SALT OVERLY SENSITIVE2 stabilizes phytochrome-interacting factors PIF4 and PIF5 to promote Arabidopsis shade avoidance.

Sun-loving plants trigger the shade avoidance syndrome (SAS) to compete against their neighbors for sunlight. Phytochromes are plant red (R) and far-red (FR) light photoreceptors that play a major role in perceiving the shading signals and triggering SAS. Shade induces a reduction in the level of active phytochrome B (phyB), thus increasing the abundance of PHYTOCHROME-INTERACTING FACTORS (PIFs), a group of growth-promoting transcription factors. However, whether other factors are involved in modulating PIF activity in the shade remains largely obscure. Here, we show that SALT OVERLY SENSITIVE2 (SOS2), a protein kinase essential for salt tolerance, positively regulates SAS in Arabidopsis thaliana. SOS2 directly phosphorylates PIF4 and PIF5 at a serine residue close to their conserved motif for binding to active phyB. This phosphorylation thus decreases their interaction with phyB and posttranslationally promotes PIF4 and PIF5 protein accumulation. Notably, the role of SOS2 in regulating PIF4 and PIF5 protein abundance and SAS is more prominent under salt stress. Moreover, phyA and phyB physically interact with SOS2 and promote SOS2 kinase activity in the light. Collectively, our study uncovers an unexpected role of salt-activated SOS2 in promoting SAS by modulating the phyB-PIF module, providing insight into the coordinated response of plants to salt stress and shade.

Reconstitution of phytochrome A-mediated light modulation of the ABA signaling pathways in yeast.

Abscisic acid (ABA), a classical plant hormone, plays an essential role in plant adaptation to environmental stresses. The ABA signaling mechanisms have been extensively investigated, and it was shown that the PYR1 (PYRABACTIN RESISTANCE1)/PYL (PYR1-LIKE)/RCAR (REGULATORY COMPONENT OF ABA RECEPTOR) ABA receptors, the PP2C coreceptors, and the SnRK2 protein kinases constitute the core ABA signaling module responsible for ABA perception and initiation of downstream responses. We recently showed that ABA signaling is modulated by light signals, but the underlying molecular mechanisms remain largely obscure. In this study, we established a system in yeast cells that was not only successful in reconstituting a complete ABA signaling pathway, from hormone perception to ABA-responsive gene expression, but also suitable for functionally characterizing the regulatory roles of additional factors of ABA signaling. Using this system, we analyzed the roles of several light signaling components, including the red and far-red light photoreceptors phytochrome A (phyA) and phyB, and the photomorphogenic central repressor COP1, in the regulation of ABA signaling. Our results showed that both phyA and phyB negatively regulated ABA signaling, whereas COP1 positively regulated ABA signaling in yeast cells. Further analyses showed that photoactivated phyA interacted with the ABA coreceptors ABI1 and ABI2 to decrease their interactions with the ABA receptor PYR1. Together, data from our reconstituted yeast ABA signaling system provide evidence that photoactivated photoreceptors attenuate ABA signaling by directly interacting with the key components of the core ABA signaling module, thus conferring enhanced ABA tolerance to light-grown plants.

Molecular mechanisms underlying coordinated responses of plants to shade and environmental stresses.

Shade avoidance syndrome (SAS) is triggered by a low ratio of red (R) to far-red (FR) light (R/FR ratio), which is caused by neighbor detection and/or canopy shade. In order to compete for the limited light, plants elongate hypocotyls and petioles by deactivating phytochrome B (phyB), a major R light photoreceptor, thus releasing its inhibition of the growth-promoting transcription factors PHYTOCHROME-INTERACTING FACTORs. Under natural conditions, plants must cope with abiotic stresses such as drought, soil salinity, and extreme temperatures, and biotic stresses such as pathogens and pests. Plants have evolved sophisticated mechanisms to simultaneously deal with multiple environmental stresses. In this review, we will summarize recent major advances in our understanding of how plants coordinately respond to shade and environmental stresses, and will also discuss the important questions for future research. A deep understanding of how plants synergistically respond to shade together with abiotic and biotic stresses will facilitate the design and breeding of new crop varieties with enhanced tolerance to high-density planting and environmental stresses.

COP1 positively regulates ABA signaling during Arabidopsis seedling growth in darkness by mediating ABA-induced ABI5 accumulation.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a well-characterized E3 ubiquitin ligase, is a central repressor of seedling photomorphogenic development in darkness. However, whether COP1 is involved in modulating abscisic acid (ABA) signaling in darkness remains largely obscure. Here, we report that COP1 is a positive regulator of ABA signaling during Arabidopsis seedling growth in the dark. COP1 mediates ABA-induced accumulation of ABI5, a transcription factor playing a key role in ABA signaling, through transcriptional and post-translational regulatory mechanisms. We further show that COP1 physically interacts with ABA-hypersensitive DCAF1 (ABD1), a substrate receptor of the CUL4-DDB1 E3 ligase targeting ABI5 for degradation. Accordingly, COP1 directly ubiquitinates ABD1 in vitro, and negatively regulates ABD1 protein abundance in vivo in the dark but not in the light. Therefore, COP1 promotes ABI5 protein stability post-translationally in darkness by destabilizing ABD1 in response to ABA. Interestingly, we reveal that ABA induces the nuclear accumulation of COP1 in darkness, thus enhancing its activity in propagating the ABA signal. Together, our study uncovers that COP1 modulates ABA signaling during seedling growth in darkness by mediating ABA-induced ABI5 accumulation, demonstrating that plants adjust their ABA signaling mechanisms according to their light environment.

Mutual upregulation of HY5 and TZP in mediating phytochrome A signaling.

Phytochrome A (phyA) is the far-red (FR) light photoreceptor in plants that is essential for seedling de-etiolation under FR-rich environments, such as canopy shade. TANDEM ZINC-FINGER/PLUS3 (TZP) was recently identified as a key component of phyA signal transduction in Arabidopsis thaliana; however, how TZP is integrated into the phyA signaling networks remains largely obscure. Here, we demonstrate that ELONGATED HYPOCOTYL5 (HY5), a well-characterized transcription factor promoting photomorphogenesis, mediates FR light induction of TZP expression by directly binding to a G-box motif in the TZP promoter. Furthermore, TZP physically interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), an E3 ubiquitin ligase targeting HY5 for 26S proteasome-mediated degradation, and this interaction inhibits COP1 interaction with HY5. Consistent with those results, TZP post-translationally promotes HY5 protein stability in FR light, and in turn, TZP protein itself is destabilized by COP1 in both dark and FR light conditions. Moreover, tzp hy5 double mutants display an additive phenotype relative to their respective single mutants under high FR light intensities, indicating that TZP and HY5 also function in largely independent pathways. Together, our data demonstrate that HY5 and TZP mutually upregulate each other in transmitting the FR light signal, thus providing insights into the complicated but delicate control of phyA signaling networks.

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.

DELAYED GREENING 409 encodes a dual-localized pentatricopeptide repeat protein required for chloroplast and mitochondrial development.

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

MYB30 Is a Key Negative Regulator of Arabidopsis Photomorphogenic Development That Promotes PIF4 and PIF5 Protein Accumulation in the Light.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants, and PHYTOCHROME-INTERACTING FACTORS (PIFs) are a group of basic helix-loop-helix family transcription factors that play central roles in repressing photomorphogenesis. Here, we report that MYB30, an R2R3-MYB family transcription factor, acts as a negative regulator of photomorphogenesis in Arabidopsis (Arabidopsis thaliana). We show that MYB30 preferentially interacts with the Pfr (active) forms of the phytochrome A (phyA) and phytochrome B (phyB) holoproteins and that MYB30 levels are induced by phyA and phyB in the light. It was previously shown that phytochromes induce rapid phosphorylation and degradation of PIFs upon R light exposure. Our current data indicate that MYB30 promotes PIF4 and PIF5 protein reaccumulation under prolonged R light irradiation by directly binding to their promoters to induce their expression and by inhibiting the interaction of PIF4 and PIF5 with the Pfr form of phyB. In addition, our data indicate that MYB30 interacts with PIFs and that they act additively to repress photomorphogenesis. In summary, our study demonstrates that MYB30 negatively regulates Arabidopsis photomorphogenic development by acting to promote PIF4 and PIF5 protein accumulation under prolonged R light irradiation, thus providing new insights into the complicated but delicate control of PIFs in the responses of plants to their dynamic light environment.

Analytical Methods for Oxalate Quantification: The Ubiquitous Organic Anion.

Oxalate is a divalent organic anion that affects many biological and commercial processes. It is derived from plant sources, such as spinach, rhubarb, tea, cacao, nuts, and beans, and therefore is commonly found in raw or processed food products. Oxalate can also be made endogenously by humans and other mammals as a byproduct of hepatic enzymatic reactions. It is theorized that plants use oxalate to store calcium and protect against herbivory. Clinically, oxalate is best known to be a major component of kidney stones, which commonly contain calcium oxalate crystals. Oxalate can induce an inflammatory response that decreases the immune system's ability to remove renal crystals. When formulated with platinum as oxaliplatin (an anticancer drug), oxalate has been proposed to cause neurotoxicity and nerve pain. There are many sectors of industry that are hampered by oxalate, and others that depend on it. For example, calcium oxalate is troublesome in the pulp industry and the alumina industry as it deposits on machinery. On the other hand, oxalate is a common active component of rust removal and cleaning products. Due to its ubiquity, there is interest in developing efficient methods to quantify oxalate. Over the past four decades, many diverse methods have been reported. These approaches include electrochemical detection, liquid chromatography or gas chromatography coupled with mass spectrometry, enzymatic degradation of oxalate with oxalate oxidase and detection of hydrogen peroxide produced, and indicator displacement-based methods employing fluorescent or UV light-absorbing compounds. Enhancements in sensitivity have been reported for both electrochemical and mass-spectrometry-based methods as recently as this year. Indicator-based methods have realized a surge in interest that continues to date. The diversity of these approaches, in terms of instrumentation, sample preparation, and sensitivity, has made it clear that no single method will work best for every purpose. This review describes the strengths and limitations of each method, and may serve as a reference for investigators to decide which approach is most suitable for their work.

Arabidopsis PP6 phosphatases dephosphorylate PIF proteins to repress photomorphogenesis.

The PHYTOCHROME-INTERACTING FACTORs (PIFs) play a central role in repressing photomorphogenesis, and phosphorylation mediates the stability of PIF proteins. Although the kinases responsible for PIF phosphorylation have been extensively studied, the phosphatases that dephosphorylate PIFs remain largely unknown. Here, we report that seedlings with mutations in FyPP1 and FyPP3, 2 genes encoding the catalytic subunits of protein phosphatase 6 (PP6), exhibited short hypocotyls and opened cotyledons in the dark, which resembled the photomorphogenic development of dark-grown pifq mutants. The hypocotyls of dark-grown sextuple mutant fypp1 fypp3 (f1 f3) pifq were shorter than those of parental mutants f1 f3 and pifq, indicating that PP6 phosphatases and PIFs function synergistically to repress photomorphogenesis in the dark. We showed that FyPPs directly interacted with PIF3 and PIF4, and PIF3 and PIF4 proteins exhibited mobility shifts in f1 f3 mutants, consistent with their hyperphosphorylation. Moreover, PIF4 was more rapidly degraded in f1 f3 mutants than in wild type after light exposure. Whole-genome transcriptomic analyses indicated that PP6 and PIFs coregulated many genes, and PP6 proteins may positively regulate PIF transcriptional activity. These data suggest that PP6 phosphatases may repress photomorphogenesis by controlling the stability and transcriptional activity of PIF proteins via regulating PIF phosphorylation.

Integration of light and temperature signaling pathways in plants.

As two of the most important environmental factors, light and temperature regulate almost all aspects of plant growth and development. Under natural conditions, light is accompanied by warm temperatures and darkness by cooler temperatures, suggesting that light and temperature are tightly associated signals for plants. Indeed, accumulating evidence shows that plants have evolved a wide range of mechanisms to simultaneously perceive and respond to dynamic changes in light and temperature. Notably, the photoreceptor phytochrome B (phyB) was recently shown to function as a thermosensor, thus reinforcing the notion that light and temperature signaling pathways are tightly associated in plants. In this review, we summarize and discuss the current understanding of the molecular mechanisms integrating light and temperature signaling pathways in plants, with the emphasis on recent progress in temperature sensing, light control of plant freezing tolerance, and thermomorphogenesis. We also discuss the questions that are crucial for a further understanding of the interactions between light and temperature signaling pathways in plants.

TANDEM ZINC-FINGER/PLUS3 Is a Key Component of Phytochrome A Signaling.

Phytochrome A (phyA) is the primary plant photoreceptor responsible for perceiving and mediating various responses to far-red (FR) light and is essential for survival in canopy shade. In this study, we identified two Arabidopsis thaliana mutants that grew longer hypocotyls in FR light. Genetic analyses showed that they were allelic and their FR phenotypes were caused by mutations in the gene named TANDEM ZINC-FINGER/PLUS3 (TZP), previously shown to encode a nuclear protein involved in blue light signaling and phyB-dependent regulation of photoperiodic flowering. We show that the expression of TZP is dramatically induced by light and that TZP proteins are differentially modified in different light conditions. Furthermore, we show that TZP interacts with both phyA and FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and regulates the abundance of phyA, FHY1, and ELONGATED HYPOCOTYL5 proteins in FR light. Moreover, our data indicate that TZP is required for the formation of a phosphorylated form of phyA in the nucleus in FR light. Together, our results identify TZP as a positive regulator of phyA signaling required for phosphorylation of the phyA photoreceptor, thus suggesting an important role of phosphorylated phyA in inducing the FR light response.

Hinge region of Arabidopsis phyA plays an important role in regulating phyA function.

Phytochrome A (phyA) is the only plant photoreceptor that perceives far-red light and then mediates various responses to this signal. Phosphorylation and dephosphorylation of oat phyA have been extensively studied, and it was shown that phosphorylation of a serine residue in the hinge region of oat phyA could regulate the interaction of phyA with its signal transducers. However, little is known about the role of the hinge region of Arabidopsis phyA. Here, we report that three sites in the hinge region of Arabidopsis phyA (i.e., S590, T593, and S602) are essential in regulating phyA function. Mutating all three of these sites to either alanines or aspartic acids impaired phyA function, changed the interactions of mutant phyA with FHY1 and FHL, and delayed the degradation of mutant phyA upon light exposure. Moreover, the in vivo formation of a phosphorylated phyA form was greatly affected by these mutations, while our data indicated that the abundance of this phosphorylated phyA form correlated well with the extent of phyA function, thus suggesting a pivotal role of the phosphorylated phyA in inducing the far-red light response. Taking these data together, our study reveals the important role of the hinge region of Arabidopsis phyA in regulating phyA phosphorylation and function, thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.

A large-scale circular RNA profiling reveals universal molecular mechanisms responsive to drought stress in maize and Arabidopsis.

Drought is a major abiotic stress that threatens global food security. Circular RNAs (circRNAs) are endogenous RNAs. How these molecules influence plant stress responses remains elusive. Here, a large-scale circRNA profiling identified 2174 and 1354 high-confidence circRNAs in maize and Arabidopsis, respectively, and most were differentially expressed in response to drought. A substantial number of drought-associated circRNA-hosting genes were involved in conserved or species-specific pathways in drought responses. Comparative analysis revealed that circRNA biogenesis was more complex in maize than in Arabidopsis. In most cases, maize circRNAs were negatively correlated with sRNA accumulation. In 368 maize inbred lines, the circRNA-hosting genes were enriched for single nucleotide polymorphisms (SNPs) associated with circRNA expression and drought tolerance, implying either important roles of circRNAs in maize drought responses or their potential use as biomarkers for breeding drought-tolerant maize. Additionally, the expression levels of circRNAs derived from drought-responsible genes encoding calcium-dependent protein kinase and cytokinin oxidase/dehydrogenase were significantly associated with drought tolerance of maize seedlings. Specifically, Arabidopsis plants overexpressing circGORK (Guard cell outward-rectifying K+ -channel) were hypersensitive to abscisic acid, but insensitive to drought, suggesting a positive role of circGORK in drought tolerance. We report the transcriptomic profiling and transgenic studies of circRNAs in plant drought responses, and provide evidence highlighting the universal molecular mechanisms involved in plant drought tolerance.

Cis-regulated alternative splicing divergence and its potential contribution to environmental responses in Arabidopsis.

Alternative splicing (AS) plays key roles in plant development and the responses of plants to environmental changes. However, the mechanisms underlying AS divergence (differential expression of transcript isoforms resulting from AS) in plant accessions and its contribution to responses to environmental stimuli remain unclear. In this study, we investigated genome-wide variation of AS in Arabidopsis thaliana accessions Col-0, Bur-0, C24, Kro-0 and Ler-1, as well as their F1 hybrids, and characterized the regulatory mechanisms for AS divergence by RNA sequencing. We found that most of the divergent AS events in Arabidopsis accessions were cis-regulated by sequence variation, including those in core splice site and splicing motifs. Many genes that differed in AS between Col-0 and Bur-0 were involved in stimulus responses. Further genome-wide association analyses of 22 environmental variables showed that single nucleotide polymorphisms influencing known splice site strength were also associated with environmental stress responses. These results demonstrate that cis-variation in genomic sequences among Arabidopsis accessions was the dominant contributor to AS divergence, and it may contribute to differences in environmental responses among Arabidopsis accessions.

Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.).

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%-67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi-allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene-free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off-target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi-dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.

Three BnaIAA7 homologs are involved in auxin/brassinosteroid-mediated plant morphogenesis in rapeseed (Brassica napus L.).

KEY MESSAGE: BnaIAA7 crosstalk with BR signaling is mediated by the interaction between BnaARF8 and BnaBZR1 to regulate rapeseed plant morphogenesis. Auxin (indole-3-acetic acid, IAA) and brassinosteroids (BRs) are essential regulators of plant morphogenesis. However, their roles in rapeseed have not been reported. Here, we identified an extremely dwarf1 (ed1) mutant of rapeseed that displays reduced stature, short hypocotyls, as well as wavy and curled leaves. We isolated ED1 by map-based cloning, and found that it encodes a protein homologous to AtIAA7. ED1 acts as a repressor of IAA signaling, and IAA induces its degradation through its degron motif. A genomic-synteny analysis revealed that ED1 has four homologs in rapeseed, but two were not expressed. Analyses of transcriptomes and of various mutant BnaIAA7s in transgenic plants revealed that the three expressed BnaIAA7 homologs had diverse expression patterns. ED1/BnaC05.IAA7 predominantly functioned in stem elongation, BnaA05.IAA7 was essential for reproduction, while BnaA03.IAA7 had the potential to reduce plant height. Physical interaction assays revealed that the three BnaIAA7 homologs interacted in different ways with BnaTIRs/AFBs and BnaARFs, which may regulate the development of specific organs. Furthermore, BnaARF8 could directly interact with the BnaIAA7s and BnaBZR1. We propose that BnaIAA7s interact with BR signaling via BnaARF8 and BnaBZR1 to regulate stem elongation in rapeseed.

Arabidopsis SAURs are critical for differential light regulation of the development of various organs.

During deetiolation of Arabidopsis seedlings, light promotes the expansion of cotyledons but inhibits the elongation of hypocotyls. The mechanism of this differential regulation of cell enlargement is unclear. Our organ-specific transcriptomic analysis identified 32 Small Auxin Up RNA (SAUR) genes whose transcripts were light-induced in cotyledons and/or repressed in hypocotyls. We therefore named these SAURs as lirSAURs Both overexpression and mutation analyses demonstrated that lirSAURs could promote cotyledon expansion and opening and enhance hypocotyl elongation, possibly by inhibiting phosphatase activity of D-clade type 2C protein phosphatases (PP2C-Ds). Light reduced auxin levels to down-regulate the expression of lirSAURs in hypocotyls. Further, phytochrome-interacting factors (PIFs) were shown to directly bind the genes encoding these SAURs and differentially regulate their expression in cotyledons and hypocotyls. Together, our study demonstrates that light mediates auxin levels and PIF stability to differentially regulate the expression of lirSAURs in cotyledons and hypocotyls, and these lirSAURs further mediate the differential growth of these two organs.

Genome-wide association study dissects the genetic bases of salt tolerance in maize seedlings.

Excess salinity is a natural stress that causes crop yield losses worldwide. The genetic bases of maize salt tolerance remain largely unknown. Here we investigated the survival rates of 445 maize natural accessions after salt treatments. A skewed distribution of the salt-tolerant phenotypes was observed in this population. Genome-wide association studies (GWAS) revealed 57 loci significantly associated with salt tolerance. Forty-nine candidate genes were detected from these loci. About 10% of these genes were co-localized with loci from QTL mapping. Forty four percent of the candidate genes were involved in stress responses, ABA signaling, stomata division, DNA binding/transcription regulation and auxin signaling, suggesting that they are key genetic mechanisms of maize salt tolerance. Transgenic studies showed that two genes, the salt-tolerance-associated-gene 4 (SAG4, GRMZM2G077295) and SAG6 (GRMZM2G106056), which encode a protein transport protein and the double-strand break repair protein MRE11, respectively, had positive roles in plant salt tolerance, and their salt-tolerant haplotypes were revealed. The genes we identified in this study provide a list of candidate targets for further study of maize salt tolerance, and of genetic markers and materials that may be used for breeding salt-tolerance in maize.

OsPKpα1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm.

Pyruvate kinase (PK) is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice (Oryza sativa) mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpα1, which encodes a plastidic isoform of PK (PKp). OsPKpα1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpα1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, at the early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein-protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts.