The organization of the microtubule cytoskeleton is critical for cell and organ morphogenesis. The evolutionarily conserved microtubule-severing enzyme KATANIN plays critical roles in microtubule organization in the plant and animal kingdoms. We previously used conical cell of Arabidopsis thaliana petals as a model system to investigate cortical microtubule organization and cell morphogenesis and determined that KATANIN promotes the formation of circumferential cortical microtubule arrays in conical cells. Here, we demonstrate that the conserved protein phosphatase PP2A interacts with and dephosphorylates KATANIN to promote the formation of circumferential cortical microtubule arrays in conical cells. KATANIN undergoes cycles of phosphorylation and dephosphorylation. Using co-immunoprecipitation coupled with mass spectrometry, we identified PP2A subunits as KATANIN-interacting proteins. Further biochemical studies showed that PP2A interacts with and dephosphorylates KATANIN to stabilize its cellular abundance. Similar to the katanin mutant, mutants for genes encoding PP2A subunits showed disordered cortical microtubule arrays and defective conical cell shape. Taken together, these findings identify PP2A as a regulator of conical cell shape and suggest that PP2A mediates KATANIN phospho-regulation during plant cell morphogenesis.

Flowering time is a fundamental factor determining the global distribution and final yield of rice (Oryza sativa). Although diverse flowering time genes have been reported in this crop, the transcriptional regulation of its key flowering genes are poorly understood. Here, we report that a basic leucine zipper transcription factor, bZIP71, functions as a flowering repressor. The overexpression of bZIP71 delays flowering, while the bzip71 mutant flowers early in both long-day and short-day conditions. A genetic analysis showed that the regulation of flowering by bZIP71 might be independent of Heading date 2 (Hd2), Hd4, and Hd5. Importantly, bZIP71 directly associates with the Early heading date 1 (Ehd1) promoter and represses its transcription, and genetically the function of bZIP71 is impaired in the ehd1 mutant. Moreover, bZIP71 interacts with major components of polycomb repressive complex 2 (PRC2), SET domain group protein 711 (SDG711), and Fertilization independent endosperm 2 (FIE2), through which bZIP71 regulates the H3K27me3 level of Ehd1. Taken together, we present a transcriptional regulatory mechanism in which bZIP71 enhances the H3K27me3 level of Ehd1 and transcriptionally represses its expression, which not only offers a novel insight into a flowering pathway, but also provides a valuable putative target for the genetic engineering and breeding of elite rice cultivars.

Drought is a major factor restricting the production of rice (Oryza sativa L.). The identification of natural variants for drought stress‐ related genes is an important step toward developing genetically improved rice varieties. Here, we characterized a member of the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE (SPL) family, OsSPL10, as a transcription factor involved in the regulation of drought tolerance in rice. OsSPL10 appears to play a vital role in drought tolerance by controlling reactive oxygen species (ROS) production and stomatal movements. Haplotype and allele frequency analyses of OsSPL10 indicated that most upland rice and improved lowland rice varieties harbor the OsSPL10^Hap1 allele, whereas the OsSPL10^Hap2 allele was mainly present in lowland and landrace rice varieties. Importantly, we demonstrated that the varieties with the OsSPL10^Hap1 allele showed low expression levels of OsSPL10 and its downstream gene, OsNAC2, which decreases the expression of OsAP37 and increases the expression of OsCOX11, thus preventing ROS accumulation and programmed cell death (PCD). Furthermore, the knockdown or knockout of OsSPL10 induced fast stomatal closure and prevented water loss, thereby improving drought tolerance in rice. Based on these observations, we propose that OsSPL10 confers drought tolerance by regulating OsNAC2 expression and that OsSPL10^Hap1 could be a valuable haplotype for the genetic improvement of drought tolerance in rice.

Anther development from stamen primordium to pollen dispersal is complex and essential to sexual reproduction. How this highly dynamic and complex developmental process is controlled genetically is not well understood, especially for genes involved in specific key developmental phases. Here we generated RNA sequencing libraries spanning 10 key stages across the entirety of anther development in maize (Zea mays). Global transcriptome analyses revealed distinct phases of cell division and expansion, meiosis, pollen maturation, and mature pollen, for which we detected 50, 245, 42, and 414 phase-specific marker genes, respectively. Phase-specific transcription factor genes were significantly enriched in the phase of meiosis. The phase-specific expression of these marker genes was highly conserved among the maize lines Chang7-2 and W23, indicating they might have important roles in anther development. We explored a desiccation-related protein gene, ZmDRP1, which was exclusively expressed in the tapetum from the tetrad to the uninucleate microspore stage, by generating knockout mutants. Notably, mutants in ZmDRP1 were completely male-sterile, with abnormal Ubisch bodies and defective pollen exine. Our work provides a glimpse into the gene expression dynamics and a valuable resource for exploring the roles of key phase-specific genes that regulate anther development.

Glycogen synthase kinase 3 (GSK3) proteins play key roles in brassinosteroid (BR) signaling during plant growth and development by phosphorylating various substrates. However, how GSK3 protein stability and activity are themselves modulated is not well understood. Here, we demonstrate in vitro and in vivo that C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (OsCPL3), a member of the RNA Pol II CTD phosphatase-like family, physically interacts with OsGSK2 in rice (Oryza sativa). OsCPL3 expression was widely detected in various tissues and organs including roots, leaves and lamina joints, and was induced by exogenous BR treatment. OsCPL3 localized to the nucleus, where it dephosphorylated OsGSK2 at the Ser-222 and Thr-284 residues to modulate its protein turnover and kinase activity, in turn affecting the degradation of BRASSINAZOLE-RESISTANT 1 (BZR1) and BR signaling. Loss of OsCPL3 function resulted in higher OsGSK2 abundance and lower OsBZR1 levels, leading to decreased BR responsiveness and alterations in plant morphology including semi-dwarfism, leaf erectness and grain size, which are of fundamental importance to crop productivity. These results reveal a previously unrecognized role for OsCPL3 and add another layer of complexity to the tightly controlled BR signaling pathway in plants.

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in response to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with OsOFP19.

Increasing plant photosynthetic capacity is a promising approach to boost yields, but it is particularly challenging in C₃ crops, such as soybean (Glycine max (L.) Merr.). Here, we identified GmFtsH25, encoding a member of the filamentation temperature‐sensitive protein H protease family, as a major gene involved in soybean photosynthesis, using linkage mapping and a genome‐wide association study. Overexpressing GmFtsH25 resulted in more grana thylakoid stacks in chloroplasts and increased photosynthetic efficiency and starch content, while knocking out GmFtsH25 produced the opposite phenotypes. GmFtsH25 interacted with photosystem I light harvesting complex 2 (GmLHCa2), and this interaction may contribute to the observed enhanced photosynthesis. GmFtsH25 overexpression lines had superior yield traits, such as yield per plant, compared to the wild type and knockout lines. Additionally, we identified an elite haplotype of GmFtsH25, generated by natural mutations, which appears to have been selected during soybean domestication. Our study sheds light on the molecular mechanism by which GmFtsH25 modulates photosynthesis and provides a promising strategy for improving the yields of soybean and other crops.

The NAC transcription factor NONRIPENING (NOR) is a master regulator of climacteric fruit ripening. Melon (Cucumis melo L.) has climacteric and non-climacteric fruit ripening varieties and is an ideal model to study fruit ripening. Two natural CmNAC-NOR variants, the climacteric haplotype CmNAC-NOR^S,N and the non-climacteric haplotype CmNAC-NOR^A,S, have effects on fruit ripening; however, their regulatory mechanisms have not been elucidated. Here, we report that a natural mutation in the transcriptional activation domain of CmNAC-NOR^S,N contributes to climacteric melon fruit ripening. CmNAC-NOR knockout in the climacteric-type melon cultivar “BYJH” completely inhibited fruit ripening, while ripening was delayed by 5–8 d in heterozygous cmnac-nor mutant fruits. CmNAC-NOR directly activated carotenoid, ethylene, and abscisic acid biosynthetic genes to promote fruit coloration and ripening. Furthermore, CmNAC-NOR mediated the transcription of the “CmNAC-NOR-CmNAC73-CmCWINV2” module to enhance flesh sweetness. The transcriptional activation activity of the climacteric haplotype CmNAC-NOR^S,N on these target genes was significantly higher than that of the non-climacteric haplotype CmNAC-NOR^A,S. Moreover, CmNAC-NOR^S,N complementation fully rescued the non-ripening phenotype of the tomato (Solanum lycopersicum) cr-nor mutant, while CmNAC-NOR^A,S did not. Our results provide insight into the molecular mechanism of climacteric and non-climacteric fruit ripening in melon.

Carbohydrate partitioning is essential for plant growth and development, and its hindrance will result in excess accumulation of carbohydrates in source tissues. Most of the related mutants in maize (Zea mays L.) display impaired whole-plant sucrose transport, but other mechanisms affecting carbohydrate partitioning have seldom been reported. Here, we characterized chlorotic leaf3 (chl3), a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts, suggesting that chl3 is defective in carbohydrate partitioning. Positional cloning revealed that the chl3 phenotype results from a frameshift mutation in ZmPHOH, which encodes starch phosphorylase 2. Two mutants in ZmPHOH exhibited the same phenotype as chl3, and both alleles failed to complement the chl3 mutant phenotype in an allelism test. Inactivation of ZmPHOH in chl3 leaves reduced the efficiency of transitory starch conversion, resulting in increased leaf starch contents and altered carbohydrate metabolism patterns. RNA-seq revealed the transcriptional downregulation of genes related to photosynthesis and carbohydrate metabolism in chl3 leaves compared to the wild type. Our results demonstrate that transitory starch remobilization is very important for cellular carbohydrate partitioning in maize, in which ZmPHOH plays an indispensable role.

Receptor-like kinases (RLKs) are a large group of plant-specific transmembrane proteins mainly acting as receptors or co-receptors of various extracellular signals. They usually turn extracellular signals into intracellular responses via altering gene expression profiles. However, recent studies confirmed that many RLKs can physically interact with diverse membrane-localized transport proteins and regulate their activities for speedy responses in limited tissues or cells. In this minireview, we highlight recent discoveries regarding how RLKs can work with membrane transport proteins collaboratively and thereby trigger cellular responses in a precise and rapid manner. It is anticipated that such regulation broadly presents in plants and more examples will be gradually revealed when in-depth analyses are conducted for the functions of RLKs.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDβ). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDβ in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDβ and CiFDα. Interestingly, CiFDα and CiFDβ were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDβ can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDβ can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

Vitamin B₁ (VB1), including thiamin, thiamin monophosphate (TMP), and thiamin pyrophosphate (TPP), is an essential micronutrient for all living organisms. Nevertheless, the precise function of VB1 in rice remains unclear. Here, we described a VB1 auxotrophic mutant, chlorotic lethal seedling (cles) from the mutation of OsTH1, which displayed collapsed chloroplast membrane system and decreased pigment content. OsTH1 encoded a phosphomethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase, and was expressed in various tissues, especially in seedlings, leaves, and young panicles. The VB1 content in cles was markedly reduced, despite an increase in the expression of VB1 synthesis genes. The decreased TPP content affected the tricarboxylic acid cycle, pentose phosphate pathway, and de novo fatty acid synthesis, leading to a reduction in fatty acids (C16:0 and C18:0) and sugars (sucrose and glucose) of cles. Additionally, irregular expression of chloroplast membrane synthesis genes led to membrane collapse. We also found that alternative splicing and translation allowed OsTH1 to be localized to both chloroplast and cytosol. Our study revealed that OsTH1 was an essential enzyme in VB1 biosynthesis and played crucial roles in seedling growth and development by participating in fatty acid and sugar metabolism, providing new perspectives on VB1 function in rice.

RICE INDETERMINATE 1 (RID1) plays a critical role in controlling floral transition in rice (Oryza sativa). However, the molecular basis for this effect, particularly the target genes and regulatory specificity, remains largely unclear. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in young leaves at the pre-floral-transition stage to identify the target genes of RID1, identifying 2,680 genes associated with RID1 binding sites genome-wide. RID1 binding peaks were highly enriched for TTTGTC, the direct binding motif of the INDETERMINATE DOMAIN protein family that includes RID1. Interestingly, CACGTG and GTGGGCCC, two previously uncharacterized indirect binding motifs, were enriched through the interactions of RID1 with the novel flowering-promoting proteins OsPIL12 and OsTCP11, respectively. Moreover, the ChIP-seq data demonstrated that RID1 bound to numerous rice heading-date genes, such as HEADING DATE 1 (HD1) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1). Notably, transcriptome sequencing (RNA-seq) analysis revealed roles of RID1 in diverse developmental pathways. Genetic analysis combined with genome-wide ChIP-seq and RNA-seq results showed that RID1 directly binds to the promoter of OsERF#136 (a repressor of rice flowering) and negatively regulates its expression. Overall, our findings provide new insights into the molecular and genetic mechanisms underlying rice floral transition and characterize OsERF#136 as a previously unrecognized direct target of RID1.

The oomycete pathogen Phytophthora sojae is a causal agent of soybean root rot. Upon colonization of soybeans, P. sojae secretes various RXLR effectors to suppress host immune responses, supporting successful infection. Previous research has demonstrated that the RXLR effector Avh94 functions as a virulence effector, but the molecular mechanism underlying its role in virulence remains unknown. Here, we demonstrate that Avh94 overexpression in plants and pathogens promotes Phytophthora infection. Avh94 interacts with soybean JAZ1/2, which is a repressor of jasmonic acid (JA) signaling. Avh94 stabilizes JAZ1/2 to inhibit JA signaling and silencing of JAZ1/2 enhances soybean resistance against P. sojae. Moreover, P. sojae lines overexpressing Avh94 inhibit JA signaling. Furthermore, exogenous application of methyl jasmonate improves plant resistance to Phytophthora. Taken together, these findings suggest that P. sojae employs an RXLR effector to hijack JA signaling and thereby promote infection.

Arabinogalactan proteins (AGPs) are widely distributed in plant cells. Fasciclin-like AGPs (FLAs) belong to a subclass of AGPs that play important roles in plant growth and development. However, little is known about the biological functions of rice FLA. Herein, we report the identification of a male-sterile mutant of DEFECTIVE EXINE AND APERTURE PATTERNING1 (DEAP1) in rice. The deap1 mutant anthers produced aberrant pollen grains with defective exine formation and a flattened aperture annulus and exhibited slightly delayed tapetum degradation. DEAP1 encodes a plasma membrane-associated member of group III plant FLAs and is specifically and temporally expressed in reproductive cells and the tapetum layer during male development. Gene expression studies revealed reduced transcript accumulation of genes related to exine formation, aperture patterning, and tapetum development in deap1 mutants. Moreover, DEAP1 may interact with two rice D6 PROTEIN KINASE-LIKE3s (OsD6PKL3s), homologs of a known Arabidopsis aperture protein, to affect rice pollen aperture development. Our findings suggested that DEAP1 is involved in male reproductive development and may affect exine formation and aperture patterning, thereby providing new insights into the molecular functions of plant FLAs in male fertility.

Receptor-like kinases (RLKs) play key roles in regulating various physiological aspects in plant growth and development. In Arabidopsis thaliana, there are at least 223 leucine-rich repeat (LRR) RLKs. The functions of the majority of RLKs in the LRR XI subfamily were previously revealed. Only three RLKs were not characterized. Here we report that two independent triple mutants of these RLKs, named ROOT ELONGATION RECEPTOR KINASES (REKs), exhibit increased cell numbers in the root apical meristem and enhanced cell size in the elongation and maturation zones. The promoter activities of a number of Quiescent Center marker genes are significantly up-regulated in the triple mutant. However, the promoter activities of several marker genes known to control root stem cell niche activities are not altered. RNA-seq analysis revealed that a number of cell wall remodeling genes are significantly up-regulated in the triple mutant. Our results suggest that these REKs play key roles in regulating root development likely via negatively regulating the expression of a number of key cell wall remodeling genes.

NAC transcription factors (TFs) play critical roles in plant immunity by modulating the expression of downstream genes via binding to specific cis-elements in promoters. Here, we report the function and regulatory network of a pathogen- and defense phytohormone-inducible NAC TF gene, ONAC083, in rice (Oryza sativa) immunity. ONAC083 localizes to the nucleus and exhibits transcriptional activation activity that depends on its C-terminal region. Knockout of ONAC083 enhances rice immunity against Magnaporthe oryzae, strengthening pathogen-induced defense responses, and boosting chitin-induced pattern-triggered immunity (PTI), whereas ONAC083 overexpression has opposite effects. We identified ONAC083-binding sites in the promoters of 82 genes, and showed that ONAC083 specifically binds to a conserved element with the core sequence ACGCAA. ONAC083 activated the transcription of the genes OsRFPH2-6, OsTrx1, and OsPUP4 by directly binding to the ACGCAA element. OsRFPH2-6, encoding a RING-H2 protein with an N-terminal transmembrane region and a C-terminal typical RING domain, negatively regulated rice immunity against M. oryzae and chitin-triggered PTI. These data demonstrate that ONAC083 negatively contributes to rice immunity against M. oryzae by directly activating the transcription of OsRFPH2-6 through the ACGCAA element in its promoter. Overall, our study provides new insight into the molecular regulatory network of NAC TFs in rice immunity.

To identify novel regulators of stem cell renewal, we mined an existing but little explored cell type-specific transcriptome dataset for the Arabidopsis root. A member of the TGA family of transcription factors, TGA8, was found to be specifically expressed in the quiescent center (QC). Mutation in TGA8 caused a subtle root growth phenotype, suggesting functional redundancy with other TGA members. Using a promoter::HGFP transgenic approach, we showed that all TGA factors were expressed in the root, albeit at different levels and with distinct spatial patterns. Mutant analyses revealed that all TGA factors examined contribute to root growth by promoting stem cell renewal, meristem activity, and cell elongation. Combining transcriptome analyses, histochemical assays, and physiological tests, we demonstrated that functional redundancy exists among members of clades II and V or those in clades I and III. These two groups of TGA factors act differently, however, as their mutants responded to oxidative stress differently and quantitative reverse transcription polymerase chain reaction assays showed they regulate different sets of genes that are involved in redox homeostasis. Our study has thus uncovered a previously unrecognized broad role and a mechanistic explanation for TGA factors in root growth and development.

Plants have developed innate immune systems to fight against pathogenic fungi by monitoring pathogenic signals known as pathogen-associated molecular patterns (PAMP) and have established endo symbiosis with arbuscular mycorrhizal (AM) fungi through recognition of mycorrhizal (Myc) factors. Chitin elicitor receptor kinase 1 of Oryza sativa subsp. Japonica (OsCERK1) plays a bifunctional role in mediating both chitin-triggered immunity and symbiotic relationships with AM fungi. However, it remains unclear whether OsCERK1 can directly recognize chitin molecules. In this study, we show that OsCERK1 binds to the chitin hexamer ((NAG)₆) and tetramer ((NAG)₄) directly and determine the crystal structure of the OsCERK1-(NAG)₆ complex at 2??. The structure shows that one OsCERK1 is associated with one (NAG)₆. Upon recognition, chitin hexamer binds OsCERK1 by interacting with the shallow groove on the surface of LysM2. These structural findings, complemented by mutational analyses, demonstrate that LysM2 is crucial for recognition of both (NAG)₆ and (NAG)₄. Altogether, these findings provide structural insights into the ability of OsCERK1 in chitin perception, which will lead to a better understanding of the role of OsCERK1 in mediating both immunity and symbiosis in rice.

Protein kinases regulate virtually all cellular processes, but it remains challenging to determine the functions of all protein kinases, collectively called the “kinome”, in any species. We developed a computational approach called EXPLICIT-Kinase to predict the functions of the Arabidopsis kinome. Because the activities of many kinases can be regulated transcriptionally, their gene expression patterns provide clues to their functions. A universal gene expression predictor for Arabidopsis was constructed to predict the expression of 30,172 non-kinase genes based on the expression of 994 kinases. The model reconstituted highly accurate transcriptomes for diverse Arabidopsis samples. It identified the significant kinases as predictor kinases for predicting the expression of Arabidopsis genes and pathways. Strikingly, these predictor kinases were often regulators of related pathways, as exemplified by those involved in cytokinesis, tissue development, and stress responses. Comparative analyses revealed that portions of these predictor kinases are shared and conserved between Arabidopsis and maize. As an example, we identified a conserved predictor kinase, RAF6, from a stomatal movement module. We verified that RAF6 regulates stomatal closure. It can directly interact with SLAC1, a key anion channel for stomatal closure, and modulate its channel activity. Our approach enables a systematic dissection of the functions of the Arabidopsis kinome.