Plants display a tremendous diversity of developmental and physiological features, resulting from gains and losses of functional innovations across the plant phylogeny. Among those, the most impactful have been undoubtedly the ones that allowed plant terrestrializations, the transitions from an aquatic to a terrestrial environment. Although the embryophyte terrestrialization has been particularly scrutinized, others occurred across the plant phylogeny with the involvement of mutualistic symbioses as a common theme. Here, we review the current pieces of evidence supporting that the repeated colonization of land by plants has been facilitated by interactions with mutualistic symbionts. In that context, we detail two of these mutualistic symbioses: the arbuscular mycorrhizal symbiosis in embryophytes and the lichen symbiosis in chlorophyte algae. We suggest that associations with bacteria should be revisited in that context, and we propose that overlooked symbioses might have facilitated the emergence of other land plant clades.

To survive in the nutrient-poor habitats, carnivorous plants capture small organisms comprising complex substances not suitable for immediate reuse. The traps of carnivorous plants, which are analogous to the digestive systems of animals, are equipped with mechanisms for the breakdown and absorption of nutrients. Such capabilities have been acquired convergently over the past tens of millions of years in multiple angiosperm lineages by modifying plant-specific organs including leaves. The epidermis of carnivorous trap leaves bears groups of specialized cells called glands, which acquire substances from their prey via digestion and absorption. The digestive glands of carnivorous plants secrete mucilage, pitcher fluids, acids, and proteins, including digestive enzymes. The same (or morphologically distinct) glands then absorb the released compounds via various membrane transport proteins or endocytosis. Thus, these glands function in a manner similar to animal cells that are physiologically important in the digestive system, such as the parietal cells of the stomach and intestinal epithelial cells. Yet, carnivorous plants are equipped with strategies that deal with or incorporate plant-specific features, such as cell walls, epidermal cuticles, and phytohormones. In this review, we provide a systematic perspective on the digestive and absorptive capacity of convergently evolved carnivorous plants, with an emphasis on the forms and functions of glands.

Different patterns of lignified cell walls are associated with diverse functions in a variety of plant tissues. These functions rely on the stiffness and hydrophobicity that lignin polymers impart to the cell wall. The precise pattern of subcellular lignin deposition is critical for the structure-function relationship in each lignified cell type. Here, we describe the role of xylem vessels as water pipes, Casparian strips as apoplastic barriers, and the role of asymmetrically lignified endocarp b cells in exploding seed pods. We highlight similarities and differences in the genetic mechanisms underpinning local lignin deposition in these diverse cell types. By bringing together examples from different developmental contexts and different plant species, we propose that comparative approaches can benefit our understanding of lignin patterning mechanisms.

Land plant life cycles are separated into distinct haploid gametophyte and diploid sporophyte stages. Indeterminate apical growth evolved independently in bryophyte (moss, liverwort, and hornwort) and fern gametophytes, and tracheophyte (vascular plant) sporophytes. The extent to which apical growth in tracheophytes co-opted conserved gametophytic gene networks, or exploited ancestral sporophytic networks, is a long-standing question in plant evolution. The recent phylogenetic confirmation of bryophytes and tracheophytes as sister groups has led to a reassessment of the nature of the ancestral land plant. Here, we review developmental genetic studies of apical regulators and speculate on their likely evolutionary history.

In winter-annual plants, exposure to cold temperatures induces cold tolerance and accelerates flowering in the following spring. However, little is known about plant adaptations to dehydration stress after winter. Here, we found that dehydration tolerance is reduced in winter-annual Arabidopsis (Arabidopsis thaliana) after vernalization. Winter-annual Arabidopsis plants with functional FRIGIDA (FRI) exhibited high dehydration tolerance, with small stomatal apertures and hypersensitivity to exogenous abscisic acid. Dehydration tolerance and FLOWERING LOCUS C (FLC) transcript levels gradually decreased with prolonged cold exposure in FRI plants. FLC directly bound to the promoter of OPEN STOMATA1 (OST1) and activated OST1 expression. Loss of FLC function resulted in decreased dehydration tolerance and reduced OST1 transcript levels. FLC and OST1 act in the same dehydration stress pathway, with OST1 acting downstream of FLC. Our study provides insights into the mechanisms by which FRI modulates dehydration tolerance through the FLC-OST1 module. Our results suggest that winter-annual Arabidopsis integrates dehydration tolerance and flowering time to adapt to environmental changes from winter to spring.

The rate of algal and cyanobacterial respiration in the light is an important ecophysiological term that remains to be completely characterized and quantified. To address this issue, we exploited process-specific decarboxylation rates from flux balance analysis and isotopically nonstationary metabolic flux analysis. Our study, based on published data, suggested that decarboxylation is about 22% of net CO2 assimilation when the tricarboxylic acid cycle is completely open (characterized by the commitment of alpha ketoglutarate to amino acid synthesis and very low rates of succinate formation). This estimate was supported by calculating the decarboxylation rates required to synthesize the major components of biomass (proteins, lipids, and carbohydrates) at their typical abundance. Of the 22 CO2 molecules produced by decarboxylation (normalized to net assimilation = 100), approximately 13 were from pyruvate and 3 were from isocitrate. The remaining six units of decarboxylation were in the amino acid synthesis pathways outside the tricarboxylic acid cycle. A small additional flux came from photorespiration, decarboxylations of six phosphogluconate in the oxidative pentose phosphate pathway, and decarboxylations in the syntheses of lower-abundance compounds, including pigments and ribonucleic acids. This general approach accounted for the high decarboxylation rates in algae and cyanobacteria compared to terrestrial plants. It prompts a simple speculation for the origin of the Kok effect and helps constrain the photoautotrophic respiration rate, in the light, in the euphotic zone of the ocean and lakes.

Leaf angle is an important agronomic trait determining maize (Zea mays) planting density and light penetration into the canopy and contributes to the yield gain in modern maize hybrids. However, little is known about the molecular mechanisms underlying leaf angle beyond the ZmLG1 (liguleless1) and ZmLG2 (Liguleless2) genes. In this study, we found that the transcription factor (TF) ZmBEH1 (BZR1/BES1 homolog gene 1) is targeted by ZmLG2 and regulates leaf angle formation by influencing sclerenchyma cell layers on the adaxial side. ZmBEH1 interacted with the TF ZmBZR1 (Brassinazole Resistant 1), whose gene expression was also directly activated by ZmLG2. Both ZmBEH1 and ZmBZR1 are bound to the promoter of ZmSCL28 (SCARECROW-LIKE 28), a third TF that influences leaf angle. Our study demonstrates regulatory modules controlling leaf angle and provides gene editing targets for creating optimal maize architecture suitable for dense planting.

The evolution of transporting tissues was an important innovation in terrestrial plants that allowed them to adapt to almost all nonaquatic environments. These tissues consist of water-conducting cells and food-conducting cells and bridge plant-soil and plant-air interfaces over long distances. The largest group of land plants, representing about 95% of all known plant species, is associated with morphologically complex transporting tissue in plants with a range of additional traits. Therefore, this entire clade was named tracheophytes, or vascular plants. However, some nonvascular plants possess conductive tissues that closely resemble vascular tissue in their organization, structure, and function. Recent molecular studies also point to a highly conserved toolbox of molecular regulators for transporting tissues. Here, we reflect on the distinguishing features of conductive and vascular tissues and their evolutionary history. Rather than sudden emergence of complex, vascular tissues, plant transporting tissues likely evolved gradually, building on pre-existing developmental mechanisms and genetic components. Improved knowledge of the intimate structure and developmental regulation of transporting tissues across the entire taxonomic breadth of extant plant lineages, combined with more comprehensive documentation of the fossil record of transporting tissues, is required for a full understanding of the evolutionary trajectory of transporting tissues.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalize the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here, we identify the obv gene and the causative mutation, a nonsynonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a polymorphism in the natural range of wild tomatoes but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other pleiotropic effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density, and fruit shape, are controlled by OBV via changes in auxin signaling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR 4 (ARF4) also results in defective BSE development, revealing an additional component of a genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection.

Crassulacean acid metabolism (CAM) is a mode of photosynthesis that evolved in response to decreasing CO2 levels in the atmosphere some 20 million years ago. An elevated ratio of O2 relative to CO2 caused many plants to face increasing stress from photorespiration, a process exacerbated for plants living under high temperatures or in water-limited environments. Today, our climate is again rapidly changing and plants' ability to cope with and adapt to these novel environments is critical for their success. This review focuses on CAM plant responses to abiotic stressors likely to dominate in our changing climate: increasing CO2 levels, increasing temperatures, and greater variability in drought. Empirical studies that have assessed CAM responses are reviewed, though notably these are concentrated in relatively few CAM lineages. Other aspects of CAM biology, including the effects of abiotic stress on the light reactions and the role of leaf succulence, are also considered in the context of climate change. Finally, more recent studies using genomic techniques are discussed to link physiological changes in CAM plants with the underlying molecular mechanism. Together, the body of work reviewed suggests that CAM plants will continue to thrive in certain environments under elevated CO2. However, how CO2 interacts with other environmental factors, how those interactions affect CAM plants, and whether all CAM plants will be equally affected remain outstanding questions regarding the evolution of CAM on a changing planet.

Seeds are highly resilient to the external environment, which allows plants to persist in unpredictable and unfavorable conditions. Some plant species have adopted a bet-hedging strategy to germinate a variable fraction of seeds in any given condition, and this could be explained by population-based threshold models. Here, in the model plant Arabidopsis (Arabidopsis thaliana), we induced secondary dormancy (SD) to address the transcriptional heterogeneity among seeds that leads to binary germination/nongermination outcomes. We developed a single-seed RNA-seq strategy that allowed us to observe a reduction in seed transcriptional heterogeneity as seeds enter stress conditions, followed by an increase during recovery. We identified groups of genes whose expression showed a specific pattern through a time course and used these groups to position the individual seeds along the transcriptional gradient of germination competence. In agreement, transcriptomes of dormancy-deficient seeds (mutant of DELAY OF GERMINATION 1) showed a shift toward higher values of the germination competence index. Interestingly, a significant fraction of genes with variable expression encoded translation-related factors. In summary, interrogating hundreds of single-seed transcriptomes during SD-inducing treatment revealed variability among the transcriptomes that could result from the distribution of population-based sensitivity thresholds. Our results also showed that single-seed RNA-seq is the method of choice for analyzing seed bet-hedging-related phenomena.

Biogenesis of ribonucleoproteins occurs in dynamic subnuclear compartments called Cajal bodies (CBs). COILIN is a critical scaffolding component essential for CB formation, composition, and activity. We recently showed that Arabidopsis (Arabidopsis thaliana) AtCOILIN is phosphorylated in response to bacterial elicitor treatment. Here, we further investigated the role of AtCOILIN in plant innate immunity. Atcoilin mutants are compromised in defense responses to bacterial pathogens. Besides confirming a role of AtCOILIN in alternative splicing (AS), Atcoilin showed differential expression of genes that are distinct from those of AS, including factors involved in RNA biogenesis, metabolism, plant immunity, and phytohormones. Atcoilin mutant plants have reduced levels of defense phytohormones. As expected, the mutant plants were more sensitive to the necrotrophic fungal pathogen Botrytis cinerea. Our findings reveal an important role for AtCOILIN in innate plant immunity.

The EPIDERMAL PATTERNING FACTOR (EPF) and EPF-LIKE (EPFL) family of small secreted peptides act to regulate many aspects of plant growth and development; however, their functions are not widely characterized in rice (Oryza sativa). Here, we used clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology to individually knockout each of 11 EPF/EPFL genes in the rice cultivar Kasalath. Loss of function of most OsEPF/EPFL genes generated no obvious phenotype alteration, while disruption of OsEPFL2 in Kasalath caused a short or no awn phenotype and reduced grain size. OsEPFL2 is strongly expressed in the young panicle, consistent with a role in regulating awn and grain development. Haplotype analysis indicated that OsEPFL2 can be classified into six major haplotypes. Nucleotide diversity and genetic differentiation analyses suggested that OsEPFL2 was positively selected during the domestication of rice. Our work to systematically investigate the function of EPF/EPFL peptides demonstrates that different members of the same gene family have been independently selected for their ability to regulate a similar biological function and provides perspective on rice domestication.

Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesized around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, we employed mass spectrometry and enzymology in a two-fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P. patens was cultured with the antibiotics fosfomycin, D-cycloserine, and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the trichloroacetic acid-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) through the uridine diphosphate N-acetylmuramic acid (UDP-MurNAc)-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram-negative bacteria, including cyanobacteria, incorporate meso-diaminopimelic acid (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram-positive bacteria. To establish the specificity of D,L-DAP incorporation into the P. patens precursors, we analyzed the recombinant protein UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (MurE) from both P. patens and the cyanobacterium Anabaena sp. (Nostoc sp. strain PCC 7120). Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram-negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.

C4 photosynthesis optimizes plant carbon and water relations, allowing high photosynthetic rates with low stomatal conductance. Stomata have long been considered a part of the C4 syndrome. However, it remains unclear how stomatal traits evolved along the path from C3 to C4. Here, we examined stomata in the Flaveria genus, a model used for C4 evolutionary study. Comparative, transgenic, and semi-in vitro experiments were performed to study the molecular basis that underlies the changes of stomatal traits in C4 evolution. The evolution from C3 to C4 species is accompanied by a gradual rather than an abrupt change in stomatal traits. The initial change appears near the Type I intermediate stage. Co-evolution of the photosynthetic pathway and stomatal traits is supported. On the road to C4, stomata tend to be fewer in number but larger in size and stomatal density dominates changes in anatomical maximum stomatal conductance (gsmax). Reduction of FSTOMAGEN expression underlies decreased gsmax in Flaveria and likely occurs in other C4 lineages. Decreased gsmax contributes to the increase in intrinsic water-use efficiency in C4 evolution. This work highlights the stomatal traits in the current C4 evolutionary model. Our study provides insights into the pattern, mechanism, and role of stomatal evolution along the road toward C4.

Plant mitochondrial fatty acid synthesis (mtFAS) appears to be important in photorespiration based on the reverse genetics research from Arabidopsis (Arabidopsis thaliana) in recent years, but its roles in plant development have not been completely explored. Here, we identified a tomato (Solanum lycopersicum) mutant, fern-like, which displays pleiotropic phenotypes including dwarfism, yellowing, curly leaves, and increased axillary buds. Positional cloning and genetic and heterozygous complementation tests revealed that the underlying gene FERN encodes a 3-hydroxyl-ACP dehydratase enzyme involved in mtFAS. FERN was causally involved in tomato morphogenesis by affecting photorespiration, energy supply, and the homeostasis of reactive oxygen species. Based on lipidome data, FERN and the mtFAS pathway may modulate tomato development by influencing mitochondrial membrane lipid composition and other lipid metabolic pathways. These findings provide important insights into the roles and importance of mtFAS in tomato development.

The ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) plays a central role in light-induced anthocyanin biosynthesis. However, the upstream regulatory factors of COP1 remain poorly understood, particularly in horticultural plants. Here, we identified an MdCOP1-interacting protein, BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC2 (MdBT2), in apple (Malus domestica). MdBT2 is a BTB protein that directly interacts with and stabilizes MdCOP1 by inhibiting self-ubiquitination. Fluorescence observation and cell fractionation assays showed that MdBT2 increased the abundance of MdCOP1 in the nucleus. Moreover, a series of phenotypic analyses indicated that MdBT2 promoted MdCOP1-mediated ubiquitination and degradation of the MdMYB1 transcription factor, inhibiting the expression of anthocyanin biosynthesis genes and anthocyanin accumulation. Overall, our findings reveal a molecular mechanism by which MdBT2 positively regulates MdCOP1, providing insight into MdCOP1-mediated anthocyanin biosynthesis.

Cysteine-rich receptor-like kinases (CRKs) play critical roles in responses to biotic and abiotic stresses. However, the molecular mechanisms of CRKs in plant defense responses remain unknown. Here, we demonstrated that two CRKs, CRK5 and CRK22, are involved in regulating defense responses to Verticillium dahliae toxins (Vd-toxins) in Arabidopsis (Arabidopsis thaliana). Biochemical and genetic analyses showed that CRK5 and CRK22 may act upstream of MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 to regulate the salicylic acid (SA)-signaling pathway in response to Vd-toxins. In addition, MPK3 and MPK6 interact with the transcription factor WRKY70 to modulate defense responses to Vd-toxins. WRKY70 directly binds the promoter domains of the SA-signaling-related transcription factor genes TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA2) and TGA6 to regulate their expression in response to Vd-toxins. Thus, our study reveals a mechanism by which CRK5 and CRK22 regulate SA signaling through the MPK3/6-WRKY70-TGA2/6 pathway in response to Vd-toxins.

Adapting to unfavorable environments is a necessary step in plant terrestrialization and radiation. The dehydration-responsive element-binding (DREB) protein subfamily plays a pivotal role in plant abiotic stress regulation. However, relationships between the origin and expansion of the DREB subfamily and adaptive evolution of land plants are still being elucidated. Here, we constructed the evolutionary history of the DREB subfamily by compiling APETALA2/ethylene-responsive element-binding protein superfamily genes from 169 representative species of green plants. Through extensive phylogenetic analyses and comparative genomic analysis, our results revealed that the DREB subfamily diverged from the ethylene-responsive factor (ERF) subfamily in the common ancestor of Zygnemophyceae and Embryophyta during the colonization of land by plants, followed by expansions to form three different ancient archetypal genes in Zygnemophyceae species, designated as groups archetype-I, archetype-II/III, and archetype-IV. Four large-scale expansions paralleling the evolution of land plants led to the nine-subgroup divergence of group archetype-II/III in angiosperms, and five whole-genome duplications during Brassicaceae and Poaceae radiation shaped the diversity of subgroup IIb-1. We identified a Poaceae-specific gene in subgroup IIb-1, ERF014, remaining in a Poaceae-specific microsynteny block and co-evolving with a small heat shock protein cluster. Expression analyses demonstrated that heat acclimation may have driven the neofunctionalization of ERF014s in Pooideae by engaging in the conserved heat-responsive module in Poaceae. This study provides insights into lineage-specific expansion and neofunctionalization in the DREB subfamily, together with evolutionary information valuable for future functional studies of plant stress biology.

Histone deacetylases (HDAs) play an important role in transcriptional regulation of multiple biological processes. In this study, we investigated the function of HDA15 in abscisic acid (ABA) responses. We used immunopurification coupled with mass spectrometry-based proteomics to identify proteins interacting with HDA15 in Arabidopsis (Arabidopsis thaliana). HDA15 interacted with the core subunits of the MOS4-associated complex (MAC), MAC3A and MAC3B, with interaction between HDA15 and MAC3B enhanced by ABA. hda15 and mac3a/mac3b mutants were ABA-insensitive during seed germination and hyposensitive to salinity. RNA sequencing analysis demonstrated that HDA15 and MAC3A/MAC3B co-regulate ABA-responsive intron retention (IR). Furthermore, HDA15 reduced the histone acetylation level of genomic regions near ABA-responsive IR sites and the association of MAC3B with ABA-responsive pre-mRNA was dependent on HDA15. Our results indicate that HDA15 is involved in ABA responses by interacting with MAC3A/MAC3B to mediate splicing of introns.

During the maturation phase of flower development, the onset of anthesis visibly marks the transition from buds to open flowers, during which petals stretch out, nectar secretion commences, and pollination occurs. Analysis of the metabolic changes occurring during this developmental transition has primarily focused on specific classes of metabolites, such as pigments and scent emission, and far less on the whole network of primary and secondary metabolites. To investigate the metabolic changes occurring at anthesis, we performed multi-platform metabolomics alongside RNA sequencing in individual florets harvested from the main inflorescence of Arabidopsis (Arabidopsis thaliana) ecotype Col-0. To trace metabolic fluxes at the level of the whole inflorescence and individual florets, we further integrated these studies with radiolabeled experiments. These extensive analyses revealed high-energy-level metabolism and transport of carbohydrates and amino acids, supporting intense metabolic rearrangements occurring at the time of this floral transition. These comprehensive data are discussed in the context of our current understanding of the metabolic shifts underlying flower opening. We envision that this analysis will facilitate the introgression of floral metabolic traits promoting pollination in crop species for which a comprehensive knowledge of flower metabolism is still limited.

In ripening grape (Vitis sp.) berries, the combination of rapid sugar import, apoplastic phloem unloading, and water discharge via the xylem creates a potential risk for apoplastic sugar to be lost from the berries. We investigated the likelihood of such sugar loss and a possible sugar retrieval mechanism in the pedicels of different Vitis genotypes. Infusion of D-glucose-1-13C or L-glucose-1-13C to the stylar end of attached berries demonstrated that both sugars can be leached from the berries, but only the nontransport sugar L-glucose moved beyond the pedicels. No 13C enrichment was found in peduncles and leaves. Genes encoding 10 sugar transporters were expressed in the pedicels throughout grape ripening. Using an immunofluorescence technique, we localized the sucrose transporter SUC27 to pedicel xylem parenchyma cells. These results indicate that pedicels possess the molecular machinery for sugar retrieval from the apoplast. Plasmodesmata were observed between vascular parenchyma cells in pedicels, and movement of the symplastically mobile dye carboxyfluorescein demonstrated that the symplastic connection is physiologically functional. Taken together, the chemical, molecular, and anatomical evidence gathered here supports the idea that some apoplastic sugar can be leached from grape berries and is effectively retrieved in a two-step process in the pedicels. First, sugar transporters may actively retrieve leached sugar from the xylem. Second, retrieved sugar may move symplastically to the pedicel parenchyma for local use or storage, or to the phloem for recycling back to the berry.

Photoperiod-sensitive plants such as soybean (Glycine max) often face threats from herbivorous insects throughout their whole growth period and especially during flowering; however, little is known about the relationship between plant flowering and insect resistance. Here, we used gene editing, multiple omics, genetic diversity and evolutionary analyses to confirm that the calcium-dependent protein kinase GmCDPK38 plays a dual role in coordinating flowering time regulation and insect resistance of soybean. Haplotype 2 (Hap2)-containing soybeans flowered later and were more resistant to the common cutworm (Spodoptera litura Fabricius) than those of Hap3. gmcdpk38 mutants with Hap3 knocked out exhibited similar flowering and resistance phenotypes as Hap2. Knocking out GmCDPK38 altered numerous flowering- and resistance-related phosphorylated proteins, genes, and metabolites. For example, the S-adenosylmethionine synthase GmSAMS1 was post-translationally upregulated in the gmcdpk38 mutants. GmCDPK38 has abundant genetic diversity in wild soybeans and was likely selected during soybean domestication. We found that Hap2 was mostly distributed at low latitudes and had a higher frequency in cultivars than in wild soybeans, while Hap3 was widely selected at high latitudes. Overall, our results elucidated that the two distinct traits (flowering time and insect resistance) are mediated by GmCDPK38.

Plant lateral roots (LRs) play vital roles in anchorage and uptake of water and nutrients. Here, we reveal that degradation of lariat intronic RNAs (lariRNAs) modulated by SICKLE (SIC) is required for LR development in Arabidopsis (Arabidopsis thaliana). Loss of SIC results in hyper-accumulation of lariRNAs and restricts the outgrowth of LR primordia, thereby reducing the number of emerged LRs. Decreasing accumulation of lariRNAs by over-expressing RNA debranching enzyme 1 (DBR1), a rate-limiting enzyme of lariRNA decay, restored LR defects in SIC-deficient plants. Mechanistically, SIC interacts with DBR1 and facilitates its nuclear accumulation, which is achieved through two functionally redundant regions (SIC1-244 and SIC252-319) for nuclear localization. Of the remaining amino acids in this region, six (SIC245-251) comprise a DBR1-interacting region while two (SICM246 and SICW251) are essential for DBR1-SIC interaction. Reducing lariRNAs restored microRNA (miRNA) levels and LR development in lariRNA hyper-accumulating plants, suggesting that these well-known regulators of LR development mainly function downstream of lariRNAs. Taken together, we propose that SIC acts as an enhancer of DBR1 nuclear accumulation by driving nuclear localization through direct interaction, thereby promoting lariRNA decay to fine-tune miRNA biogenesis and modulating LR development.

Erratum in Plant Physiol. 2022 Nov 28;190(4):2868.

Different environmental stresses often evoke similar physiological disorders such as growth retardation; however, specific consequences reported among individual stresses indicate potential mechanisms to distinguish different stress types in plants. Here, we examined mechanisms to differentiate between stress types in Arabidopsis (Arabidopsis thaliana). Gene expression patterns recapitulating several abiotic stress responses suggested abscisic acid (ABA) as a mediator of the common stress response, while stress type-specific responses were related to metabolic adaptations. Transcriptome and metabolome analyses identified Arabidopsis Gβ (AGB1) mediating the common stress-responsive genes and primary metabolisms under nitrogen excess. AGB1 regulated the expressions of multiple WRKY transcription factors. Gene Ontology and mutant analyses revealed different roles among WRKYs: WRKY40 is involved in ABA and common stress responses, while WRKY75 regulates metabolic processes. The AGB1-WRKY signaling module controlled developmental plasticity in roots under nitrogen excess. Signal transmission from AGB1 to a selective set of WRKYs would be essential to evoke unique responses to different types of stresses.

Understanding gene expression and regulation requires insights into RNA transcription, processing, modification, and translation. However, the relationship between the epitranscriptome and the proteome under drought stress remains undetermined in poplar (Populus trichocarpa). In this study, we used Nanopore direct RNA sequencing and tandem mass tag-based proteomic analysis to examine epitranscriptomic and proteomic regulation induced by drought treatment in stem-differentiating xylem (SDX). Our results revealed a decreased full-length read ratio under drought treatment and, especially, a decreased association between transcriptome and proteome changes in response to drought. Epitranscriptome analysis of cellulose- and lignin-related genes revealed an increased N6-Methyladenosine (m6A) ratio, which was accompanied by decreased RNA abundance and translation, under drought stress. Interestingly, usage of the distal poly(A) site increased during drought stress. Finally, we found that transcripts of highly expressed genes tend to have shorter poly(A) tail length (PAL), and drought stress increased the percentage of transcripts with long PAL. These findings provide insights into the interplay among m6A, polyadenylation, PAL, and translation under drought stress in P. trichocarpa SDX.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

Angiosperm genome evolution was marked by many clade-specific whole-genome duplication events. The Microlepidieae is one of the monophyletic clades in the mustard family (Brassicaceae) formed after an ancient allotetraploidization. Postpolyploid cladogenesis has resulted in the extant c. 17 genera and 60 species endemic to Australia and New Zealand (10 species). As postpolyploid genome diploidization is a trial-and-error process under natural selection, it may proceed with different intensity and be associated with speciation events. In Microlepidieae, different extents of homoeologous recombination between the two parental subgenomes generated clades marked by slow ("cold") versus fast ("hot") genome diploidization. To gain a deeper understanding of postpolyploid genome evolution in Microlepidieae, we analyzed phylogenetic relationships in this tribe using complete chloroplast sequences, entire 35S rDNA units, and abundant repetitive sequences. The four recovered intra-tribal clades mirror the varied diploidization of Microlepidieae genomes, suggesting that the intrinsic genomic features underlying the extent of diploidization are shared among genera and species within one clade. Nevertheless, even congeneric species may exert considerable morphological disparity (e.g. in fruit shape), whereas some species within different clades experience extensive morphological convergence despite the different pace of their genome diploidization. We showed that faster genome diploidization is positively associated with mean morphological disparity and evolution of chloroplast genes (plastid-nuclear genome coevolution). Higher speciation rates in perennials than in annual species were observed. Altogether, our results confirm the potential of Microlepidieae as a promising subject for the analysis of postpolyploid genome diploidization in Brassicaceae.

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. More than 25 acylsugar variants could be produced in a single enzyme assay with four N. acuminata acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites.

Ubiquitination is a major mechanism of eukaryotic posttranslational protein turnover that has been implicated in abscisic acid (ABA)-mediated drought stress response. Here, we isolated T-DNA insertion mutant lines in which ABA-insensitive RING protein 5 (AtAIRP5) was suppressed, resulting in hyposensitive ABA-mediated germination compared to wild-type Arabidopsis (Arabidopsis thaliana) plants. A homology search revealed that AtAIRP5 is identical to gibberellin (GA) receptor RING E3 ubiquitin (Ub) ligase (GARU), which downregulates GA signaling by degrading the GA receptor GID1, and thus AtAIRP5 was renamed AtAIRP5/GARU. The atairp5/garu knockout progeny were impaired in ABA-dependent stomatal closure and were markedly more susceptible to drought stress than wild-type plants, indicating a positive role for AtAIRP5/GARU in the ABA-mediated drought stress response. Yeast two-hybrid, pull-down, target ubiquitination, and in vitro and in planta degradation assays identified serine carboxypeptidase-like1 (AtSCPL1), which belongs to the clade 1A AtSCPL family, as a ubiquitinated target protein of AtAIRP5/GARU. atscpl1 single and atairp5/garu-1 atscpl1-2 double mutant plants were more tolerant to drought stress than wild-type plants in an ABA-dependent manner, suggesting that AtSCPL1 is genetically downstream of AtAIRP5/GARU. After drought treatment, the endogenous ABA levels in atscpl1 and atairp5/garu-1 atscpl1-2 mutant leaves were higher than those in wild-type and atairp5/garu leaves. Overall, our results suggest that AtAIRP5/GARU RING E3 Ub ligase functions as a positive regulator of the ABA-mediated drought response by promoting the degradation of AtSCPL1.

The protocorm is a structure that is formed upon germination of an orchid seed. It lacks cotyledons and is ovoid in shape. The protocorm-like body (PLB), on the other hand, is a protocorm-like organ induced from somatic tissues. PLBs have been widely used for orchid micropropagation. Because of its unique structure and its application in the orchid industry, PLB development has drawn considerable interest from orchid and developmental biologists. Our previous genome-wide comparative transcriptome study demonstrated that protocorms and PLBs share similar molecular signatures and suggested that SHOOT MERISTEMLESS (STM)-dependent organogenesis is important for PLB development. Here, we show that overexpression of Phalaenopsis aphrodite STM (PaSTM) greatly enhances PLB regeneration from vegetative tissue-based explants of Phalaenopsis orchids, confirming its regulatory role in PLB development. Expression of PaSTM restored shoot meristem function of the Arabidopsis (Arabidopsis thaliana) stm-2 mutant. Moreover, we identified class S11 MYB transcription factors (TFs) as targets downstream of PaSTM. A cis-acting element, TTGACT, identified in the promoters of S11 MYB TFs was found to be important for PaSTM binding and activation. Overexpression of PaSTM or its downstream targets, PaMYB13, PaMYB14, and PaMYB17, enhanced de novo shoot regeneration in Arabidopsis, indicating the active role of the PaSTM-S11 PaMYB module in organogenesis. In summary, our data demonstrate that PaSTM is important for PLB development. The STM-S11 MYB regulatory module is evolutionarily conserved and may regulate shoot or shoot-related organ development in plants.

Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the protein factors involved and the underlying molecular mechanisms are still not well understood. Here, we report on the function of the MITOCHONDRIAL STABILITY FACTOR 3 (MTSF3) protein, previously named EMBRYO DEFECTIVE 2794 (EMB2794), and show that it is essential for accumulation of the mitochondrial NADH dehydrogenase subunit 2 (nad2) transcript in Arabidopsis (Arabidopsis thaliana) but not for splicing of nad2 intron 2 as previously proposed. The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality, but viable mtsf3 mutant plants can be generated through partial complementation with the developmentally regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses revealed growth retardation in rescued mtsf3 plants owing to the specific destabilization of mature nad2 mRNA and a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3' terminus of nad2. Destabilization of nad2 mRNA induces a substantial decrease in complex I assembly and activity and overexpression of the alternative respiratory pathway. Our results support a role for MTSF3 protein in protecting two nad2 transcripts from degradation by mitochondrial exoribonucleases by binding to their 3' extremities.

Botrytis cinerea is one of the most widely distributed and harmful pathogens worldwide. Both the phytohormone jasmonate (JA) and the VQ motif-containing proteins play crucial roles in plant resistance to B. cinerea. However, their crosstalk in resistance to B. cinerea is unclear, especially in tomato (Solanum lycopersicum). In this study, we found that the tomato VQ15 was highly induced upon B. cinerea infection and localized in the nucleus. Silencing SlVQ15 using virus-induced gene silencing reduced resistance to B. cinerea. Overexpression of SlVQ15 enhanced resistance to B. cinerea, while disruption of SlVQ15 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) technology increased susceptibility to B. cinerea. Furthermore, SlVQ15 formed homodimers. Additionally, SlVQ15 interacted with JA-ZIM domain proteins, repressors of the JA signaling pathway, and SlWRKY31. SlJAZ11 interfered with the interaction between SlVQ15 and SlWRKY31 and repressed the SlVQ15-increased transcriptional activation activity of SlWRKY31. SlVQ15 and SlWRKY31 synergistically regulated tomato resistance to B. cinerea, as silencing SlVQ15 enhanced the sensitivity of slwrky31 to B. cinerea. Taken together, our findings showed that the SlJAZ-interacting protein SlVQ15 physically interacts with SlWRKY31 to cooperatively control JA-mediated plant defense against B. cinerea.

Chloroplasts and mitochondria are subcellular organelles that evolved from cyanobacteria and α-proteobacteria, respectively. Although they have their own genomes, the majority of their proteins are encoded by nuclear genes, translated by cytosolic ribosomes, and imported via outer and inner membrane translocon complexes. The unfolding of mature regions of proteins is thought to be a prerequisite for the import of the proteins into these organelles. However, it is not fully understood how protein folding properties affect their import into these organelles. In this study, we examined the import behavior of chloroplast and mitochondrial reporters with normal green fluorescent protein (GFP) and two GFP variants with enhanced folding propensity, superfolder GFP (sfGFP) and extra-superfolder GFP (esGFP), which is folded better than sfGFP. sfGFP and esGFP were less dependent on the sequence motifs of the transit peptide (TP) and import machinery during protein import into Arabidopsis (Arabidopsis thaliana) chloroplasts, compared with normal GFP. sfGFP and esGFP were efficiently imported into chloroplasts by a mutant TP with an alanine substitution in the N-terminal MLM motif, whereas the same mutant TP showed a defect in importing normal GFP into chloroplasts. Moreover, sfGFP and esGFP were efficiently imported into plastid protein import 2 (ppi2) and heat shock protein 93-V (hsp93-V) plants, which have mutations in atToc159 and Hsp93-V, respectively. In contrast, the presequence-mediated mitochondrial import of sfGFP and esGFP was severely impaired. Based on these results, we propose that the chloroplast import machinery is more tolerant to different folding states of preproteins, whereas the mitochondrial machinery is more specialized in the translocation of unfolded preproteins.

The bulbil is an important vegetative reproductive organ in triploid tiger lily (Lilium lancifolium). Based on our previously obtained transcriptome data, we screened two WUSCHEL-related homeobox (WOX) genes closely related to bulbil formation, LlWOX9 and LlWOX11. However, the biological functions and regulatory mechanisms of LlWOX9 and LlWOX11 are unclear. In this study, we cloned the full-length coding sequences of LlWOX9 and LlWOX11. Transgenic Arabidopsis (Arabidopsis thaliana) showed increased branch numbers, and the overexpression of LlWOX9 and LlWOX11 in stem segments promoted bulbil formation, while the silencing of LlWOX9 and LlWOX11 inhibited bulbil formation, indicating that LlWOX9 and LlWOX11 are positive regulators of bulbil formation. Cytokinin type-B response regulators could bind to the promoters of LlWOX9 and LlWOX11 and promote their transcription. LlWOX11 could enhance cytokinin pathway signaling by inhibiting the transcription of type-A LlRR9. Our study enriches the understanding of the regulation of plant development by the WOX gene family and lays a foundation for further research on the molecular mechanism of bulbil formation in lily.

Spikelets are highly specialized and short-lived branches and function as a constitutional unit of the complex grass inflorescences. A series of genetic, genomic, and developmental studies across different clades of the family have called for and permitted a synthesis on the regulation and evolution of spikelets, and hence inflorescence diversity. Here, we have revisited the identity specification of a spikelet, focusing on the diagnostic features of a spikelet from morphological, developmental, and molecular perspectives. Particularly, recent studies on a collection of barley (Hordeum vulgare L.), wheat (Triticum spp.), and rice (Oryza sativa L.) mutants have highlighted a set of transcription factors that are important in the control of spikelet identity and the patterning of floral parts of a spikelet. In addition, we have endeavored to clarify some puzzling issues on the (in)determinacy and modifications of spikelets over the course of evolution. Meanwhile, genomes of two sister taxa of the remaining grass species have again demonstrated the importance of genome duplication and subsequent gene losses on the evolution of spikelets. Accordingly, we argue that changes in the orthologs of spikelet-related genes could be critical for the development and evolution of the spikelet, an evolutionary innovation in the grass family. Likewise, the conceptual discussions on the regulation of a fundamental unit of compound inflorescences could be translated into other organismal groups where compound structures are similarly formed, permitting a comparative perspective on the control of biological complexity.

Cyanobacteria are phototrophic bacteria that perform oxygenic photosynthesis. They use a supermolecular light-harvesting antenna complex, the phycobilisome (PBS), to capture and transfer light energy to photosynthetic reaction centers. Certain cyanobacteria alter the absorption maxima and/or overall structure of their PBSs in response to the ambient light wavelength-a process called chromatic acclimation (CA). One of the most well-known CA types is the response to green and red light, which is controlled by either the RcaEFC or CcaSR photosensory system. Here, we characterized a hybrid type of CA in the cyanobacterium Pleurocapsa sp. Pasteur Culture Collection (PCC) 7319 that uses both RcaEFC and CcaSR systems. In vivo spectroscopy suggested that strain PCC 7319 alters the relative composition of green-absorbing phycoerythrin and red-absorbing phycocyanin in the PBS. RNA sequencing and promoter motif analyses suggested that the RcaEFC system induces a gene operon for phycocyanin under red light, whereas the CcaSR system induces a rod-membrane linker gene under green light. Induction of the phycoerythrin genes under green light may be regulated through a yet unidentified photosensory system called the Cgi system. Spectroscopy analyses of the isolated PBSs suggested that hemidiscoidal and rod-shaped PBSs enriched with phycoerythrin were produced under green light, whereas only hemidiscoidal PBSs enriched with phycocyanin were produced under red light. PCC 7319 uses the RcaEFC and CcaSR systems to regulate absorption of green or red light (CA3) and the amount of rod-shaped PBSs (CA1), respectively. Cyanobacteria can thus flexibly combine diverse CA types to acclimate to different light environments.

The timing of flowering is a crucial factor for successful grain production at a wide range of latitudes. Domestication of rice (Oryza sativa) included selection for natural alleles of flowering-time genes that allow rice plants to adapt to broad geographic areas. Here, we describe the role of natural alleles of CIRCADIAN CLOCK ASSOCIATED1 (OsCCA1) in cultivated rice based on analysis of single-nucleotide polymorphisms deposited in the International Rice Genebank Collection Information System database. Rice varieties harboring japonica-type OsCCA1 alleles (OsCCA1a haplotype) flowered earlier than those harboring indica-type OsCCA1 alleles (OsCCA1d haplotype). In the japonica cultivar "Dongjin", a T-DNA insertion in OsCCA1a resulted in late flowering under long-day and short-day conditions, indicating that OsCCA1 is a floral inducer. Reverse transcription quantitative PCR analysis showed that the loss of OsCCA1a function induces the expression of the floral repressors PSEUDO-RESPONSE REGULATOR 37 (OsPRR37) and Days to Heading 8 (DTH8), followed by repression of the Early heading date 1 (Ehd1)-Heading date 3a (Hd3a)-RICE FLOWERING LOCUS T 1 (RFT1) pathway. Binding affinity assays indicated that OsCCA1 binds to the promoter regions of OsPRR37 and DTH8. Naturally occurring OsCCA1 alleles are evolutionarily conserved in cultivated rice (O. sativa). Oryza rufipogon-I (Or-I) and Or-III type accessions, representing the ancestors of O. sativa indica and japonica, harbored indica- and japonica-type OsCCA1 alleles, respectively. Taken together, our results demonstrate that OsCCA1 is a likely domestication locus that has contributed to the geographic adaptation and expansion of cultivated rice.

Long noncoding RNAs (lncRNAs) are widely involved in the regulation of plant growth and development, but their mechanism of action in response to cold stress in plants remains unclear. Here, we found an lncRNA transcribed from the antisense strand of DgTCP1 (class I Teosinte branched1/Cycloidea/Proliferating [TCP] transcription factor) of chrysanthemum (Chrysanthemum morifolium Ramat.), named DglncTCP1. During the response of chrysanthemum to cold stress, overexpression of DgTCP1 improved the cold tolerance of chrysanthemum, while the DgTCP1 editing line (dgtcp1) showed decreased tolerance to cold stress. Overexpression of DglncTCP1 also increased the cold tolerance of chrysanthemum, while the DglncTCP1 amiRNA lines (DglncTCP1 amiR-18/38) also showed decreased tolerance to cold stress. Additionally, the overexpression of DglncTCP1 upregulated the expression of DgTCP1. This indicated that DglncTCP1 may play a cis-regulatory role in the regulatory process of DgTCP1 in cold tolerance. DglncTCP1 acts as a scaffold to recruit the histone modification protein DgATX (ARABIDOPSIS TRITHORAX from chrysanthemum) to DgTCP1 to enhance H3K4me3 levels, thereby activating DgTCP1 expression. Moreover, DgTCP1 can directly target DgPOD (peroxidase gene from chrysanthemum) to promote its expression and reduce reactive oxygen species accumulation, thereby improving the cold tolerance of chrysanthemum. In conclusion, these results suggest that natural antisense lncRNA plays a key role in improving the cold tolerance of chrysanthemum.

Pre-mRNA splicing is an important step in the posttranscriptional processing of transcripts and a key regulator of development. The heterotrimeric retention and splicing (RES) complex plays vital roles in the growth and development of yeast, zebrafish, and humans by mediating pre-mRNA splicing of multiple genes. However, whether the RES complex is conserved in plants and what specific functions it has remain unknown. In this study, we identified Arabidopsis (Arabidopsis thaliana) BUD13 (AtBUD13), GROWTH, DEVELOPMENT AND SPLICING 1 (GDS1), and DAWDLE (DDL) as the counterparts of the yeast RES complex subunits Bud site selection protein 13 (Bud13), U2 snRNP component Snu17 (Snu17), and Pre-mRNA leakage protein 1, respectively. Moreover, we showed that RES is an ancient complex evolutionarily conserved in eukaryotes. GDS1 directly interacts with both AtBUD13 and DDL in nuclear speckles. The BUD13 domain of AtBUD13 and the RNA recognition motif domain of GDS1 are necessary and sufficient for AtBUD13-GDS1 interaction. Mutants of AtBUD13, GDS1, and DDL failed to properly splice multiple genes involved in cell proliferation and showed defects in early embryogenesis and root development. In addition, we found that GDS1 and DDL interact, respectively, with the U2 small nuclear ribonucleoproteins auxiliary factor AtU2AF65B and the NineTeen Complex-related splicing factor SKIP, which are essential for early steps of spliceosome assembly and recognition of splice sites. Altogether, our work reveals that the Arabidopsis RES complex is important for root and early embryo development by modulating pre-mRNA splicing.

Phosphorus (P) is a mineral nutrient essential for plant growth and development, but most P in the soil is unavailable for plants. To understand the genetic basis of P acquisition regulation, we performed genome-wide association studies (GWASs) on a diversity panel of Arabidopsis (Arabidopsis thaliana). Two primary determinants of P acquisition were considered, namely, phosphate (Pi)-uptake activity and PHOSPHATE TRANSPORTER 1 (PHT1) protein abundance. Association mapping revealed a shared significant peak on chromosome 5 (Chr5) where the PHT1;1/2/3 genes reside, suggesting a connection between the regulation of Pi-uptake activity and PHT1 protein abundance. Genes encoding transcription factors, kinases, and a metalloprotease associated with both traits were also identified. Conditional GWAS followed by statistical analysis of genotype-dependent PHT1;1 expression and transcriptional activity assays revealed an epistatic interaction between PHT1;1 and MYB DOMAIN PROTEIN 52 (MYB52) on Chr1. Further, analyses of F1 hybrids generated by crossing two subgroups of natural accessions carrying specific PHT1;1- and MYB52-associated single nucleotide polymorphisms (SNPs) revealed strong effects of these variants on PHT1;1 expression and Pi uptake activity. Notably, the soil P contents in Arabidopsis habitats coincided with PHT1;1 haplotype, emphasizing how fine-tuned P acquisition activity through natural variants allows environmental adaptation. This study sheds light on the complex regulation of P acquisition and offers a framework to systematically assess the effectiveness of GWAS approaches in the study of quantitative traits.

Comment in Plant Physiol. 2022 Aug 29;190(1):182-184.

Comment in Plant Physiol. 2022 Aug 29;190(1):185-187.

C4 photosynthesis allows faster photosynthetic rates and higher water and nitrogen use efficiency than C3 photosynthesis, but at the cost of lower quantum yield due to the energy requirement of its biochemical carbon concentration mechanism. It has also been suspected that its operation may be impaired in low irradiance. To investigate fluxes under moderate and low irradiance, maize (Zea mays) was grown at 550 µmol photons m-2 s-l and 13CO2 pulse-labeling was performed at growth irradiance or several hours after transfer to 160 µmol photons m-2 s-1. Analysis by liquid chromatography/tandem mass spectrometry or gas chromatography/mass spectrometry provided information about pool size and labeling kinetics for 32 metabolites and allowed estimation of flux at many steps in C4 photosynthesis. The results highlighted several sources of inefficiency in low light. These included excess flux at phosphoenolpyruvate carboxylase, restriction of decarboxylation by NADP-malic enzyme, and a shift to increased CO2 incorporation into aspartate, less effective use of metabolite pools to drive intercellular shuttles, and higher relative and absolute rates of photorespiration. The latter provides evidence for a lower bundle sheath CO2 concentration in low irradiance, implying that operation of the CO2 concentration mechanism is impaired in this condition. The analyses also revealed rapid exchange of carbon between the Calvin-Benson cycle and the CO2-concentration shuttle, which allows rapid adjustment of the balance between CO2 concentration and assimilation, and accumulation of large amounts of photorespiratory intermediates in low light that provides a major carbon reservoir to build up C4 metabolite pools when irradiance increases.

The pollen wall is important for protecting the male gametophyte and for fertilization. The lipid components of the pollen wall are mainly synthesized and transported from the sporophytic tapetum. Although several factors related to lipid biosynthesis have been characterized, the molecular mechanisms underlying lipid biosynthesis during pollen development in rice (Oryza sativa L.) remain elusive. Here, we showed that mutation in the SWOLLEN TAPETUM AND STERILITY 1 (STS1) gene causes delayed tapetum degradation and aborted pollen wall formation in rice. STS1 encodes an endoplasmic reticulum (ER)-localized protein that contains domain of unknown function (DUF) 726 and exhibits lipase activity. Lipidomic and transcriptomic analyses showed that STS1 is involved in anther lipid homeostasis. Moreover, STS1 interacts with Polyketide Synthase 2 (OsPKS2) and Acyl-CoA Synthetase 12 (OsACOS12), two enzymes crucial in lipidic sporopollenin biosynthesis in pollen wall formation, suggesting a potentially lipidic metabolon for sporopollenin biosynthesis in rice. Collectively, our results indicate that STS1 is an important factor for lipid biosynthesis in reproduction, providing a target for the artificial control of male fertility in hybrid rice breeding and insight into the function of DUF726-containing protein in plants.

Plant plastids generate signals, including some derived from lipids, that need to be mobilized to effect signaling. We used informatics to discover potential plastid membrane proteins involved in microbial responses in Arabidopsis (Arabidopsis thaliana). Among these are proteins co-regulated with the systemic immunity component AZELAIC ACID INDUCED 1, a hybrid proline-rich protein (HyPRP), and HyPRP superfamily members. HyPRPs have a transmembrane domain, a proline-rich region (PRR), and a lipid transfer protein domain. The precise subcellular location(s) and function(s) are unknown for most HyPRP family members. As predicted by informatics, a subset of HyPRPs has a pool of proteins that target plastid outer envelope membranes via a mechanism that requires the PRR. Additionally, two HyPRPs may be associated with thylakoid membranes. Most of the plastid- and nonplastid-localized family members also have pools that localize to the endoplasmic reticulum, plasma membrane, or plasmodesmata. HyPRPs with plastid pools regulate, positively or negatively, systemic immunity against the pathogen Pseudomonas syringae. HyPRPs also regulate the interaction with the plant growth-promoting rhizobacteria Pseudomonas simiae WCS417 in the roots to influence colonization, root system architecture, and/or biomass. Thus, HyPRPs have broad and distinct roles in immunity, development, and growth responses to microbes and reside at sites that may facilitate signal molecule transport.

The genomes of Gramineae plants have been preferentially sequenced owing to their economic value. These genomes are often quite complex, for example harboring many duplicated genes, and are the main source of genetic innovation and often the result of recurrent polyploidization. Deciphering these complex genome structures and linking duplicated genes to specific polyploidization events are important for understanding the biology and evolution of plants. However, efforts have been hampered by the complexity of analyzing these genomes. Here, we analyzed 29 well-assembled and up-to-date Gramineae genome sequences by hierarchically relating duplicated genes in collinear regions to specific polyploidization or speciation events. We separated duplicated genes produced by each event, established lists of paralogous and orthologous genes, and ultimately constructed an online database, GGDB (http://www.grassgenome.com/). Homologous gene lists from each plant and between plants can be displayed, searched, and downloaded from the database. Interactive comparison tools are deployed to demonstrate homology among user-selected plants and to draw genome-scale or local alignment figures and gene-based phylogenetic trees corrected by exploiting gene collinearity. Using these tools and figures, users can easily detect structural changes in genomes and explore the effects of paleo-polyploidy on crop genome structure and function. The GGDB will provide a useful platform for improving our understanding of genome changes and functional innovation in Gramineae plants.

Leaves and flowers are produced by the shoot apical meristem (SAM) at a certain distance from its center, a process that requires the hormone auxin. The amount of auxin and the pattern of its distribution in the initiation zone determine the size and spatial arrangement of organ primordia. Auxin gradients in the SAM are formed by PIN-FORMED (PIN) auxin efflux carriers whose polar localization in the plasma membrane depends on the protein kinase PINOID (PID). Previous work determined that ERECTA (ER) family genes (ERfs) control initiation of leaves. ERfs are plasma membrane receptors that enable cell-to-cell communication by sensing extracellular small proteins from the EPIDERMAL PATTERNING FACTOR/EPF-LIKE (EPF/EPFL) family. Here, we investigated whether ERfs regulate initiation of organs by altering auxin distribution or signaling in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological data suggested that ERfs do not regulate organogenesis through PINs while transcriptomics data showed that ERfs do not alter primary transcriptional responses to auxin. Our results indicated that in the absence of ERf signaling the peripheral zone cells inefficiently initiate leaves in response to auxin signals and that increased accumulation of auxin in the er erecta-like1 (erl1) erl2 SAM can partially rescue organ initiation defects. We propose that both auxin and ERfs are essential for leaf initiation and that they have common downstream targets. Genetic data also indicated that the role of PID in initiation of cotyledons and leaves cannot be attributed solely to regulation of PIN polarity and PID is likely to have other functions in addition to regulation of auxin distribution.

Comment in Plant Physiol. 2022 Aug 29;190(1):188-189.

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.

Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS C (FLC) acts as a key flowering regulator by repressing the expression of the floral integrator FLOWERING LOCUS T (FT). Prolonged exposure to cold (vernalization) induces flowering by reducing FLC expression. The long noncoding RNAs (lncRNAs) COOLAIR and COLDAIR, which are transcribed from the 3' end and the first intron of FLC, respectively, are important for FLC repression under vernalization. However, the molecular mechanism of how COOLAIR and COLDAIR are transcriptionally activated remains elusive. In this study, we found that the group-III WRKY transcription factor WRKY63 can directly activate FLC. wrky63 mutant plants display an early flowering phenotype and are insensitive to vernalization. Interestingly, we found that WRKY63 can activate the expression of COOLAIR and COLDAIR by binding to their promoters.WRKY63 therefore acts as a dual regulator that activates FLC directly under non-vernalization conditions but represses FLC indirectly during vernalization through inducing COOLAIR and COLDAIR. Furthermore, genome-wide occupancy profile analyses indicated that the binding of WRKY63 to vernalization-induced genes increases after vernalization. In addition, WRKY63 binding is associated with decreased levels of the repressive marker Histone H3 Lysine 27 trimethylation (H3K27me3). Collectively, our results indicate that WRKY63 is an important flowering regulator involved in vernalization-induced transcriptional regulation.

Sodium (Na+) and potassium (K+) homeostasis is essential for plant survival in saline soils. A member of the High-Affinity K+ Transporter (HKT) family in rice (Oryza sativa), OsHKT1;1, is a vital regulator of Na+ exclusion from shoots and is bound by a MYB transcription factor (OsMYBc). Here, we generated transgenic rice lines in the oshkt1;1 mutant background for genetic complementation using genomic OsHKT1;1 containing a native (Com) or mutated (mCom) promoter that cannot be bound by OsMYBc. In contrast to wild-type (WT) or Com lines, the mCom lines were not able to recover the salt-sensitive phenotype of oshkt1;1. The OsMYBc-overexpressing plants were more tolerant to salt stress than WT plants. A yeast two-hybrid screen using the OsMYBc N-terminus as bait identified a rice MYBc stress-related RING finger protein (OsMSRFP). OsMSRFP is an active E3 ligase that ubiquitinated OsMYBc in vitro and mediated 26S proteasome-mediated degradation of OsMYBc under semi-in vitro and in vivo conditions. OsMSRFP attenuated OsMYBc-mediated OsHKT1;1 expression, and knockout of OsMSRFP led to rice salt tolerance. These findings uncover a regulatory mechanism of salt response that fine-tunes OsHKT1;1 transcription by ubiquitination of OsMYBc.

Evidence suggests that anthropogenically-mediated global warming results in accelerated flowering for many plant populations. However, the fact that some plants are late flowering or unaffected by warming, underscores the complex relationship between phase change, temperature, and phylogeny. In this review, we present an emerging picture of how plants sense temperature changes, and then discuss the independent recruitment of ancient flowering pathway genes for the evolution of ambient, low, and high temperature-regulated reproductive development. As well as revealing areas of research required for a better understanding of how past thermal climates have shaped global patterns of plasticity in plant phase change, we consider the implications for these phenological thermal responses in light of climate change.

Comment in Plant Physiol. 2022 Aug 1;189(4):1896-1897.

Castor bean (Ricinus communis) seed oil (triacylglycerol [TAG]) is composed of ∼90% of the industrially important ricinoleoyl (12-hydroxy-9-octadecenoyl) groups. Here, phosphatidylcholine (PC):diacylglycerol (DAG) cholinephosphotransferase (PDCT) from castor bean was biochemically characterized and compared with camelina (Camelina sativa) PDCT. DAGs with ricinoleoyl groups were poorly used by Camelina PDCT, and their presence inhibited the utilization of DAG with "common" acyl groups. In contrast, castor PDCT utilized DAG with ricinoleoyl groups similarly to DAG with common acyl groups and showed a 10-fold selectivity for DAG with one ricinoleoyl group over DAG with two ricinoleoyl groups. Castor DAG acyltransferase2 specificities and selectivities toward different DAG and acyl-CoA species were assessed and shown to not acylate DAG without ricinoleoyl groups in the presence of ricinoleoyl-containing DAG. Eighty-five percent of the DAG species in microsomal membranes prepared from developing castor endosperm lacked ricinoleoyl groups. Most of these species were predicted to be derived from PC, which had been formed by PDCT in exchange with DAG with one ricinoleoyl group. A scheme of the function of PDCT in castor endosperm is proposed where one ricinoleoyl group from de novo-synthesized DAG is selectivity transferred to PC. Nonricinoleate DAG is formed and ricinoleoyl groups entering PC are re-used either in de novo synthesis of DAG with two ricinoleoyl groups or in direct synthesis of triricinoleoyl TAG by PDAT. The PC-derived DAG is not used in TAG synthesis but is proposed to serve as a substrate in membrane lipid biosynthesis during oil deposition.

The parasitic plant Striga (Striga hermonthica) invades the host root through the formation of a haustorium and has detrimental impacts on cereal crops. The haustorium results from the prehaustorium, which is derived directly from the differentiation of the Striga radicle. The molecular mechanisms leading to radicle differentiation shortly after germination remain unclear. In this study, we determined the developmental programs that regulate terminal prehaustorium formation in S. hermonthica at cellular resolution. We showed that shortly after germination, cells in the root meristem undergo multiplanar divisions. During growth, the meristematic activity declines and associates with reduced expression of the stem cell regulator PLETHORA1 and the cell cycle genes CYCLINB1 and HISTONE H4. We also observed a basal localization of the PIN-FORMED (PIN) proteins and a decrease in auxin levels in the meristem. Using the structural layout of the root meristem and the polarity of outer-membrane PIN proteins, we constructed a mathematical model of auxin transport that explains the auxin distribution patterns observed during S. hermonthica root growth. Our results reveal a fundamental molecular and cellular framework governing the switch of S. hermonthica roots to form the invasive prehaustoria.

Comment in doi: 10.1093/plphys/kiac350.

Selenium-binding proteins (SBPs) represent a ubiquitous protein family implicated in various environmental stress responses, although the exact molecular and physiological role of the SBP family remains elusive. In this work, we report the identification and characterization of CrSBD1, an SBP homolog from the model microalgae Chlamydomonas reinhardtii. Growth analysis of the C. reinhardtii sbd1 mutant strain revealed that the absence of a functional CrSBD1 resulted in increased growth under mild oxidative stress conditions, although cell viability rapidly declined at higher hydrogen peroxide (H2O2) concentrations. Furthermore, a combined global transcriptomic and metabolomic analysis indicated that the sbd1 mutant exhibited a dramatic quenching of the molecular and biochemical responses upon H2O2-induced oxidative stress when compared to the wild-type. Our results indicate that CrSBD1 represents a cell regulator, which is involved in the modulation of C. reinhardtii early responses to oxidative stress. We assert that CrSBD1 acts as a member of an extensive and conserved protein-protein interaction network including Fructose-bisphosphate aldolase 3, Cysteine endopeptidase 2, and Glutaredoxin 6 proteins, as indicated by yeast two-hybrid assays.

Understanding mass transport of photosynthates in the phloem of plants is necessary for predicting plant carbon allocation, productivity, and responses to water and thermal stress. Several hypotheses about optimization of phloem structure and function and limitations of phloem transport under drought have been proposed and tested with models and anatomical data. However, the true impact of radial water exchange of phloem conduits with their surroundings on mass transport of photosynthates has not been addressed. Here, the physics of the Munch mechanism of sugar transport is re-evaluated to include local variations in viscosity resulting from the radial water exchange in two dimensions (axial and radial) using transient flow simulations. Model results show an increase in radial water exchange due to a decrease in sap viscosity leading to increased sugar front speed and axial mass transport across a wide range of phloem conduit lengths. This increase is around 40% for active loaders (e.g. crops) and around 20% for passive loaders (e.g. trees). Thus, sugar transport operates more efficiently than predicted by previous models that ignore these two effects. A faster front speed leads to higher phloem resiliency under drought because more sugar can be transported with a smaller pressure gradient.

Comment in Plant Physiol. 2022 Aug 1;189(4):1890-1892.

Plant adjustment to environmental changes involves complex crosstalk between extrinsic and intrinsic cues. In the past two decades, extensive research has elucidated the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and the phytohormone auxin in thermomorphogenesis. In this study, we identified a previously unexplored role of jasmonate (JA) signaling components, the Mediator complex, and their integration with auxin signaling during thermomorphogenesis in Arabidopsis (Arabidopsis thaliana). Warm temperature induces expression of JA signaling genes including MYC2, but, surprisingly, this transcriptional activation is not JA dependent. Warm temperature also promotes accumulation of the JA signaling receptor CORONATINE INSENSITIVE1 (COI1) and degradation of the JA signaling repressor JASMONATE-ZIM-DOMAIN PROTEIN9, which probably leads to de-repression of MYC2, enabling it to contribute to the expression of MEDIATOR SUBUNIT17 (MED17). In response to warm temperature, MED17 occupies the promoters of thermosensory genes including PIF4, YUCCA8 (YUC8), INDOLE-3-ACETIC ACID INDUCIBLE19 (IAA19), and IAA29. Moreover, MED17 facilitates enrichment of H3K4me3 on the promoters of PIF4, YUC8, IAA19, and IAA29 genes. Interestingly, both occupancy of MED17 and enrichment of H3K4me3 on these thermomorphogenesis-related promoters are dependent on PIF4 (or PIFs). Altered accumulation of COI1 under warm temperature in the med17 mutant suggests the possibility of a feedback mechanism. Overall, this study reveals the role of the Mediator complex as an integrator of JA and auxin signaling pathways during thermomorphogenesis.

Plant organ abscission, a process that is important for development and reproductive success, is inhibited by the phytohormone auxin and promoted by another phytohormone, jasmonic acid (JA). However, the molecular mechanisms underlying the antagonistic effects of auxin and JA in organ abscission are unknown. We identified a tomato (Solanum lycopersicum) class III homeodomain-leucine zipper transcription factor, HOMEOBOX15A (SlHB15A), which was highly expressed in the flower pedicel abscission zone and induced by auxin. Knocking out SlHB15A using clustered regularly interspaced short palindromic repeats-associated protein 9 technology significantly accelerated abscission. In contrast, overexpression of microRNA166-resistant SlHB15A (mSlHB15A) delayed abscission. RNA sequencing and reverse transcription-quantitative PCR analyses showed that knocking out SlHB15A altered the expression of genes related to JA biosynthesis and signaling. Furthermore, functional analysis indicated that SlHB15A regulates abscission by depressing JA-isoleucine (JA-Ile) levels through inhabiting the expression of JASMONATE-RESISTANT1 (SlJAR1), a gene involved in JA-Ile biosynthesis, which could induce abscission-dependent and abscission-independent ethylene signaling. SlHB15A bound directly to the SlJAR1 promoter to silence SlJAR1, thus delaying abscission. We also found that flower removal enhanced JA-Ile content and that application of JA-Ile severely impaired the inhibitory effects of auxin on abscission. These results indicated that SlHB15A mediates the antagonistic effect of auxin and JA-Ile during tomato pedicel abscission, while auxin inhibits abscission through the SlHB15A-SlJAR1 module.

In vascular plants, roots anchor themselves into the soil and take up water and nutrients to provide them to the shoots. Therefore, continuous growth and development of the roots are important for plant life. To achieve this, photosynthesizing leaves must be able to supply sufficient photoassimilates to the roots. However, the mechanisms by which plants maintain carbon levels in roots remain elusive. Here, we focused on the Arabidopsis (Arabidopsis thaliana) CLAVATA3/ESR-related 2 (CLE2) peptide, which was detected in Arabidopsis xylem exudate, and its homologs. CLE2 and CLE3 genes responded to carbon-deficient conditions. Loss- and gain-of-function mutant analyses showed that CLE genes positively affected root sucrose level. Mutations in the CLE genes resulted in a high shoot/root ratio under sucrose-free conditions. Grafting experiments demonstrated the systemic effect of CLE peptide genes. These findings provide insights into the molecular basis for the relationship between roots and leaves in maintenance of the root sucrose levels and growth.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

High temperature (HT) causes male sterility and decreases crop yields. Our previous works have demonstrated that sugar and auxin signaling pathways, Gossypium hirsutum Casein kinase I (GhCKI), and DNA methylation are all involved in HT-induced male sterility in cotton. However, the signaling mechanisms leading to distinct GhCKI expression patterns induced by HT between HT-tolerant and HT-sensitive cotton anthers remain largely unknown. Here, we identified a GhCKI promoter (ProGhCKI) region that functions in response to HT in anthers and found the transcription factor GhMYB4 binds to this region to act as an upstream positive regulator of GhCKI. In the tapetum of early-stage cotton anthers, upregulated expression of GhMYB4 under HT and overexpressed GhMYB4 under normal temperature both led to severe male sterility phenotypes, coupled with enhanced expression of GhCKI. We also found that GhMYB4 interacts with GhMYB66 to form a heterodimer to enhance its binding to ProGhCKI. However, GhMYB66 showed an expression pattern similar to GhMYB4 under HT but did not directly bind to ProGhCKI. Furthermore, HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter, which enhanced the expression of GhMYB4 in tetrad stage anthers and promoted the formation of the GhMYB4/GhMYB66 heterodimer, which in turn elevated the transcription of GhCKI in the tapetum, leading to male sterility. Overall, we shed light on the GhMYB66-GhMYB4-GhCKI regulatory pathway in response to HT in cotton anthers.

Despite the known critical regulatory functions of microRNAs, histone modifications, and DNA methylation in reprograming plant epigenomes in response to pathogen infection, the molecular mechanisms underlying the tight coordination of these components remain poorly understood. Here, we show how Arabidopsis (Arabidopsis thaliana) miR778 coordinately modulates the root transcriptome, histone methylation, and DNA methylation via post-transcriptional regulation of the H3K9 methyltransferases SU(var)3-9 homolog 5 (SUVH5) and SUVH6 upon infection by the beet cyst nematode Heterodera schachtii. miR778 post-transcriptionally silences SUVH5 and SUVH6 upon nematode infection. Manipulation of the expression of miR778 and its two target genes significantly altered plant susceptibility to H. schachtii. RNA-seq analysis revealed a key role of SUVH5 and SUVH6 in reprograming the transcriptome of Arabidopsis roots upon H. schachtii infection. In addition, chromatin immunoprecipitation (ChIP)-seq analysis established SUVH5 and SUVH6 as the main enzymes mediating H3K9me2 deposition in Arabidopsis roots in response to nematode infection. ChIP-seq analysis also showed that these methyltransferases possess distinct DNA binding preferences in that they are targeting transposable elements under noninfected conditions and protein-coding genes in infected plants. Further analyses indicated that H3K9me2 deposition directed by SUVH5 and SUVH6 contributes to gene expression changes both in roots and in nematode feeding sites and preferentially associates with CG DNA methylation. Together, our results uncovered multi-layered epigenetic regulatory mechanisms coordinated by miR778 during Arabidopsis-H. schachtii interactions.

Comment in Plant Physiol. 2022 Aug 1;189(4):1893-1895.

The shoot apical meristem (SAM), which is formed during embryogenesis, generates leaves, stems, and floral organs during the plant life cycle. SAM development is controlled by SHOOT MERISTEMLESS (STM), a conserved Class I KNOX transcription factor that interacts with another subclass homeodomain protein, BELL, to form a heterodimer, which regulates gene expression at the transcriptional level in Arabidopsis (Arabidopsis thaliana). Meanwhile, SKI-INTERACTING PROTEIN (SKIP), a conserved protein in eukaryotes, works as both a splicing factor and as a transcriptional regulator in plants to control gene expression at the transcriptional and posttranscriptional levels by interacting with distinct partners. Here, we show that, similar to plants with a loss of function of STM, a loss of function of SKIP or the specific knockout of SKIP in the SAM region resulted in failed SAM development and the inability of the mutants to complete their life cycle. In comparison, Arabidopsis mutants that expressed SKIP specifically in the SAM region formed a normal SAM and were able to generate a shoot system, including leaves and floral organs. Further analysis confirmed that SKIP interacts with STM in planta and that SKIP and STM regulate the expression of a similar set of genes by binding to their promoters. In addition, STM also interacts with EARLY FLOWERING 7 (ELF7), a component of Polymerase-Associated Factor 1 complex, and mutation in ELF7 exhibits similar SAM defects to that of STM and SKIP. This work identifies a component of the STM transcriptional complex and reveals the mechanism underlying SKIP-mediated SAM formation in Arabidopsis.

Potassium (K+) is one of the essential macronutrients for plant growth and development. However, the available K+ concentration in soil is relatively low. Plant roots can perceive low K+ (LK) stress, then enhance high-affinity K+ uptake by activating H+-ATPases in root cells, but the mechanisms are still unclear. Here, we identified the receptor-like protein kinase Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) that is involved in LK response by regulating the Arabidopsis (Arabidopsis thaliana) plasma membrane H+-ATPase isoform 2 (AHA2). The bak1 mutant showed leaf chlorosis phenotype and reduced K+ content under LK conditions, which was due to the decline of K+ uptake capacity. BAK1 could directly interact with the AHA2 C terminus and phosphorylate T858 and T881, by which the H+ pump activity of AHA2 was enhanced. The bak1 aha2 double mutant also displayed a leaf chlorosis phenotype that was similar to their single mutants. The constitutively activated form AHA2Δ98 and phosphorylation-mimic form AHA2T858D or AHA2T881D could complement the LK sensitive phenotypes of both aha2 and bak1 mutants. Together, our data demonstrate that BAK1 phosphorylates AHA2 and enhances its activity, which subsequently promotes K+ uptake under LK conditions.

Comment in Plant Physiol. 2022 Aug 1;189(4):1887-1889.

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.

Comment in Plant Physiol. 2022 Aug 29;190(1):190-192.

Mangroves are frequently inundated with saline water and have evolved different anatomical and physiological mechanisms to filter and, in some species, excrete excess salt from the water they take up. Because salts impose osmotic stress, interspecific differences in salt tolerance and salt management strategy may influence physiological responses to drought throughout the entire plant hydraulic pathway, from roots to leaves. Here, we characterized embolism vulnerability simultaneously in leaves, stems, and roots of seedlings of two mangrove species (Avicennia marina and Bruguiera gymnorrhiza) along with turgor-loss points in roots and leaves and xylem anatomical traits. In both species, the water potentials causing 50% of total embolism were less negative in roots and leaves than they were in stems, but the water potentials causing incipient embolism (5%) were similar in roots, stems, and leaves. Stomatal closure in leaves and turgor loss in both leaves and roots occurred at water potentials only slightly less negative than the water potentials causing 5% of total embolism. Xylem anatomical traits were unrelated to vulnerability to embolism. Vulnerability segmentation may be important in limiting embolism spread into stems from more vulnerable roots and leaves. Interspecific differences in salt tolerance affected hydraulic traits from roots to leaves: the salt-secretor A. marina lost turgor at more negative water potentials and had more embolism-resistant xylem than the salt-excluder B. gymnorrhiza. Characterizing physiological thresholds of roots may help to explain recent mangrove mortality after drought and extended saltwater inundation.

Aluminum (Al) toxicity is one of the key factors limiting crop production in acid soils; however, little is known about its transcriptional regulation in plants. In this study, we characterized the role of a NAM, ATAF1/2, and cup-shaped cotyledon 2 (NAC) transcription factors (TFs), ANAC017, in the regulation of Al tolerance in Arabidopsis (Arabidopsis thaliana). ANAC017 was localized in the nucleus and exhibited constitutive expression in the root, stem, leaf, flower, and silique, although its expression and protein accumulation were repressed by Al stress. Loss of function of ANAC017 enhanced Al tolerance when compared with wild-type Col-0 and was accompanied by lower root and root cell wall Al content. Furthermore, both hemicellulose and xyloglucan content decreased in the anac017 mutants, indicating the possible interaction between ANAC017 and xyloglucan endotransglucosylase/hydrolase (XTH). Interestingly, the expression of XTH31, which is responsible for xyloglucan modification, was downregulated in the anac017 mutants regardless of Al supply, supporting the possible interaction between ANAC017 and XTH31. Yeast one-hybrid, dual-luciferase reporter assay, and chromatin immunoprecipitation-quantitative PCR analysis revealed that ANAC017 positively regulated the expression of XTH31 through directly binding to the XTH31 promoter region, and overexpression of XTH31 in the anac017 mutant background rescued its Al-tolerance phenotype. In conclusion, we identified that the tTF ANAC017 acts upstream of XTH31 to regulate Al tolerance in Arabidopsis.

Photosynthesis holds the promise of sustainable generation of useful products using light energy. Key to realizing this potential is the ability to rationally design photosynthesis to redirect energy and reductant derived from photons to desired products. Cytochrome P450s (P450s), which catalyze a broad array of reactions, have been engineered into a variety of photosynthetic organisms, where their activity has been shown to be photosynthesis-dependent, thus acting as heterologous sinks of electrons derived from photosynthesis. Furthermore, the addition of P450s can increase the photosynthetic capacity of the host organism. In this study, we developed this technology further using a P450 (CYP1A1) expressed in the cyanobacterium Synechococcus sp. PCC 7002. We show that rationally engineering photosynthesis by the removal of a competing electron sink, the respiratory terminal oxidase cytochrome c oxidase, increased the activity of CYP1A1. We provide evidence that this enhanced CYP1A1 activity was facilitated via an increase in the flux of electrons through Photosystem I. We also conducted a transcriptomic analysis on the designed strains to gain a more holistic understanding of how the cell responds to rational engineering. We describe a complex response including changes in expression of genes involved in photosynthesis and electron transfer linked to respiration. Specifically, the expression of CYP1A1 resulted in the reduction in expression of other natural electron dissipation pathways. This study emphasizes the potential for engineering photosynthetic organisms in biotechnology but also highlights the need to consider the broader impacts on cellular metabolism of any rationally induced changes.

Comment in Plant Physiol. 2022 Aug 29;190(1):193-195.

Comment in Plant Physiol. 2022 Aug 1;189(4):1904-1905.

Comment in Plant Physiol. 2022 Aug 1;189(4):1884-1886.

At the transition from vegetative to reproductive growth in rice (Oryza sativa), a developmental program change occurs, resulting in panicle (rice inflorescence) formation. The initial event of the transition is the change of the shoot apical meristem to an inflorescence meristem (IM), accompanied by a rapid increase in the meristem size. Suppression of leaf growth also occurs, resulting in the formation of bracts. The IM generates branch meristems (BMs), indeterminate meristems that reiteratively generate next-order meristems. All meristems eventually acquire a determinate spikelet meristem identity and terminate after producing a floret. ABERRANT PANICLE ORGANIZATION2 (APO2) is the rice ortholog of Arabidopsis (Arabidopsis thaliana) LEAFY (LFY), a plant-specific transcription factor (TF). APO2 is a positive regulator of panicle branch formation. Here, we show that APO2 is also required to increase the meristem size of the IM and suppress bract outgrowth. We identified genes directly and indirectly regulated by APO2 and identified APO2-binding sites. These analyses showed that APO2 directly controls known regulators of panicle development, including SQUAMOSA PROMOTER BINDING PROTEIN LIKE14 and NECK LEAF1. Furthermore, we revealed that a set of genes act as downstream regulators of APO2 in controlling meristem cell proliferation during reproductive transition, bract suppression, and panicle branch formation. Our findings indicate that APO2 acts as a master regulator of rice panicle development by regulating multiple steps in the reproductive transition through directly controlling a set of genes.

Erratum in Plant Physiol. 2022 Oct 27;190(3):2059.

Chinese jujube (Ziziphus jujuba) is an important fruit tree in China, and soil salinity is the main constraint affecting jujube production. It is unclear how arbuscular mycorrhizal (AM) symbiosis supports jujube adaptation to salt stress. Herein, we performed comparative physiological, ion flux, fatty acid (FA) metabolomic, and transcriptomic analyses to examine the mechanism of AM jujube responding to salt stress. AM seedlings showed better performance during salt stress. AM symbiosis altered phytohormonal levels: indole-3-acetic acid and abscisic acid contents were significantly increased in AM roots and reduced by salt stress. Mycorrhizal colonization enhanced root H+ efflux and K+ influx, while inducing expression of plasma membrane-type ATPase 7 (ZjAHA7) and high-affinity K+ transporter 2 (ZjHAK2) in roots. High K+/Na+ homeostasis was maintained throughout salt exposure. FA content was elevated in AM leaves as well as roots, especially for palmitic acid, oleic acid, trans oleic acid, and linoleic acid, and similar effects were also observed in AM poplar (Populus. alba × Populus. glandulosa cv. 84K) and Medicago truncatula, indicating AM symbiosis elevating FA levels could be a conserved physiological effect. Gene co-expression network analyses uncovered a core gene set including 267 genes in roots associated with AM symbiosis and conserved transcriptional responses, for example, FA metabolism, phytohormone signal transduction, SNARE interaction in vesicular transport, and biotin metabolism. In contrast to widely up-regulated genes related to FA metabolism in AM roots, limited genes were affected in leaves. We propose a model of AM symbiosis-linked reprogramming of FA metabolism and provide a comprehensive insight into AM symbiosis with a woody species adaptation to salt stress.

Cystathionine-β-synthase (CBS) domains are found in proteins of all living organisms and have been proposed to play a role as energy sensors regulating protein activities through their adenosyl ligand binding capacity. In plants, members of the CBSX protein family carry a stand-alone pair of CBS domains. In Arabidopsis (Arabidopsis thaliana), CBSX1 and CBSX2 are targeted to plastids where they have been proposed to regulate thioredoxins (TRXs). TRXs are ubiquitous cysteine thiol oxido-reductases involved in the redox-based regulation of numerous enzymatic activities as well as in the regeneration of thiol-dependent peroxidases. In Arabidopsis, 10 TRX isoforms have been identified in plastids and divided into five sub-types. Here, we show that CBSX2 specifically inhibits the activities of m-type TRXs toward two chloroplast TRX-related targets. By testing activation of NADP-malate dehydrogenase and reduction of 2-Cys peroxiredoxin, we found that TRXm1/2 inhibition by CBSX2 was alleviated in the presence of AMP or ATP. We also determined, by pull-down assays, a direct interaction of CBSX2 with reduced TRXm1 and m2 that was abolished in the presence of adenosyl ligands. In addition, we report that, compared with wild-type plants, the Arabidopsis T-DNA double mutant cbsx1 cbsx2 exhibits growth and chlorophyll accumulation defects in cold conditions, suggesting a function of plastidial CBSX proteins in plant stress adaptation. Together, our results show an energy-sensing regulation of plastid TRX m activities by CBSX, possibly allowing a feedback regulation of ATP homeostasis via activation of cyclic electron flow in the chloroplast, to maintain a high energy level for optimal growth.

Eukaryotic precursor mRNAs often harbor noncoding introns that must be removed prior to translation. Accurate splicing of precursor messenger RNA depends on placement and assembly of small nuclear ribonucleoprotein (snRNP) sub-complexes of the spliceosome. Yeast (Saccharomyces cerevisiae) studies established a role in splice-site selection for PRE-RNA PROCESSING8 (PRP8), a conserved spliceosome scaffolding protein of the U5 snRNP. However, analogous splice-site selection studies in multicellular eukaryotes are lacking. Such studies are crucial for a comprehensive understanding of alternative splicing, which is extensive in plants and animals but limited in yeast. In this work, we describe an Arabidopsis (Arabidopsis thaliana) prp8a mutant that modulates splice-site selection. We isolated prp8a-14 from a screen for suppressors of pex14-6, which carries a splice-site mutation in the PEROXIN14 (PEX14) peroxisome biogenesis gene. To elucidate Arabidopsis PRP8A function in spliceosome fidelity, we combined prp8a-14 with various pex14 splice-site mutations and monitored the double mutants for physiological and molecular consequences of dysfunctional and functional peroxisomes that correspond to impaired and recovered splicing, respectively. prp8a-14 restored splicing and PEX14 function to alleles with mutations in the exonic guanine of the 5'-splice site but did not restore splicing or function to alleles with mutations in the intronic guanine of 5'- or 3'-splice sites. We used RNA-seq to reveal the systemic impact of prp8a-14 and found hundreds of differentially spliced transcripts and thousands of transcripts with significantly altered levels. Among differentially spliced transcripts, prp8a-14 significantly altered 5'- and 3'-splice-site utilization to favor sites resulting in shorter introns. This study provides a genetic platform for probing splicing in plants and hints at a role for plant PRP8 in splice-site selection.

Conjugation of the small ubiquitin-like modifier (SUMO) peptide to target proteins is an important post-translational modification. SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (MdSIZ1) is an apple (Malus domestica Borkh). SUMO E3 ligase that mediates sumoylation of its targets during plant growth and development under adverse environmental conditions. However, it is unclear how MdSIZ1 senses the various environmental signals and whether sumoylation is regulated at the transcriptional level. In this study, we analyzed the MdSIZ1 promoter and found that it contained an MYB binding site (MBS) motif that was essential for the response of MdSIZ1 to low temperature (LT) and drought. Subsequently, we used yeast one-hybridization screening to demonstrate that a MYB transcription factor, MdMYB2, directly bound to the MBS motif in the MdSIZ1 promoter. Phenotypic characterization of MdMYB2 and MdSIZ1 suggested that the expression of both MdMYB2 and MdSIZ1 substantially improved cold tolerance in plants. MdMYB2 was induced by LT and further activated the expression of MdSIZ1, thereby promoting the sumoylation of MdMYB1, a key regulator of anthocyanin biosynthesis in apple. MdMYB2 promoted anthocyanin accumulation in apple fruits, apple calli, and Arabidopsis (Arabidopsis thaliana) in an MdSIZ1-dependent manner. In addition, the interaction of MdMYB2 and the MdSIZ1 promoter substantially improved plant tolerance to cold stress. Taken together, our findings reveal an important role for transcriptional regulation of sumoylation and provide insights into plant anthocyanin biosynthesis regulation mechanisms and stress response.

The formation of diploid gametes through chromosome doubling is a major mechanism of polyploidization, diversification, and speciation in plants. Unfavorable climate conditions can induce or stimulate the production of diploid gametes during meiosis. Here, we demonstrated that heat shock stress (38°C for 3 or 6 h) induced 2n pollen formation, and we generated 42 triploids derived from heat shock-induced 2n pollen of Populus canescens. Meiotic analysis of treated pollen mother cells revealed that induced 2n pollen originated from the complete loss of meiosis II (MII). Among the 42 triploids, 38 triploids derived from second division restitution (SDR)-type 2n pollen and 4 triploids derived from first division restitution-type 2n pollen were verified using simple sequence repeats (SSR) molecular markers. Twenty-two differentially expressed genes related to the cell cycle were identified and characterized by expression profile analysis. Among them was POPTR_0002s08020g (PtCYCA1;2), which encodes a type A Cyclin CYCA1;2 that is required for the meiosis I (MI) to MII transition. After male flower buds were exposed to heat shock, a significant reduction was detected in PtCYCA1;2 expression. We inferred that the failure of MI-to-MII transitions might be associated with downregulated expression of PtCYCA1;2, leading to the formation of SDR-type 2n pollen. Our findings provide insights into mechanisms of heat shock-induced 2n pollen formation in a woody plant and verify that sensitivity to environmental stress has evolutionary importance in terms of polyploidization.

Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gβ, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gβ1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the daytime and remobilize it to support maintenance and growth at night. Starch accumulation is increased when carbon is in short supply, for example, in short photoperiods. Mobilization is paced to exhaust starch around dawn, as anticipated by the circadian clock. This diel pattern of turnover is largely robust against loss of day, dawn, dusk, or evening clock components. Here, we investigated diel starch turnover in the triple circadian clock mutant lhy cca1 elf3, which lacks the LATE ELONGATED HYPOCOTYL and the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) dawn components and the EARLY FLOWERING3 (ELF3) evening components of the circadian clock. The diel oscillations of transcripts for the remaining clock components and related genes like REVEILLE and PHYTOCHROME-INTERACING FACTOR family members exhibited attenuated amplitudes and altered peak time, weakened dawn dominance, and decreased robustness against changes in the external light-dark cycle. The triple mutant was unable to increase starch accumulation in short photoperiods. However, it was still able to pace starch mobilization to around dawn in different photoperiods and growth irradiances and to around 24 h after the previous dawn in T17 and T28 cycles. The triple mutant was able to slow down starch mobilization after a sudden low-light day or a sudden early dusk, although in the latter case it did not fully compensate for the lengthened night. Overall, there was a slight trend to less linear mobilization of starch. Thus, starch mobilization can be paced rather robustly to dawn despite a major disruption of the transcriptional clock. It is proposed that temporal information can be delivered from clock components or a semi-autonomous oscillator.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

Brassinosteroids (BRs) are an important group of plant steroid hormones that regulate growth and development. Several members of the SMALL AUXIN UP RNA (SAUR) family have roles in BR-regulated hypocotyl elongation and root growth. However, the mechanisms are unclear. Here, we show in Arabidopsis (Arabidopsis thaliana) that SAUR15 interacts with cell surface receptor-like kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) in BR-treated plants, resulting in enhanced BRI1 phosphorylation status and recruitment of the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1. Genetic and phenotypic assays indicated that the SAUR15 effect on BRI1 can be uncoupled from BRASSINOSTEROID INSENSITIVE 2 activity. Instead, we show that SAUR15 promotes BRI1 direct activation of plasma membrane H+-ATPase (PM H+-ATPase) via phosphorylation. Consequently, SAUR15-BRI1-PM H+-ATPase acts as a direct, PM-based mode of BR signaling that drives cell expansion to promote the growth and development of various organs. These data define an alternate mode of BR signaling in plants.

Comment in Plant Physiol. 2022 Aug 1;189(4):1898-1900.

Comment in Plant Physiol. 2022 Aug 1;189(4):1901-1903.