonstrated that SlEIL2 functions in controlling β-carotene and AsA levels, providing a potential strategy for genetic engineering to improve the nutritional value and quality of tomato fruit.

Plant water uptake is determined by the root system architecture and its hydraulic capacity, which together define the root hydraulic architecture. The current research aims at understanding the water uptake capacities of maize (Zea mays), a model organism and major crop. We explored the genetic variations within a collection of 224 maize inbred Dent lines and successively defined core genotype subsets to access multiple architectural, anatomical, and hydraulic parameters in the primary root (PR) and seminal roots (SR) of hydroponically grown seedlings. We found 9-, 3.5-, and 12.4-fold genotypic differences for root hydraulics (Lpr), PR size, and lateral root size, respectively, that shaped wide and independent variations of root structure and function. Within genotypes, PR and SR showed similarities in hydraulics and, to a lesser extent, in anatomy. They had comparable aquaporin activity profiles that, however, could not be explained by aquaporin expression levels. Genotypic variations in the size and number of late meta xylem vessels were positively correlated with Lpr. Inverse modeling further revealed dramatic genotypic differences in the xylem conductance profile. Thus, tremendous natural variation of maize root hydraulic architecture underlies a high diversity of water uptake strategies and paves the way to quantitative genetic dissection of its elementary traits.

Plant architecture is one of the most important factors that determines crop yield potential and productivity. In apple (Malus domestica), genetic improvement of tree architecture has been challenging due to a long juvenile phase and growth as complex trees composed of a distinct scion and a rootstock. To better understand the genetic control of apple tree architecture, the dominant weeping growth phenotype was investigated. We report the identification of MdLAZY1A (MD13G1122400) as the genetic determinant underpinning the Weeping (W) locus that largely controls weeping growth in Malus. MdLAZY1A is one of the four paralogs in apple that are most closely related to AtLAZY1 involved in gravitropism in Arabidopsis (Arabidopsis thaliana). The weeping allele (MdLAZY1A-W) contains a single nucleotide mutation c.584T > C that leads to a leucine to proline (L195P) substitution within a predicted transmembrane domain that co-localizes with Region III, one of the five conserved regions in LAZY1-like proteins. Subcellular localization revealed that MdLAZY1A localizes to the plasma membrane and nucleus in plant cells. Over-expressing the weeping allele in apple cultivar Royal Gala (RG) with standard growth habit impaired its gravitropic response and altered the growth to weeping-like. Suppressing the standard allele (MdLAZY1A-S) by RNA interference (RNAi) in RG similarly changed the branch growth direction to downward. Overall, the L195P mutation in MdLAZY1A is genetically causal for weeping growth, underscoring not only the crucial roles of residue L195 and Region III in MdLAZY1A-mediated gravitropic response, but also a potential DNA base editing target for tree architecture improvement in Malus and other crops.

Heat stress has a deleterious effect on male fertility in rice (Oryza sativa), but mechanisms to protect against heat stress in rice male gametophytes are poorly understood. Here, we have isolated and characterized a heat-sensitive male-sterile rice mutant, heat shock protein60-3b (oshsp60-3b), that shows normal fertility at optimal temperatures but decreasing fertility as temperatures increase. High temperatures interfered with pollen starch granule formation and reactive oxygen species (ROS) scavenging in oshsp60-3b anthers, leading to cell death and pollen abortion. In line with the mutant phenotypes, OsHSP60-3B was rapidly upregulated in response to heat shock and its protein products were localized to the plastid. Critically, overexpression of OsHSP60-3B enhanced the heat tolerance of pollen in transgenic plants. We demonstrated that OsHSP60-3B interacted with FLOURY ENDOSPERM6(FLO6) in plastids, a key component involved in the starch granule formation in the rice pollen. Western blot results showed that FLO6 level was substantially decreased in oshsp60-3b anthers at high temperature, indicating that OsHSP60-3B is required to stabilize FLO6 when temperatures exceed optimal conditions. We suggest that in response to high temperature, OsHSP60-3B interacts with FLO6 to regulate starch granule biogenesis in rice pollen and attenuates ROS levels in anthers to ensure normal male gametophyte development in rice.

Enhancing fruit sugar contents, especially for high-flavonoid apples with a sour taste, is one of the main goals of horticultural crop breeders. This study analyzed sugar accumulation and the underlying mechanisms in the F2 progenies of a hybridization between the high-sugar apple (Malus × domestica) variety "Gala" and high-flavonoid apple germplasm "CSR6R6". We revealed that MdSWEET9b (sugars will eventually be exported transporter) helps mediate sugar accumulation in fruits. Functional characterization of MdSWEET9b in yeast mutants lacking sugar transport as well as in overexpressing and CRISPR/Cas9 knockdown apple calli revealed MdSWEET9b could transport sucrose specifically, ultimately promoting normal yeast growth and accumulation of total sugar contents. Moreover, MdWRKY9 bound to the MdSWEET9b promoter and regulated its activity, which responded to abscisic acid (ABA) signaling. Furthermore, MdWRKY9 interacted with MdbZIP23 (basic leucine zipper) and MdbZIP46, key ABA signal transducers, at the protein and DNA levels to enhance its regulatory effect on MdSWEET9b expression, thereby influencing sugar accumulation. Based on the contents of ABA in lines with differing sugar contents and the effects of ABA treatments on fruits and calli, we revealed ABA as one of the main factors responsible for the diversity in apple fruit sugar content. The results of this study have clarified how MdSWEET9b influences fruit sugar accumulation, while also further elucidating the regulatory effects of the ABA-signaling network on fruit sugar accumulation. This work provides a basis for future explorations of the crosstalk between hormone and sugar metabolism pathways.

The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation. Hall's panicgrass (Panicum hallii, P. hallii) is a wild perennial grass that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the 2 major ecotypes in P. hallii, the upland ecotype (P. hallii var hallii, HAL2 genotype) with compact inflorescence and large seed and the lowland ecotype (P. hallii var filipes, FIL2 genotype) with an open inflorescence and small seed. Here we conducted a comparative analysis of the transcriptome and DNA methylome, an epigenetic mark that influences gene expression regulation, across different stages of inflorescence development using genomic references for each ecotype. Global transcriptome analysis of differentially expressed genes (DEGs) and co-expression modules underlying the inflorescence divergence revealed the potential role of cytokinin signaling in heterochronic changes. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of P. hallii inflorescence. We found that a large proportion of differentially methylated regions (DMRs) were located in the flanking regulatory regions of genes. Intriguingly, we observed a substantial bias of CHH hypermethylation in the promoters of FIL2 genes. The integration of DEGs, DMRs, and Ka/Ks ratio results characterized the evolutionary features of DMR-associated DEGs that contribute to the divergence of the P. hallii inflorescence. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a genomic resource for perennial grass biology.

The circadian oscillator allows organisms to synchronize their cellular and physiological activities with diurnal environmental changes. In plants, the circadian clock is primarily composed of multiple transcriptional-translational feedback loops. Regulators of post-transcriptional events, such as precursor messenger RNAs (pre-mRNA) splicing factors, are also involved in controlling the pace of the clock. However, in most cases the underlying mechanisms remain unclear. We have previously identified XAP5 CIRCADIAN TIMEKEEPER (XCT) as an Arabidopsis thaliana circadian clock regulator with uncharacterized molecular functions. Here, we report that XCT physically interacts with components of the spliceosome, including members of the Nineteen Complex (NTC). PacBio Iso-Seq data show that xct mutants have transcriptome-wide pre-mRNA splicing defects, predominantly aberrant 3' splice site selection. Expression of a genomic copy of XCT fully rescues those splicing defects, demonstrating that functional XCT is important for splicing. Dawn-expressed genes are significantly enriched among those aberrantly spliced in xct mutants, suggesting that the splicing activity of XCT may be circadian regulated. Furthermore, we show that loss-of-function mutations in PRP19A or PRP19B, 2 homologous core NTC components, suppress the short circadian period phenotype of xct-2. However, we do not see rescue of the splicing defects of core clock genes in prp19 xct mutants. Therefore, our results suggest that XCT may regulate splicing and the clock function through genetically separable pathways.

Phytohormones are naturally occurring small organic molecules found at low concentrations in plants. They perform essential functions in growth and developmental processes, from organ initiation to senescence, including fruit ripening. These regulatory molecules are studied using different experimental approaches, such as performing exogenous applications, evaluating endogenous levels, and/or obtaining genetically modified lines. Here, we discuss the advantages and limitations of current experimental approaches used to study active biomolecules modulating fruit ripening, focusing on melatonin. Although melatonin has been implicated in fruit ripening in several model fruit crops, current knowledge is affected by the different experimental approaches used, which have given different and sometimes even contradictory results. The methods of application and the doses used have produced different results in studies based on exogenous applications, while different measurement methods and ways of expressing results explain most of the variability in studies using correlative analyses. Furthermore, studies on genetically modified crops have focused on tomato (Solanum lycopersicum L.) plants only. However, TILLING and CRISPR methodologies are becoming essential tools to complement the results from the experimental approaches described above. This will not only help the scientific community better understand the role of melatonin in modulating fruit ripening, but it will also help develop technological advances to improve fruit yield and quality in major crops. The combination of various experimental approaches will undoubtedly lead to a complete understanding of the function of melatonin in fruit ripening in the near future, so that this knowledge can be effectively transferred to the field.

Mycorrhizae are ubiquitous symbioses established between fungi and plant roots. Orchids, in particular, require compatible mycorrhizal fungi for seed germination and protocorm development. Unlike arbuscular mycorrhizal fungi, which have wide host ranges, orchid mycorrhizal fungi are often highly specific to their host orchids. However, the molecular mechanism of orchid mycorrhizal symbiosis is largely unknown compared to that of arbuscular mycorrhizal and rhizobial symbiosis. Here, we report that an endophytic Sebacinales fungus, Serendipita indica, promotes seed germination and the development of protocorms into plantlets in several epiphytic Epidendroideae orchid species (6 species in 2 genera), including Dendrobium catenatum, a critically endangered orchid with high medicinal value. Although plant-pathogen interaction and high meristematic activity can induce the hypoxic response in plants, it has been unclear whether interactions with beneficial fungi, especially mycorrhizal ones, also involve the hypoxic response. By studying the symbiotic relationship between D. catenatum and S. indica, we determined that hypoxia-responsive genes, such as those encoding alcohol dehydrogenase (ADH), are highly induced in symbiotic D. catenatum protocorms. In situ hybridization assay indicated that the ADH gene is predominantly expressed in the basal mycorrhizal region of symbiotic protocorms. Additionally, the ADH inhibitors puerarin and 4-methylpyrazole both decreased S. indica colonization in D. catenatum protocorms. Thus, our study reveals that S. indica is widely compatible with orchids and that ADH and its related hypoxia-responsive pathway are involved in establishing successful symbiotic relationships in germinating orchids.

Terpenes are volatile compounds responsible for aroma and the postharvest quality of commercially important xiangfei (Torreya grandis) nuts, and there is interest in understanding the regulation of their biosynthesis. Here, a transcriptomics analysis of xiangfei nuts after harvest identified 156 genes associated with the terpenoid metabolic pathway. A geranyl diphosphate (GPP) synthase (TgGPPS) involved in production of the monoterpene precursor GPP was targeted for functional characterization, and its transcript levels positively correlated with terpene levels. Furthermore, transient overexpression of TgGPPS in tobacco (Nicotiana tabacum) leaves or tomato (Solanum lycopersicum) fruit led to monoterpene accumulation. Analysis of differentially expressed transcription factors identified one basic helix-loop-helix protein (TgbHLH95) and one basic leucine zipper protein (TgbZIP44) as potential TgGPPS regulators. TgbHLH95 showed significant transactivation of the TgGPPS promoter, and its transient overexpression in tobacco leaves led to monoterpene accumulation, whereas TgbZIP44 directly bound to an ACGT-containing element in the TgGPPS promoter, as determined by yeast one-hybrid test and electrophoretic mobility shift assay. Bimolecular fluorescence complementation, firefly luciferase complementation imaging, co-immunoprecipitation and GST pull-down assays confirmed a direct protein-protein interaction between TgbHLH95 and TgbZIP44 in vivo and in vitro, and in combination these proteins induced the TgGPPS promoter up to 4.7-fold in transactivation assays. These results indicate that a TgbHLH95/TgbZIP44 complex activates the TgGPPS promoter and up-regulates terpene biosynthesis in xiangfei nuts after harvest, thereby contributing to its aroma.

Plant lignocellulosic biomass, i.e., secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan, but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation, increases lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene co-expression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was not only regulated by the secondary cell wall hierarchical regulatory network, but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification, and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

Roots anchor plants in soil, and the failure of anchorage (i.e. root lodging) is a major cause of crop yield loss. Anchorage is often assumed to be driven by root system architecture (RSA). We made use of a natural experiment to measure the overlap between the genetic regulation of RSA and anchorage. After one of the most devastating derechos ever recorded in August 2020, we phenotyped root lodging in a maize (Zea mays) diversity panel consisting of 369 genotypes grown in 6 environments affected by the derecho. Genome-wide and transcriptome-wide association studies identified 118 candidate genes associated with root lodging. Thirty-four percent (40/118) of these were homologs of genes from Arabidopsis (Arabidopsis thaliana) that affect traits such as root morphology and lignin content, expected to affect root lodging. Finally, gene ontology enrichment analysis of the candidate genes and their predicted interaction partners at the transcriptional and translational levels revealed the complex regulatory networks of physiological and biochemical pathways underlying root lodging in maize. Limited overlap between genes associated with lodging resistance and RSA in this diversity panel suggests that anchorage depends in part on factors other than the gross characteristics of RSA.

Sorbitol is a major photosynthate produced in leaves and transported through the phloem of apple (Malus domestica) and other tree fruits in Rosaceae. Sorbitol stimulates its own metabolism, but the underlying molecular mechanism remains unknown. Here, we show that sucrose nonfermenting 1 (SNF1)-related protein kinase 1 (SnRK1) is involved in regulating the sorbitol-responsive expression of both SORBITOL DEHYDROGENASE 1 (SDH1) and ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), encoding 2 key enzymes in sorbitol metabolism. SnRK1 expression is increased by feeding of exogenous sorbitol but decreased by sucrose. SnRK1 interacts with and phosphorylates the basic leucine zipper (bZIP) transcription factor bZIP39. bZIP39 binds to the promoters of both SDH1 and A6PR and activates their expression. Overexpression of SnRK1 in 'Royal Gala' apple increases its protein level and activity, upregulating transcript levels of both SDH1 and A6PR without altering the expression of bZIP39. Of all the sugars tested, sorbitol is the only 1 that stimulates SDH1 and A6PR expression, and this stimulation is blocked by RNA interference (RNAi)-induced repression of either SnRK1 or bZIP39. These findings reveal that sorbitol acts as a signal regulating its own metabolism via SnRK1-mediated phosphorylation of bZIP39, which integrates sorbitol signaling into the SnRK1-mediated sugar signaling network to modulate plant carbohydrate metabolism.

Apple (Malus domestica) trees often experience various abiotic and biotic stresses. However, due to the long juvenile period of apple and its high degree of genetic heterozygosity, only limited progress has been made in developing cold-hardy and disease-resistant cultivars through traditional approaches. Numerous studies reveal that biotechnology is a feasible approach to improve stress tolerance in woody perennial plants. HYPONASTIC LEAVES1 (HYL1), a double-stranded RNA-binding protein, is a key regulator involved in apple drought stress response. However, whether HYL1 participates in apple cold response and pathogen resistance remains unknown. In this study, we revealed that MdHYL1 plays a positive role in cold tolerance and pathogen resistance in apple. MdHYL1 acted upstream to positively regulate freezing tolerance and Alternaria alternata resistance by positively modulating transcripts of MdMYB88 and MdMYB124 in response to cold stress or A. alternata infection. In addition, MdHYL1 regulated the biogenesis of several miRNAs responsive to cold and A. alternata infection in apple. Furthermore, we identified Mdm-miRNA156 (Mdm-miR156) as a negative regulator of cold tolerance and Mdm-miRNA172 (Mdm-miR172) as a positive regulator of cold tolerance, and that Mdm-miRNA160 (Mdm-miR160) decreased plant resistance to infection by A. alternata. In summary, we highlight the molecular role of MdHYL1 regarding cold tolerance and A. alternata infection resistance, thereby providing candidate genes for breeding apple with freezing tolerance and A. alternata resistance using biotechnology.

The primary cell wall is a fundamental plant constituent that is flexible but sufficiently rigid to support the plant cell shape. Although many studies have demonstrated that reactive oxygen species (ROS) serve as important signaling messengers to modify the cell wall structure and affect cellular growth, the regulatory mechanism underlying the spatial-temporal regulation of ROS activity for cell wall maintenance remains largely unclear. Here, we demonstrate the role of the Arabidopsis (Arabidopsis thaliana) multicopper oxidase-like protein skewed 5 (SKU5) and its homolog SKU5-similar 1 (SKS1) in root cell wall formation through modulating ROS homeostasis. Loss of SKU5 and SKS1 function resulted in aberrant division planes, protruding cell walls, ectopic deposition of iron, and reduced nicotinamide adeninedinucleotide phosphate (NADPH) oxidase-dependent ROS overproduction in the root epidermis-cortex and cortex-endodermis junctions. A decrease in ROS level or inhibition of NADPH oxidase activity rescued the cell wall defects of sku5 sks1 double mutants. SKU5 and SKS1 proteins were activated by iron treatment, and iron over-accumulated in the walls between the root epidermis and cortex cell layers of sku5 sks1. The glycosylphosphatidylinositol-anchored motif was crucial for membrane association and functionality of SKU5 and SKS1. Overall, our results identified SKU5 and SKS1 as regulators of ROS at the cell surface for regulation of cell wall structure and root cell growth.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

Lignified stone cell content is a key factor used to evaluate fruit quality, influencing the economic value of pear (Pyrus pyrifolia) fruits. However, our understanding of the regulatory networks of stone cell formation is limited due to the complex secondary metabolic pathway. In this study, we used a combination of co-expression network analysis, gene expression profiles, and transcriptome analysis in different pear cultivars with varied stone cell content to identify a hub MYB gene, PbrMYB24. The relative expression of PbrMYB24 in fruit flesh was significantly correlated with the contents of stone cells, lignin, and cellulose. We then verified the function of PbrMYB24 in regulating lignin and cellulose formation via genetic transformation in homologous and heterologous systems. We constructed a high-efficiency verification system for lignin and cellulose biosynthesis genes in pear callus. PbrMYB24 transcriptionally activated multiple target genes involved in stone cell formation. On the one hand, PbrMYB24 activated the transcription of lignin and cellulose biosynthesis genes by binding to different cis-elements [AC-I (ACCTACC) element, AC-II (ACCAACC) element and MYB-binding sites (MBS)]. On the other hand, PbrMYB24 bound directly to the promoters of PbrMYB169 and NAC STONE CELL PROMOTING FACTOR (PbrNSC), activating the gene expression. Moreover, both PbrMYB169 and PbrNSC activated the promoter of PbrMYB24, enhancing gene expression. This study improves our understanding of lignin and cellulose synthesis regulation in pear fruits through identifying a regulator and establishing a regulatory network. This knowledge will be useful for reducing the stone cell content in pears via molecular breeding.

Drought and cold stresses seriously affect tree growth and fruit yield during apple (Malus domestica) production, with combined stress causing injury such as shoot shriveling. However, the molecular mechanism underlying crosstalk between responses to drought and cold stress remains to be clarified. In this study, we characterized the zinc finger transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10) through comparative analysis of shoot-shriveling tolerance between tolerant and sensitive apple rootstocks. MhZAT10 responded to both drought and cold stresses. Heterologous expression of MhZAT10 in the sensitive rootstock 'G935' from domesticated apple (Malus domestica) promoted shoot-shriveling tolerance, while silencing of MhZAT10 expression in the tolerant rootstock 'SH6' of Malus honanensis reduced stress tolerance. We determined that the apple transcription factor DEHYDRATION RESPONSE ELEMENT-BINDING PROTEIN 2A (DREB2A) is a direct regulator activating the expression of MhZAT10 in response to drought stress. Apple plants overexpressing both MhDREB2A and MhZAT10 genes exhibited enhanced tolerance to drought and cold stress, while plants overexpressing MhDREB2A but with silenced expression of MhZAT10 showed reduced tolerance, suggesting a critical role of MhDREB2A-MhZAT10 in the crosstalk between drought and cold stress responses. We further identified drought-tolerant MhWRKY31 and cold-tolerant MhMYB88 and MhMYB124 as downstream regulatory target genes of MhZAT10. Our findings reveal a MhDREB2A-MhZAT10 module involved in crosstalk between drought and cold stress responses, which may have applications in apple rootstock breeding programs aimed at developing shoot-shriveling tolerance.

Suboptimal nitrogen availability is a primary constraint to plant growth. We used OpenSimRoot, a functional-structural plant/soil model, to test the hypothesis that larger root cortical cell size (CCS), reduced cortical cell file number (CCFN), and their interactions with root cortical aerenchyma (RCA) and lateral root branching density (LRBD) are useful adaptations to suboptimal soil nitrogen availability in maize (Zea mays). Reduced CCFN increased shoot dry weight over 80%. Reduced respiration, reduced nitrogen content, and reduced root diameter accounted for 23%, 20%, and 33% of increased shoot biomass, respectively. Large CCS increased shoot biomass by 24% compared with small CCS. When simulated independently, reduced respiration and reduced nutrient content increased the shoot biomass by 14% and 3%, respectively. However, increased root diameter resulting from large CCS decreased shoot biomass by 4% due to an increase in root metabolic cost. Under moderate N stress, integrated phenotypes with reduced CCFN, large CCS, and high RCA improved shoot biomass in silt loam and loamy sand soils. In contrast, integrated phenotypes composed of reduced CCFN, large CCS, and reduced LRBD had the greatest growth in silt loam, while phenotypes with reduced CCFN, large CCS, and high LRBD were the best performers in loamy sands. Our results support the hypothesis that larger CCS, reduced CCFN, and their interactions with RCA and LRBD could increase nitrogen acquisition by reducing root respiration and root nutrient demand. Phene synergisms may exist between CCS, CCFN, and LRBD. CCS and CCFN merit consideration for breeding cereal crops with improved nitrogen acquisition, which is critical for global food security.

Acidity is a key determinant of fruit organoleptic quality. Here, a candidate gene for fruit acidity, designated MdMYB123, was identified from a comparative transcriptome study of two Ma1Ma1 apple (Malus domestica) varieties, "Qinguan (QG)" and "Honeycrisp (HC)" with different malic acid content. Sequence analysis identified an A→T SNP, which was located in the last exon, resulting in a truncating mutation, designated mdmyb123. This SNP was significantly associated with fruit malic acid content, accounting for 9.5% of the observed phenotypic variation in apple germplasm. Differential MdMYB123- and mdmyb123-mediated regulation of malic acid accumulation was observed in transgenic apple calli, fruits, and plantlets. Two genes, MdMa1 and MdMa11, were up- and down-regulated in transgenic apple plantlets overexpressing MdMYB123 and mdmyb123, respectively. MdMYB123 could directly bind to the promoter of MdMa1 and MdMa11, and induce their expression. In contrast, mdmyb123 could directly bind to the promoters of MdMa1 and MdMa11, but with no transcriptional activation of both genes. In addition, gene expression analysis in 20 different apple genotypes based on SNP locus from "QG" × "HC" hybrid population confirmed a correlation between A/T SNP with expression levels of MdMa1 and MdMa11. Our finding provides valuable functional validation of MdMYB123 and its role in the transcriptional regulation of both MdMa1 and MdMa11, and apple fruit malic acid accumulation.

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall properties might help improve photosynthesis and crop water-use efficiency. We tested this using two independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters cell wall hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, cell wall composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll cell wall thickness (-14%), and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic water-use efficiency (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in above-ground biomass (+12.5%) and canopy-level water-use efficiency (+8.8%; ratio of above-ground biomass to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in cell wall composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic water-use efficiency will need to enhance mesophyll conductance and photosynthesis whilst maintaining or decreasing stomatal conductance.

Fruit color is a very important external commodity factor for consumers. Compared to the most typical red octoploid strawberry (Fragaria × ananassa), the pink strawberry often sells for a more expensive price and has a higher economic benefit due to its outstanding color. However, few studies have examined the molecular basis of pink colored strawberry fruit. Through an EMS mutagenesis of woodland strawberry (Fragaria vesca), we identified a mutant with pink fruits and green petioles. BSA-Seq analysis and gene function verification confirmed that the responsible mutation resides in a gene encoding flavanone-3-hydroxylase (F3H) in the anthocyanin synthesis pathway. This nonsynonymous mutation results in an arginine to histidine change at position 130 of F3H. Molecular docking experiments showed that the arginine to histidine mutation results in a reduction of intermolecular force-hydrogen bonding between the F3H protein and its substrates. Enzymatic experiments showed a greatly reduced ability of the mutated F3H protein to catalyze the conversion of the substrates, and hence a blockage of the anthocyanin synthesis pathway. The discovery of a key residue in the F3H gene controlling anthocyanin synthesis provides a clear target of modification for the molecular breeding of strawberry varieties with pink-colored fruits, which may be of great commercial value.

Sweetness and appearance of fresh fruits are key palatable and preference attributes for consumers and are often controlled by multiple genes. However, fine-mapping the key loci or genes of interest by single genome-based genetic analysis is challenging. Herein, we present the chromosome-level genome assembly of one landrace melon accession (Cucumis melo ssp. agrestis) with wild morphologic features and thus construct a melon pan-genome atlas via integrating sequenced melon genome datasets. Our comparative genomic analysis reveals a total of 3.4 million genetic variations, of which the presence/absence variations (PAVs) are mainly involved in regulating the function of genes for sucrose metabolism during melon domestication and improvement. We further resolved several loci that are accountable for sucrose contents, flesh color, rind stripe and suture using a structural variation (SV)-based genome-wide association study (GWAS). Furthermore, via BSA-seq and map-based cloning, we uncovered that a single gene, (CmPIRL6), determines the edible or inedible characteristics of melon fruit exocarp. These findings provide important melon pan-genome information and provide a powerful toolkit for future pan-genome-informed cultivar breeding of melon.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

Norisoprenoids and flavonols are important secondary metabolites in grape berries (Vitis vinifera L.). The former is a class of ubiquitous flavor and fragrance compounds produced by the cleavage of carotenoids, and the latter, which is derived from the flavonoid metabolic pathway, has been proposed as a general quality marker for red grapes. However, the transcriptional regulatory mechanisms underlying norisoprenoid and flavonol production are still not fully understood. In this study, we characterized a transcription factor, VvWRKY70, as a repressor of both norisoprenoid and flavonol biosynthesis in grape berries, and its expression was down-regulated by light and high-temperature treatment. Overexpressing VvWRKY70 in grape calli reduced norisoprenoid and flavonol production, particularly under light exposure or at high temperature, by repressing the expression of several related genes in the isoprenoid and flavonoid metabolic pathways. VvWRKY70 downregulated β-CAROTENE HYDROXYLASE 2 (VvBCH2) and CHALCONE SYNTHASE 3 (VvCHS3) expression based on yeast one-hybrid analysis combined with electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR. We discuss the role of VvWRKY70 in the coordinated regulatory network of isoprenoid and flavonoid metabolism. These findings provide a theoretical basis to improve flavor, color and other comprehensive qualities of fruit crops and their processing products.

Ammonium (NH4+) is a key inorganic nitrogen source in cellular amino acid biosynthesis. The coupling of transcriptional and posttranslational regulation of AMMONIUM TRANSPORTER (AMT) ensures that NH4+ acquisition by plant roots is properly balanced, which allows for rapid adaptation to a variety of nitrogen conditions. Here, we report that phospholipase D (PLD)-derived phosphatidic acid (PA) interacts with AMT1; 1 to mediate NH4+ uptake in Arabidopsis (Arabidopsis thaliana). We examined pldα1 pldδ-knockout mutants and found that a reduced PA level increased seedling growth under nitrogen deficiency and inhibited root growth upon NH4+ stress, which was consistent with the enhanced accumulation of cellular NH4+. PA directly bound to AMT1; 1 and inhibited its transport activity. Mutation of AMT1; 1 R487 to Gly (R487G) resulted in abolition of PA suppression and, subsequently, enhancement of ammonium transport activity in vitro and in vivo. Observations of AMT1; 1-GFP showed suppressed endocytosis under PLD deficiency or by mutation of the PA-binding site in AMT1; 1. Endocytosis was rescued by PA in the pldα1 pldδ mutant but not in the mutant AMT1; 1R487G-GFP line. Together, these findings demonstrated PA-based shutoff control of plant NH4+ transport and point to a broader paradigm of lipid-transporter function.

Rice (Oryza sativa L.) is a cold-sensitive species that often faces cold stress, which adversely affects yield productivity and quality. However, the genetic basis for low-temperature adaptation in rice remains unclear. Here, we demonstrate that two functional polymorphisms in Oryza sativa SEC13 Homolog 1 (OsSEH1), encoding a WD40-repeat nucleoporin, between the two subspecies Oryza sativa japonica and Oryza sativa indica rice may have facilitated cold adaptation in japonica rice. We show that OsSEH1 of the japonica variety expressed in OsSEH1MSD plants (transgenic line overexpressing the OsSEH1 allele from Mangshuidao [MSD], cold-tolerant landrace) has a higher affinity for metallothionein 2b (OsMT2b) than that of OsSEH1 of indica. This high affinity of OsSEH1MSD for OsMT2b results in inhibition of OsMT2b degradation, with decreased accumulation of reactive oxygen species and increased cold tolerance. Transcriptome analysis indicates that OsSEH1 positively regulates the expression of the genes encoding dehydration-responsive element-binding transcription factors, i.e., OsDREB1 genes, and induces the expression of multiple cold-regulated genes to enhance cold tolerance. Our findings highlight a breeding resource for improving cold tolerance in rice.

Carotenoids are plastidial isoprenoids required for photosynthesis and phytohormone production in all plants. In tomato (Solanum lycopersicum), carotenoids also provide color to flowers and ripe fruit. Phytoene synthase (PSY) catalyzes the first and main flux-controlling step of the carotenoid pathway. Three genes encoding PSY isoforms are present in tomato, PSY1 to PSY3. Mutants have shown that PSY1 is the isoform providing carotenoids for fruit pigmentation, but it is dispensable in photosynthetic tissues. No mutants are available for PSY2 or PSY3, but their expression profiles suggest a main role for PSY2 in leaves and PSY3 in roots. To further investigate isoform specialization with genetic tools, we created gene-edited lines defective in PSY1 and PSY2 in the MicroTom background. The albino phenotype of lines lacking both PSY1 and PSY2 confirmed that PSY3 does not contribute to carotenoid biosynthesis in shoot tissues. Our work further showed that carotenoid production in tomato shoots relies on both PSY1 and PSY2 but with different contributions in different tissues. PSY2 is the main isoform for carotenoid biosynthesis in leaf chloroplasts, but PSY1 is also important in response to high light. PSY2 also contributes to carotenoid production in flower petals and, to a lesser extent, fruit chromoplasts. Most interestingly, our results demonstrate that fruit growth is controlled by abscisic acid (ABA) specifically produced in the pericarp from PSY1-derived carotenoid precursors, whereas PSY2 is the main isoform associated with ABA synthesis in seeds and salt-stressed roots.

Although brassinolide (BR) and jasmonic acid (JA) play essential roles in the regulation of cold stress responses, the molecular basis of their crosstalk remains elusive. Here, we show a key component of BR signaling in apple (Malus × domestica), BR INSENSITIVE1 (BRI1)-EMS-SUPPRESSOR1 (BES1)-INTERACTING MYC-LIKE PROTEIN1 (MdBIM1), increases cold tolerance by directly activating expression of C-REPEAT BINDING FACTOR1 (MdCBF1) and forming a complex with C-REPEAT BINDING FACTOR2 (MdCBF2) to enhance MdCBF2-activated transcription of cold-responsive genes. Two repressors of JA signaling, JAZMONATE ZIM-DOMAIN1 (MdJAZ1) and JAZMONATE ZIM-DOMAIN2 (MdJAZ2), interact with MdBIM1 to integrate BR and JA signaling under cold stress. MdJAZ1 and MdJAZ2 reduce MdBIM1-promoted cold stress tolerance by attenuating transcriptional activation of MdCBF1 expression by MdBIM1 and interfering with the formation of the MdBIM1-MdCBF2 complex. Furthermore, the E3 ubiquitin ligase ARABIDOPSIS TÓXICOS en LEVADURA73 (MdATL73) decreases MdBIM1-promoted cold tolerance by targeting MdBIM1 for ubiquitination and degradation. Our results not only reveal crosstalk between BR and JA signaling mediated by a JAZ-BIM1-CBF module, but also provide insights into the post-translational regulatory mechanism of BR signaling.

An apical hook is a special structure formed during skotomorphogenesis in dicotyledonous plant species. It is critical for protecting the shoot apical meristem from mechanical damage during seed germination and hypocotyl elongation in soil. Brassinosteroid (BR) and jasmonate (JA) phytohormones antagonistically regulate apical hook formation. However, the interrelationship between BRs and JAs in this process has not been well elucidated. Here, we reveal that JAs repress BRs to regulate apical hook development in Arabidopsis (Arabidopsis thaliana). Exogenous application of methyl jasmonate (MeJA) repressed the expression of the rate-limiting BR biosynthetic gene DWARF4 (DWF4) in a process relying on 3 key JA-dependent transcription factors, MYC2, MYC3, and MYC4. We demonstrated that MYC2 interacts with the critical BR-activated transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), disrupting the association of BZR1 with its partner transcription factors, such as those of the PHYTOCHROME INTERACTING FACTOR (PIF) family and downregulating the expression of their target genes, such as WAVY ROOT GROWTH 2 (WAG2), encoding a protein kinase essential for apical hook development. Our results indicate that JAs not only repress the expression of BR biosynthetic gene DWF4 but, more importantly, attenuate BR signaling by inhibiting the transcriptional activation of BZR1 by MYC2 during apical hook development.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines, but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28°C (instead of 22°C) did not appear to require PDX3 function, and we observed that the consequent drive towards C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.

Belonging to Rosaceae, red raspberry (Rubus idaeus) and wild strawberry (Fragaria vesca) are closely related species with distinct fruit types. While the numerous ovaries become the juicy drupelet fruits in raspberry, their strawberry counterparts become dry and tasteless achenes. In contrast, while the strawberry receptacle, the stem tip, enlarges to become a red fruit, the raspberry receptacle shrinks and dries. The distinct fruit-forming ability of homologous organs in these two species allows us to investigate fruit type determination. We assembled and annotated the genome of red raspberry (Rubus idaeus) and characterized its fruit development morphologically and physiologically. Subsequently, transcriptomes of dissected and staged raspberry fruit tissues were compared to those of strawberry from a prior study. Class B MADS box gene expression was negatively associated with fruit-forming ability, which suggested a conserved inhibitory role of class B heterodimers, PISTILLATA/TM6 or PISTILLATA/APETALA3, for fruit formation. Additionally, the inability of strawberry ovaries to develop into fruit flesh was associated with highly expressed lignification genes and extensive lignification of the ovary pericarp. Finally, co-expressed gene clusters preferentially expressed in the dry strawberry achenes were enriched in "cell wall biosynthesis" and "ABA signaling", while co-expressed clusters preferentially expressed in the fleshy raspberry drupelets were enriched in "protein translation". Our work provides extensive genomic resources as well as several potential mechanisms underlying fruit type specification. These findings provide the framework for understanding the evolution of different fruit types, a defining feature of angiosperms.

Composite generalist herbivores are comprised of host-adapted populations that retain the ability to shift hosts. The degree and overlap of mechanisms used by host-adapted generalist and specialist herbivores to overcome the same host plant defences are largely unknown. Tetranychidae mites are exceptionally suited to address the relationship between host-adaptation and specialization in herbivores as this group harbours closely related species with remarkably different host ranges - an extreme generalist the two spotted spider mite (Tetranychus urticae Koch, Tu) and the Solanaceous specialist Tetranychus evansi (Te). Here, we used tomato-adapted two spotted spider mite (Tu-A) and Te populations to compare mechanisms underlying their host adaptation and specialization. We show that both mites attenuate induced tomato defenses, including protease inhibitors (PIs) that target mite cathepsin L digestive proteases. While Te solely relies on transcriptional attenuation of PI induction, Tu and Tu-A have elevated constitutive activity of cathepsin L proteases, making them less susceptible to plant anti-digestive proteins. Tu-A and Te also rely on detoxification of tomato constitutive defenses. Te uses esterase and P450 activities, while Tu-A depends on the activity of all major detoxification enzymatic classes to disarm tomato defensive compounds to a lesser extent. Thus, even though both Tu-A and Te use similar mechanisms to counteract tomato defenses, Te can better cope with them. This finding is congruent with the ecological and evolutionary times required to establish mite adaptation and specialization states, respectively.

Photorespiration begins with the oxygenation reaction catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase and is the second highest metabolic flux in plants after photosynthesis. Although the core biochemical pathway of photorespiration has been well characterized, little is known about the underlying regulatory mechanisms. Some rate-limiting regulation of photorespiration has been suggested to occur at both the transcriptional and post-translational levels, but experimental evidence is scarce. Here, we found that Mitogen-Activated Protein Kinase 2 (MAPK2) interacts with photorespiratory glycolate oxidase and hydroxypyruvate reductase, and the activities of these photorespiratory enzymes were regulated via phosphorylation modifications in rice (Oryza sativa L.). Gas exchange measurements revealed that the photorespiration rate decreased in rice mapk2 mutants under normal growth conditions, without disturbing photosynthesis. Due to decreased photorespiration, the levels of some key photorespiratory metabolites, such as 2-phosphoglycolate, glycine, and glycerate, significantly decreased in mapk2 mutants, but those of photosynthetic metabolites were not altered. Transcriptome assays also revealed that the expression levels of some flux-controlling genes in photorespiration were significantly downregulated in mapk2 mutants. Our findings provide molecular evidence for the association between MAPK2 and photorespiration and suggest that MAPK2 regulates the key enzymes of photorespiration at both the transcriptional and post-translational phosphorylation levels in rice.

Phytophthora capsici deploys effector proteins to manipulate host immunity and facilitate its colonization. However, the underlying mechanisms remain largely unclear. In this study, we demonstrated that a Sne-like (Snel) RxLR effector gene PcSnel4 is highly expressed at the early stages of P. capsici infection in Nicotiana benthamiana. Knocking out both alleles of PcSnel4 attenuated the virulence of P. capsici, while expression of PcSnel4 promoted its colonization in N. benthamiana. PcSnel4B could suppress the hypersensitive reaction (HR) induced by Avr3a-R3a and RESISTANCE TO PSEUDOMONAS SYRINGAE 2 (AtRPS2), but it did not suppress cell death elicited by Phytophthora infestin 1 (INF1) and Crinkler 4 (CRN4). COP9 signalosome 5 (CSN5) in N. benthamiana was identified as a host target of PcSnel4. Silencing NbCSN5 compromised the cell death induced by (AtRPS2). PcSnel4B impaired the interaction and co-localization of Cullin1 (CUL1) and CSN5 in vivo. Expression of AtCUL1 promoted the degradation of AtRPS2 and disrupted HR, while AtCSN5a stabilized AtRPS2 and promoted HR, regardless of the expression of AtCUL1. PcSnel4 counteracted the effect of AtCSN5 and enhanced the degradation of AtRPS2, resulting in HR suppression. This study deciphered the underlying mechanism of PcSnel4-mediated suppression of HR induced by AtRPS2.

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery (Apium graveolens) and parsley (Petroselinum crispum) in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified UGT94AX1 as an Apium graveolens apiosyltransferase (AgApiT) responsible for catalyzing the last sugar modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. Identification of this plant apiosyltransferase gene will enhance our understanding of the physio-ecological functions of apiose and apiose-containing compounds.

Numerous endogenous and environmental signals regulate the intricate and highly orchestrated process of plant senescence. Ethylene (ET), which accumulates as senescence progresses, is a major promoter of leaf senescence.The master transcription activator ETHYLENE INSENSITIVE3 (EIN3) activates the expression of a wide range of downstream genes during leaf senescence. Here, we found that a unique EIN3-LIKE 1 (EIL1) gene, Cotton LINT YIELD INCREASING (GhLYI), encodes a truncated EIN3 protein in upland cotton (Gossypium hirsutum L.) that functions as an ET signal response factor and a positive regulator of senescence. Ectopic expression or overexpression of GhLYI accelerated leaf senescence in both Arabidopsis (Arabidopsis thaliana) and cotton. Cleavage under targets and tagmentation (CUT&Tag) analyses revealed SENESCENCE-ASSOCIATED GENE 20 (SAG20) was a target of GhLYI. Electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H), and dual-luciferase transient expression assay confirmed that GhLYI directly bound the promoter of SAG20 to activate its expression. Transcriptome analysis revealed that transcript levels of a series of senescence related genes, SAG12, NAC LIKE, ACTIVATED by APETALA 3/PISTILLATA (NAP/ANAC029), and WRKY53, are substantially induced in GhLYI overexpression plants compared with wild-type plants. VIGS(Virus-induced gene silencing) preliminarily confirmed that knockdown of GhSAG20 delayed leaf senescence. Collectively, our findings provide a regulatory module involving GhLYI-GhSAG20 in controlling senescence in cotton.

The molecular innovation underpinning efficient carbon and energy metabolism during evolution of land plants remains largely unknown. Invertase-mediated sucrose cleavage into hexoses is central to fuel growth. Why some cytoplasmic invertases (CINs) function in the cytosol, whereas others operate in chloroplasts and mitochondria, is puzzling. We attempted to shed light on this question from an evolutionary perspective. Our analyses indicated that plant CINs originated from an putatively orthologous ancestral gene in cyanobacteria and formed the plastidic CIN (α1 clade) through endo-symbiotic gene transfer, while its duplication in algae with a loss of its signal peptide produced the β clade CINs in the cytosol. The mitochondrial CINs (α2) derived from duplication of the plastidic CINs and co-evolved with vascular plants. Importantly, the copy number of mitochondrial and plastidic CINs increased upon the emergence of seed plants, corresponding with the rise of respiratory, photosynthetic and growth rates. The cytosolic CIN (β subfamily) kept expanding from algae to gymnosperm, indicating its role in supporting the increase in carbon use efficiency during evolution. Affinity purification mass spectrometry identified a cohort of proteins interacting with α1 and 2 CINs, which points to their roles in plastid and mitochondrial glycolysis, oxidative stress tolerance and the maintenance of subcellular sugar homeostasis. Collectively, the findings indicate evolutionary roles of α1 and α2 CINs in chloroplasts and mitochondria for achieving high photosynthetic and respiratory rates, respectively, which, together with the expanding of cytosolic CINs, likely underpin the colonization of land plants through fueling rapid growth and biomass production.

In the Rhizobium-Legume symbiosis, the nodulation outer protein P (NopP) effector is one of the key regulators for rhizobial infection and nodule organogenesis. However, the molecular mechanism through which host legume plants sense NopP remains largely unknown. Here, we constructed an nopP deletion mutant of Mesorhizobium huakuii and found that nopP negatively regulates nodulation on Chinese milk vetch (Astragalus sinicus). Screening for NopP interacting proteins in host plants using the yeast 2-hybrid system identified NopP interacting protein 43 (AsNIP43), which encodes a G-type receptor-like kinase (LecRLK). The B-lectin domain at the N terminus of AsNIP43 was essential in mediating its interaction with NopP, which was confirmed in vitro and in vivo. Subcellular localization, co-localization, and gene expression analyses showed that AsNIP43 and NopP function tightly associated with earlier infection events. RNA interference (RNAi) knockdown of AsNIP43 expression by hairy root transformation led to decreased nodule formation. AsNIP43 plays a positive role in symbiosis, which was further verified in the model legume Medicago truncatula. Transcriptome analysis indicated that MtRLK (a homolog of AsNIP43 in M. truncatula) may function to affect defense gene expression and thus to regulate early nodulation. Taken together, we show that LecRLK AsNIP43 is a legume host target that interacts with rhizobia effector NopP is essential for rhizobial infection and nodulation.

β-1,3-glucanase functions in plant physiological and developmental processes. However, how β-1,3-glucanase participates in cell wall development remains largely unknown. Here, we answered this question by examining the role of GhGLU18, a β-1,3-glucanase, in cotton (Gossypium hirsutum) fibers, in which the content of β-1,3-glucan changes dynamically from 10% of the cell wall mass at the onset of secondary wall deposition to < 1% at maturation. GhGLU18 was specifically expressed in cotton fiber with higher expression in late fiber elongation and secondary cell wall (SCW) synthesis stages. GhGLU18 largely localized to the cell wall and was able to hydrolyze β-1, 3-glucan in vitro. Overexpression of GhGLU18 promoted polysaccharide accumulation, cell wall reconstruction and cellulose synthesis, which led to increased fiber length and strength with thicker cell walls and shorter pitch of the fiber helix. However, GhGLU18-suppressed cotton resulted in opposite phenotypes. Additionally, GhGLU18 was directly activated by GhFSN1 (fiber secondary cell wall-related NAC1), a NAC transcription factor reported previously as the master regulator in SCW formation during fiber development. Our results demonstrate that cell wall-localized GhGLU18 promotes fiber elongation and SCW thickening by degrading callose and enhancing polysaccharide metabolism and cell wall synthesis.

The water caltrop (Trapa natans) develops unique woody fruits with unusually large seeds among aquatic plants. During fruit development, the inner fruit wall (endocarp) sclerifies and forms a protective layer for the seed. Endocarp sclerification also occurs in many land plants with large seeds; however, in T. natans the processes of fruit formation, endocarp hardening, and seed storage take place entirely underwater. To identify potential chemical and structural adaptations for the aquatic environment, we investigated the cell wall composition in the endocarp at a young developmental stage, as well as at fruit maturity. Our work shows that hydrolyzable tannins - specifically gallotannins -flood the endocarp tissue during secondary wall formation and are integrated into cell walls along with lignin during maturation. Within the secondary walls of mature tissue, we identified unusually strong spectroscopic features of ester linkages, suggesting that the gallotannins and their derivatives are cross-linked to other wall components via ester bonds, leading to unique cell wall properties. The synthesis of large amounts of water-soluble, defensive aromatic metabolites during secondary wall formation might be a fast way to defend seeds within the insufficiently lignified endocarp of T. natans.

Wurfbainia longiligularis and Wurfbainia villosa are both rich in volatile terpenoids and are two primary plant sources of Fructus Amomi used for curing gastrointestinal diseases. Metabolomic profiling has demonstrated that bornyl diphosphate (BPP)-related terpenoids are more abundant in the W. villosa seeds and have a wider tissue distribution in W. longiligularis. To explore the genetic mechanisms underlying the volatile terpenoids divergence, a high-quality chromosome-level genome of W. longiligularis (2.29 Gb, contig N50 of 80.39 Mb) was assembled. Functional characterization of 17 terpene synthases (WlTPSs) revealed that WlBPPS, along with WlTPS 24/26/28 with bornyl diphosphate synthase (BPPS) activity, contribute to the wider tissue distribution of BPP-related terpenoids in W. longiligularis compared to W. villosa. Furthermore, transgenic Nicotiana tabacum showed that the GCN4-motif element positively regulates seed expression of WvBPPS and thus promotes the enrichment of BPP-related terpenoids in W. villosa seeds. Systematic identification and analysis of candidate TPS in 29 monocot plants from 16 families indicated that substantial expansion of TPS-a and TPS-b subfamily genes in Zingiberaceae may have driven increased diversity and production of volatile terpenoids. Evolutionary analysis and functional identification of BPPS genes showed that BPP-related terpenoids may be distributed only in the Zingiberaceae of monocot plants. This research provides valuable genomic resources for breeding and improving Fructus Amomi with medicinal and edible value and sheds light on the evolution of terpenoid biosynthesis in Zingiberaceae.

Legume roots can be symbiotically colonized by arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria. In Lotus japonicus, the latter occurs intracellularly by the cognate rhizobial partner Mesorhizobium loti or intercellularly with the Agrobacterium pusense strain IRBG74. Although these symbiotic programs show distinctive cellular and transcriptome signatures, some molecular components are shared. In this study, we demonstrate that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 1 (DAHPS1), the first enzyme in the biosynthetic pathway of aromatic amino acids (AAAs), plays a critical role in root hair development and for AM and rhizobial symbioses in Lotus. Two homozygous DAHPS1 mutants (dahps1-1 and dahps1-2) showed drastic alterations in root hair morphology, associated with alterations in cell wall dynamics and a progressive disruption of the actin cytoskeleton. The altered root hair structure was prevented by pharmacological and genetic complementation. dahps1-1 and dahps1-2 showed significant reductions in rhizobial infection (intracellular and intercellular) and nodule organogenesis and a delay in AM colonization. RNAseq analysis of dahps1-2 roots suggested that these phenotypes are associated with downregulation of several cell wall-related genes, and with an attenuated signaling response. Interestingly, the dahps1 mutants showed no detectable pleiotropic effects, suggesting a more selective recruitment of this gene in certain biological processes. This work provides robust evidence linking AAA metabolism to root hair development and successful symbiotic associations.

The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3-ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localised SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by two further observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and secondly, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.

Allene oxide cyclase (AOC) is a key enzyme in the biosynthesis of jasmonic acid (JA), which is involved in plant growth and development as well as adaptation to environmental stresses. We identified the cold- and pathogen-responsive AOC2 gene from Medicago sativa subsp. falcata (MfAOC2) and its homolog MtAOC2 from Medicago truncatula. Heterologous expression of MfAOC2 in M. truncatula enhanced cold tolerance and resistance to the fungal pathogen Rhizoctonia solani, with greater accumulation of JA and higher transcript levels of JA downstream genes than in wild-type plants. By contrast, mutation of MtAOC2 reduced cold tolerance and pathogen resistance, with less accumulation of JA and lower transcript levels of JA downstream genes in the aoc2 mutant than in wild-type plants. The aoc2 phenotype and low levels of cold-responsive C-repeat-binding factor (CBF) transcripts could be rescued by expressing MfAOC2 in aoc2 plants or exogenous application of methyl jasmonate. Compared with wild-type plants, higher levels of CBF transcripts were observed in lines expressing MfAOC2 but lower levels of CBF transcripts were observed in the aoc2 mutant under cold conditions; superoxide dismutase, catalase, and ascorbate-peroxidase activities as well as proline concentrations were higher in MfAOC2-expressing lines but lower in the aoc2 mutant. These results suggest that expression of MfAOC2 or MtAOC2 promotes biosynthesis of JA, which positively regulates expression of CBF genes and antioxidant defense under cold conditions and expression of JA downstream genes after pathogen infection, leading to greater cold tolerance and pathogen resistance.