One mechanism plants use to tolerate high salinity is the deposition of cutin and suberin to form apoplastic barriers that limit the influx of ions. However, the mechanism underlying barrier formation under salt stress is unclear. Here, we characterized the glycerol-3-phosphate acyltransferase (GPAT) family gene TaGPAT6, encoding a protein involved in cutin and suberin biosynthesis for apoplastic barrier formation in wheat (Triticum aestivum). TaGPAT6 has both acyltransferase and phosphatase activities, which are responsible for the synthesis of sn-2-monoacylglycerol (sn-2 MAG), the precursor of cutin and suberin. Overexpressing TaGPAT6 promoted the deposition of cutin and suberin in the seed coat and the outside layers of root tip cells and enhanced salt tolerance by reducing sodium ion accumulation within cells. By contrast,TaGPAT6 knockout mutants showed increased sensitivity to salt stress due to reduced cutin and suberin deposition and enhanced sodium ion accumulation. Yeast-one-hybrid and electrophoretic mobility shift assays identified TaABI5 as the upstream regulator of TaGPAT6. TaABI5 knockout mutants showed suppressed expression of TaGPAT6 and decreased barrier formation in the seed coat. These results indicate that TaGPAT6 is involved in cutin and suberin biosynthesis and the resulting formation of an apoplastic barrier that enhances salt tolerance in wheat.

Members of the cyclic nucleotide-gated channel (CNGC) proteins are reportedly involved in a variety of biotic and abiotic responses and stomatal movement. However, it is unknown if and how a single member could regulate multiple responses. Here we characterized three closely related CNGC genes in rice,OsCNGC14,OsCNGC15 and OsCNGC16, to determine whether they function in multiple abiotic stresses. The loss-of-function mutants of each of these three genes had reduced calcium ion (Ca²⁺) influx and slower stomatal closure in response to heat, chilling, drought and the stress hormone abscisic acid (ABA). These mutants also had reduced tolerance to heat, chilling and drought compared with the wild-type. Conversely, overexpression of OsCNGC16 led to a more rapid stomatal closure response to stresses and enhanced tolerance to heat, chilling and drought. The tight association of stomatal closure and stress tolerance strongly suggests that tolerance to multiple abiotic stresses conferred by these OsCNGC genes results at least partially from their regulation of stomatal movement. In addition, physical interactions were observed among the three OsCNGC proteins but not with a distantly related CNGC, suggesting the formation of hetero-oligomers among themselves. This study unveils the crucial role of OsCNGC14, 15 and 16 proteins in stomatal response and tolerance to multiple stresses, suggesting a mechanism of tolerance to multiple stresses that involves calcium influx and stomatal movement regulation.

Lodging reduces grain yield and quality in cereal crops. Lodging resistance is affected by the strength of the culm, which is influenced by the culm diameter, culm wall thickness, and cell wall composition. To explore the genetic architecture of culm diameter in rice (Oryza sativa), we conducted a genome-wide association study (GWAS). We identified STRONG CULM 2 (STRONG2), which encodes the mannan synthase CSLA5, and showed that plants that overexpressed this gene had increased culm diameter and improved lodging resistance. STRONG2 appears to increase the levels of cell wall components, such as mannose and cellulose, thereby enhancing sclerenchyma development in stems. SNP14931253 in the STRONG2 promoter contributes to variation in STRONG2 expression in natural germplasms and the transcription factor MYB61 directly activates STRONG2 expression. Furthermore,STRONG2 overexpressing plants produced significantly more grains per panicle and heavier grains than the wild-type plants. These results demonstrate that the MYB61–STRONG2 module positively regulates culm diameter and lodging resistance, information that could guide breeding efforts for improved yield in rice.

Stomata are epidermal pores that are essential for water evaporation and gas exchange in plants. Stomatal development is orchestrated by intrinsic developmental programs, hormonal controls, and environmental cues. The steroid hormone brassinosteroid (BR) inhibits stomatal lineage progression by regulating BIN2 and BSL proteins in leaves. Notably, BR is known to promote stomatal development in hypocotyls as opposed to leaves; however, its molecular mechanism remains elusive. Here, we show that BR signaling has a dual regulatory role in controlling stomatal development in Arabidopsis hypocotyls. We found that brassinolide (BL; the most active BR) regulates stomatal development differently in a concentration-dependent manner. At low and moderate concentrations, BL promoted stomatal formation by upregulating the expression of SPEECHLESS (SPCH) and its target genes independently of BIN2 regulation. In contrast, high concentrations of BL and bikinin, which is a specific inhibitor of BIN2 and its homologs, significantly reduced stomatal formation. Genetic analyses revealed that BIN2 regulates stomatal development in hypocotyls through molecular mechanisms distinct from the regulatory mechanism of the cotyledons. In hypocotyls, BIN2 promoted stomatal development by inactivating BZR1, which suppresses the expression of SPCH and its target genes. Taken together, our results suggest that BR precisely coordinates the stomatal development of hypocotyls using an antagonistic control of SPCH expression via BZR1-dependent and BZR1-independent transcriptional regulation.

The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus. However, due to their complex genetic backgrounds, the origins and evolution of many citrus species remain unclear. We assembled de novo the 294-Mbp chromosome-level genome of a more than 200-year-old primitive papeda (DYC002). Comparison between the two sets of homologous chromosomes of the haplotype-resolved genome revealed 1.2% intragenomic variations, including 1.75 million SNPs, 149, 471 insertions and 154, 215 deletions. Using this genome as a reference, we resequenced and performed population and phylogenetic analyses of 378 representative citrus accessions. Our study confirmed that the primary origin center of core Citrus species is in South China, particularly in the Himalaya–Hengduan Mountains. Papeda species are an ancient Citrus type compared with C. ichangensis. We found that the evolution of the Citrus genus followed two radiations through two routes (to East China and Southeast Asia) along river systems. Evidence for the origin and evolution of some individual citrus species was provided. Papeda probably played an important role in the origins of Australian finger lime, citrons, Honghe papeda and pummelos; Ichang papeda originated from Yuanjiang city of Yunnan Province, China, and C. mangshanensis has a close relationship with kumquat and Ichang papeda. Moreover, the Hunan and Guangdong Provinces of China are predicted to be the origin center of mandarin, sweet orange and sour orange. Additionally, our study revealed that fruit bitterness was significantly selected against during citrus domestication. Taken together, this study provides new insight into the origin and evolution of citrus species and may serve as a valuable genomic resource for citrus breeding and improvement.

Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2-type zinc-finger transcription factor,CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α-branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB,CitHYD, and CitNCED2, promoting β-branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4-mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4-mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops.

Phosphorus (P) is an essential macronutrient for plant growth and development. Vacuoles play a crucial role in inorganic phosphate (Pi) storage and remobilization in plants. However, the physiological function of vacuolar phosphate efflux transporters in plant Pi remobilization remains obscure. Here, we identified three ZmVPE genes (ZmVPE1,ZmVPE2a,ZmVPE2b) by combining them with transcriptome and quantitative real-time polymerase chain reaction (PCR) analyses, showing a relatively higher expression in older leaves than in younger leaves in maize. Moreover, the expression of the ZmVPEs was triggered by Pi deficiency and abscisic acid. ZmVPEs were localized to the vacuolar membrane and responsible for vacuolar Pi efflux. Compared with the wild-type, Pi remobilization from older to younger leaves was enhanced in ZmVPE-overexpression lines. zmvpe2a mutants displayed an increase in the total P and Pi concentrations in older leaves, but a decrease in younger leaves. In rice, Pi remobilization was impaired in the osvpe1osvpe2 double mutant and enhanced in OsVPE-overexpression plants, suggesting conserved functions of VPEs in modulating Pi homeostasis and remobilization in crop plants. Taken together, our findings revealed a novel mechanism underlying Pi remobilization from older to younger leaves mediated by plant vacuolar Pi efflux transporters, facilitating the development of Pi-efficient crop plants.

Fruit taste quality is greatly influenced by the content of soluble sugars, which are predominantly stored in the vacuolar lumen. However, the accumulation and regulation mechanisms of sugars in most fruits remain unclear. Recently, we established the citrus fruit vacuole proteome and discovered the major transporters localized in the vacuole membrane. Here, we demonstrated that the expression of tonoplast sugar transporter 2 (CsTST2) is closely associated with sugar accumulation during sweet orange (Citrus sinensis) ripening. It was further demonstrated that CsTST2 had the function of transporting hexose and sucrose into the vacuole. Overexpression of CsTST2 resulted in an elevation of sugar content in citrus juice sac, calli, and tomato fruit, whereas the downregulation of its expression led to the reduction in sugar levels. CsTST2 was identified as interacting with CsCIPK23, which binds to the upstream calcium signal sensor protein CsCBL1. The phosphorylation of the three serine residues (Ser277, Ser337, and Ser354) in the loop region of CsTST2 by CsCIPK23 is crucial for maintaining the sugar transport activity of CsTST2. Additionally, the expression of CsCIPK23 is positively correlated with sugar content. Genetic evidence further confirmed that calcium and CsCIPK23-mediated increase in sugar accumulation depends on CsTST2 and its phosphorylation level. These findings not only unveil the functional mechanism of CsTST2 in sugar accumulation, but also explore a vital calcium signal regulation module of CsCBL1/CIPK23 for citrus sweetness quality.

Besides playing a crucial role in plant immunity via the nonexpressor of pathogenesis-related (NPR) proteins, increasing evidence shows that salicylic acid (SA) can also regulate plant root growth. However, the transcriptional regulatory network controlling this SA response in plant roots is still unclear. Here, we found that NPR1 and WRKY45, the central regulators of SA response in rice leaves, control only a reduced sector of the root SA signaling network. We demonstrated that SA attenuates root growth via a novel NPR1/WRKY45-independent pathway. Furthermore, using regulatory network analysis and mutant characterization, we identified a set of new NPR1/WRKY45-independent regulators that conservedly modulate the root development and root-associated microbiota composition in both Oryza sativa (monocot) and Arabidopsis thaliana (dicot) in response to SA. Our results established the SA signaling as a central element regulating plant root functions under ecologically relevant conditions. These results provide new insights to understand how regulatory networks control plant responses to abiotic and biotic stresses.

Starch biosynthesis is a critical factor in wheat (Triticum aestivum L.) quality and yield. However, the full scope of its regulation is not fully understood. Here we report that TaDL interacts with TaB3 and TaNF-YB1 to synergistically regulate starch biosynthesis and quality in wheat. Genome-edited tadl mutant lines had smaller and lighter grains with lower total starch and amylose contents compared to wild type (WT). Correspondingly, the transcript levels of starch biosynthesis-related genes, including TaSUS1,TaSUS2,TaAGPL2,TaSBEIIa,TaGBSSII, and TaSWEET2a, were markedly lower at 15 d after flowering (DAF) in tadl mutants. TaDL physically interacted with TaB3 and TaNF-YB1 and activated the transcription of TaSUS2 and TaAGPL2 through direct binding to their promoter regions. A null mutant of TaB3 also affected grain filling, with phenotypes similar to those of tadl mutants, whereas overexpression of TaNF-YB1 promoted grain filling. Our study demonstrated that TaDL plays an essential role in starch biosynthesis and identified an elite allele (TaDL-BI) associated with starch content, providing insights into the underlying molecular mechanism of wheat grain filling, which may be useful in breeding of high-yielding wheat and quality improvement.

Light is a vital environmental signal that regulates the expression of plastid genes. Plastids are crucial organelles that respond to light, but the effects of light on plastid RNA processing following transcription remain unclear. In this study, we systematically examined the influence of light exposure on plastid RNA processing, focusing on RNA splicing and RNA editing. We demonstrated that light promotes the splicing of transcripts from the plastid genes rps12,ndhA,atpF,petB, and rpl2. Additionally, light increased the editing rate of the accD transcript at nucleotide 794 (accD-794) and the ndhF transcript at nucleotide 290 (ndhF-290), while decreasing the editing rate of the clpP transcript at nucleotide 559 (clpP-559). We have identified key regulators of signaling pathways, such as CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), ELONGATED HYPOCOTYL 5 (HY5), and PHYTOCHROME-INTERACTING FACTORs (PIFs), as important players in the regulation of plastid RNA splicing and editing. Notably, COP1 was required for GENOMES UNCOUPLED1 (GUN1)-dependent repression of clpP-559 editing in the light. We showed that HY5 and PIF1 bind to the promoters of nuclear genes encoding plastid-localized RNA processing factors in a light-dependent manner. This study provides insight into the mechanisms underlying light-mediated post-transcriptional regulation of plastid gene expression.

Glycosylation, a prevalent post-translational modification in eukaryotic secreted and membrane-associated proteins, plays a pivotal role in diverse physiological and pathological processes. Although UDP-N-acetylglucosamine (UDP-GlcNAc) is essential for this modification, the specific glycosylation mechanisms during plant leaf senescence and defense responses remain poorly understood. In our research, we identified a novel rice mutant named rbb1 (resistance to blast and bacterial blight1), exhibiting broad-spectrum disease resistance. This mutant phenotype results from a loss-of-function mutation in the gene encoding glucosamine-6-phosphate acetyltransferase, an important enzyme in D-glucosamine 6-phosphate acetylation. The rbb1 mutant demonstrates enhanced defense responses, evident in increased resistance to rice blast and bacterial blight, along with the upregulation of defense-response genes. Various biochemical markers indicate an activated defense mechanism in the rbb1 mutant, such as elevated levels of reactive oxygen species and malondialdehyde, reduced enzyme activity and UDP-GlcNAc content, and decreased expression of N-glycan and O-glycan modifying proteins. Moreover, proteome analysis of N-glycosylation modifications reveals alterations in the N-glycosylation of several disease-resistance-related proteins, with a significant reduction in Prx4 and Prx13 in rbb1-1. Additionally, the knockout of Prx4 or Prx13 also enhances resistance to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (M. oryzae). This study uncovers a novel mechanism of defense response in rice, suggesting potential targets for the development of disease-resistant varieties.

The phytohormone jasmonates (JAs) regulate plant growth and defense responses. The reproductive organs of flowers are devastated by insect herbivores. However, the molecular mechanisms of floral defense remain largely unknown. Here, we found that the Arabidopsis JA receptor CORONATINE INSENSITIVE1 (COI1) and its substrates JA ZIM-domain (JAZ) repressors, and the mediator subunit MEDIATOR25-based MED25–MYC–MYB (MMM) complexes, including MYC2/3/4/5 and MYB28/29/76, mediated floral defense against the insects Helicoverpa armigera,Spodoptera exigua, and Spodoptera frugiperda. The flower-specific IIIa bHLH factors ABORTED MICROSPORES (AMS) and DYSFUNCTIONAL TAPETUM 1 (DYT1) were JAZ-interaction proteins. They interacted with members of the MMM complexes, inhibited the transcriptional activity of MYC2 and MYB28, and repressed floral defense against insects. AMS and DYT1 recruited the flower-specific MYB21/24, and these MYBs interacted with members of MMM complexes, inhibited the MYC2–MYB28 function, and suppressed floral defense against insects. Our study revealed that the JA–COI1–JAZ–MMM pathway mediated flower defense, and the AMS/DYT1–MYB21/24 module antagonized the MMM complexes to repress floral defense against insects.