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In cereal plants, the size of the panicle (inflorescence) is a critical factor for yield. Panicle size is determined by a complex interplay of genetic and environmental factors, but the mechanisms underlying adaptations to temperature stress during panicle development remain largely unknown. We identify the rice THERMOSENSITIVE BARREN PANICLE (TAP) gene, which encodes a transposase-derived FAR1-RELATED SEQUENCE (FRS) protein and is responsible for regulating panicle and spikelet development at high ambient temperature. The tap mutants display high temperature-dependent reproductive abnormalities, including compromised secondary branch and spikelet initiation and pleiotropic floral organ defects. Consistent with its thermosensitive phenotype, TAP expression is induced by high temperature. TAP directly promotes the expression of OsYABBY3 (OsYAB3), OsYAB4, and OsYAB5, which encode key transcriptional regulators in panicle and spikelet development. In addition, TAP physically interacts with OsYAB4 and OsYAB5 proteins; phenotypic analysis of osyab4 tap-1 and osyab5 tap-1 double mutants indicates that TAP-OsYAB4/OsYAB5 complexes act to maintain normal panicle and spikelet development. Taken together, our study reveals the novel role of a TE-derived transcription factor in controlling rice panicle development under high ambient temperatures, shedding light on the molecular mechanism underlying the adaptation of cereal crops to increasing environmental temperatures.

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

Environmental signals, especially daylength, play important roles in determining fertility in photoperiod-sensitive genic male sterile (PGMS) lines that are critical to sustain production of high-yielding hybrid rice (Oryza sativa) varieties. However, the mechanisms by which PGMS lines perceive changes in photoperiod and transmit those signals to elicit downstream effects are not well understood. In this study, we compared the transcriptomes from the leaves and anthers of carbon starved anther (csa), a PGMS line, to wild-type (WT) tissues under different photoperiods. Components of circadian clock in the leaves, including Circadian Clock-Associated 1 and Pseudo-Response Regulator (PRR95), played vital roles in sensing the photoperiod signals. Photoperiod signals were weakly transduced to anthers, where gene expression was mainly controlled by the CSA allele. CSA played a critical role in regulating sugar metabolism and cell wall synthesis in anthers under short-day conditions, and transcription of key genes inducing csa-directed sterility was upregulated under long-day (LD) conditions though not to WT levels, revealing a mechanism to explain the partial restoration of fertility in rice under LD conditions. Eight direct targets of CSA regulation were identified, all of which were genes involved in sugar metabolism and transport (cell wall invertases, SWEETs, and monosaccharide transporters) expressed only in reproductive tissues. Several hub genes coordinating the effects of CSA regulation were identified as critical elements determining WT male fertility and further analysis of these and related genes will reveal insights into how CSA coordinates sugar metabolism, cell wall biosynthesis, and photoperiod sensing in rice anther development.

In plants, non-green plastids in heterotrophic tissues are sites for starch and fatty acids biosynthesis, which are essential for plant development and reproduction. Distinct from chloroplasts, the metabolites for these processes in non-green plastids have to be imported through specific transporters. Glucose 6-Phosphate/Phosphate Translocator 1 is required for the uptake of cytosolic Glucose 6-Phosphate into non-green plastids. In Arabidopsis, GPT1 has been demonstrated to play essential roles in male, female gametophyte and embryo development. However, the roles of GPTs in other species are yet largely unknown. Here, we reported that rice OsGPT1 is indispensable for normal tapetal degeneration and pollen exine formation during anther and pollen development. OsGPT1 is localized in the plastid and distributed in the anther wall layers and late-stage pollen grains. Different from the gametic defects caused by mutation in AtGPT1, disruption of OsGPT1 does not affect male and female gamete transmission as well as embryo development. On the contrary, osgpt1 mutant exhibits delayed tapetum degeneration, decreased Ubisch bodies formation and thinner pollen exine, leading to pollen abortion at the mature stage. Furthermore, the expression of several genes involved in tapetal programmed cell death (PCD) and sporopollenin formation is decreased in osgpt1. Our study suggests that OsGPT1 coordinates the development of anther sporophytic tissues and the male gametophyte by integrating carbohydrate and fatty acid metabolism in the plastid.

The spatiotemporal control of meristem identity is critical for determining inflorescence architecture, and thus yield, of cereal plants. However, the precise mechanisms underlying inflorescence and spikelet meristem determinacy in cereals are still largely unclear. We have generated loss-of-function and overexpression mutants of the paralogous OsMADS5 and OsMADS34 genes in rice (Oryza sativa), and analysed their panicle phenotypes. Using chromatin immunoprecipitation, electrophoretic mobility-shift and dual-luciferase assays, we have also identified RICE CENTRORADIALIS 4 (RCN4), a TFL1-like gene, as a direct downstream target of both OsMADS proteins, and have analysed RCN4 mutants. The osmads5 osmads34 mutant lines had significantly enhanced panicle branching with increased secondary, and even tertiary and quaternary, branches, compared to wild type and osmads34 plants. The osmads34 mutant phenotype could largely be rescued by also knocking out RCN4. Moreover, transgenic panicles overexpressing RCN4 had significantly increased branching, and initiated development of ~7× more spikelets than wild type. Our results reveal a role for OsMADS5 in panicle development, and show that OsMADS5 and OsMADS34 play similar functions in limiting branching and promoting the transition to spikelet meristem identity, in part by repressing RCN4 expression. These findings provide new insights to better understand the molecular regulation of rice inflorescence architecture.

Plant Rho small GTPases (Rop/Rac) are versatile molecular switches regulating many plant developmental processes. Particularly, their important functions in regulating pollen development have been demonstrated in Arabidopsis. A group of conserved Rop/Rac activators RopGEFs were recently reported to regulate rice (Oryza sativa) pollen tube germination, indicating that rice and Arabidopsis may have a conserved Rop/Rac mediated signaling pathway in regulating pollen tube growth. However, the Rop/Rac activated by the rice pollen specific RopGEFs remains to be identified. Here we demonstrated a Rop/Rac gene, OsRacB, co-expressed with the mature pollen expressed OsRopGEF2/3/6/8. The knockout mutants were normal in anther and pollen development but defective in the pollen grain germination, suggesting a specific and non-redundant role of OsRacB in the mature pollen. We further demonstrated that OsRacB is directly activated by the pollen specific expressing OsRopGEFs in vitro. Together with the previous study, we establish a RopGEF-Rop/Rac regulon which plays essential roles in rice pollen grain germination. Our data encourage further identification of the upstream and downstream players of RopGEF-Rop/Rac signaling in pollen germination and have agricultural implications for breeding robust seed yielding cultivars.

Seed germination is critical for plant survival and agricultural production, which is affected by both internal seed factors and external environmental conditions. However, the genetic basis and underlying molecular mechanisms of early seed germination in crops remain largely unclear. Here, we report that R2R3 MYB transcription factor Carbon Starved Anther (CSA) is expressed specifically in Oryza sativa embryo and aleurone in response to seed imbibition, peaking at 3–6 h and undetectable by 24-h post-imbibition. CSA seeds germinated more quickly than wild-type rice seeds and had higher levels of amylase activity, glucose, and inactive abscisic acid-glucose ester (ABA-GE), but lower levels of ABA. Through analyzing the CSA-associated transcriptome and CSA binding to downstream target genes, we identified two glycolytic genes as direct CSA targets. CSA inhibits Amylase 3A expression to limit glucose production from starch and activates Os3BGlu6 expression to promote de-conjugation of ABA-GE to ABA; these functions serve to slow germination and improve seedling resilience to abiotic stress in the first 3 weeks of growth. Therefore, this study unveils a protection mechanism conferred by CSA during early seed germination by balancing glucose and ABA metabolism to optimize seed germination and stress response fitness.

Several QTLs and genes responsible for seed dormancy were detected and SNP candidates were shown to cause changes in seed germination. Seed dormancy is a key agricultural trait to prevent pre-harvest sprouting in crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum), and barley (Hordeum vulgare L.). However, our knowledge of seed dormancy is hampered by the complexities of studying a trait that changes over time after seed harvest, and is complicated by interactions between phytohormones, seed coat components and the environment. Here, we have conducted a genome-wide association study using a panel of 311 natural accessions of cultivated rice, examining a total of 519,158 single nucleotide polymorphisms (SNPs). Eight quantitative trait loci (QTLs) were found to associate with seed dormancy in the whole panel and five in the Japonica and Indica subpanel; expression of candidate genes within 100 kb of each QTL was examined in two published, germination-specific transcriptomic datasets. Ten candidate genes, differentially expressed within the first four days post-imbibition, were identified. Five of these genes had previously been associated with awn length, heading date, yield, and spikelet length phenotypes. Two candidates were validated using Quantitative Reverse Transcription (qRT)-PCR. In addition, previously identified genes involved in hormone signaling during germination were found to be differentially expressed between a japonica and an indica line; SNPs in the promoter of Os9BGlu33 were associated with germination index, with qRT-PCR validation. Collectively, our results are useful for future characterization of seed dormancy mechanism and crop improvement, and suggest haplotypes for further analysis that may be of use to boost PHS resistance in rice.

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanisms underpinning how plant inflorescence architecture responds to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature-dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognized role of plant MADS-box proteins in floral development.

Photoperiod-dependent male fertility is a critical enabler of modern hybrid breeding. A MYB transcription factor, CSA, is a key regulator of sugar partitioning in rice anthers, disruption of which causes photoperiod-sensitive male sterility. However, little is known about the molecular mechanisms governing plant fertility in response to photoperiod. Here, we have obtained another rice photoperiod-sensitive male sterile mutant, csa2, which exhibits semi-sterility under long-day (LD) conditions, with normal fertility under short-day (SD) conditions. CSA2 specifically expressed in anthers, and here is shown to be indispensable for sugar partitioning to anthers under LD conditions. The CSA2 protein can restore the fertility of csa mutants under SD conditions when expressed in a CSA-specific pattern, indicating that the two proteins share common downstream regulatory targets. Transcriptomic analyses also reveal discrete regulatory targets in anthers. Furthermore, the regulatory role of CSA2 in sugar transport was influenced by the photoperiod conditions during floral initiation, not simply during anther development. Collectively, we propose that rice evolved at least two MYB proteins, CSA2 and CSA, that regulate sugar transport in anthers under LD and SD conditions, respectively. This finding provides insight into the molecular mechanisms that regulate male fertility in response to photoperiod.

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct. ispartof: PLANT JOURNAL vol:106 issue:1 pages:159-173 ispartof: location:England status: published

This study assessed the protective effects of paeoniflorin against preeclampsia-related endothelial damage (ED). Human umbilical vein endothelial cells (HUVECs) isolated from healthy puerperae were identified by immunofluorescence assay. After paeoniflorin treatment, HUVECs were induced by soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) to establish ED. Cell viability, migration, invasion, tube formation, and apoptosis were assessed by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium MTT assay, Scratch assay, Transwell assay, tube formation assay, and flow cytometry. VEGFA expression in HUVECs was analyzed by Western blot. HUVECs were successfully isolated and identified as Von Willebrand factor (vWF) positive. Individual treatment or cotreatment of sFlt-1 and sEng inhibited migration, invasion and tube formation, enhanced apoptosis, and decreased VEGFA expression in HUVECs. Paeoniflorin pretreatment partially reversed the effects delivered by cotreatment of sFlt-1 and sEng in HUVECs. Paeoniflorin alleviated preeclampsia-related ED caused by overexpression of sFlt-1 and sEng by upregulating VEGFA.

Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial-temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive. Here, through histological and high-throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis. AGO1d preferentially binds to miR2118-triggered 21-nucleotide (nt) phasiRNAs with a 5'-terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21- and 24-nt phasiRNAs and temperature-sensitive male sterility. At lower temperatures, anthers of the ago1d mutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism. These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.

Rice tiller angle is a key agronomic trait determining rice grain yield. Several quantitative trait loci (QTLs) affecting rice tiller angle have been mapped in the past decades. little is known about the genetic base of tiller angle in rice, because rice tiller angle is a complex polygenic trait. In this study, we performed genome-wide association study (GWAS) on tiller angle in rice using a population of 164 japonica varieties derived from the 3K Rice Genomes Project (3K RGP). We detected a total of 18 QTLs using 1,135,519 single-nucleotide polymorphisms (SNP) based on three GWAS models (GLM, FastLMM and FarmCPU). Among them, two identified QTLs, qTA8.3 and qTA8.4, overlapped with PAY1 and TIG1, respectively, and additional 16 QTLs were identified for the first time. Combined with haplotype and expression analyses, we further revealed that PAY1 harbors one non-synonymous variation at its coding region, likely leading to variable tiller angle in the population, and that nature variations in the promoter of TIG1 significantly affect its expression, closely correlating with tiller angle phenotypes observed. Similarly, using qRT-PCR and haplotype analysis, we identified 1 and 7 candidate genes in qTA6.1 and qTA8.1 that were commonly detected by two GWAS models, respectively. In addition, we identified 3 more candidate genes in the remaining 14 novel QTLs after filtering by transcriptome analysis and qRT-PCR. In summary, this study provides new insights into the genetic architecture of rice tiller angle and candidate genes for rice breeding.

The shape of an inflorescence varies among cereals, ranging from a highly branched panicle in rice to a much more compact spike in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). However, little is known about the molecular basis of cereal inflorescence architecture. We profiled transcriptomes at three developmental stages of the barley main shoot apex — spikelet initiation, floral organ differentiation, and floral organ growth — and compared them with those from vegetative seedling tissue. Transcript analyses identified 3688 genes differentially transcribed between the three meristem stages, with a further 1394 genes preferentially expressed in reproductive compared with vegetative tissue. Co-expression assembly and Gene Ontology analysis classified these 4888 genes into 28 clusters, revealing distinct patterns for genes such as transcription factors, histone modification, and cell-cycle progression specific for each stage of inflorescence development. We also compared expression patterns of VRS (SIX-ROWED SPIKE) genes and auxin-, gibberellic acid- and cytokinin-associated genes between two-rowed and six-rowed barley to describe regulators of lateral spikelet fertility. Our findings reveal barley inflorescence phase-specific gene expression, identify new candidate genes that regulate barley meristem activities and flower development, and provide a new genetic resource for further dissection of the molecular mechanisms of spike development. Keywords: Inflorescence meristem, Transcriptome, Gene expression, Hormones, Barley

Rice inflorescence development determines yield and relies on the activity of axillary meristems (AMs); however, high-resolution analysis of its early development is lacking. Here, we have used high-throughput single-cell RNA sequencing to profile 37 571 rice inflorescence cells and constructed a genome-scale gene expression resource covering the inflorescence-to-floret transition during early reproductive development. The differentiation trajectories of florets and AMs were reconstructed, and discrete cell types and groups of regulators in the highly heterogeneous young inflorescence were identified and then validated by in situ hybridization and with fluorescent marker lines. Our data demonstrate that a WOX transcription factor, DWARF TILLER1, regulates flower meristem activity, and provide evidence for the role of auxin in rice inflorescence branching by exploring the expression and biological role of the auxin importer OsAUX1. Our comprehensive transcriptomic atlas of early rice inflorescence development, supported by genetic evidence, provides single-cell-level insights into AM differentiation and floret development.

Soil compaction represents a major agronomic challenge, inhibiting root elongation and impacting crop yields. Roots use ethylene to sense soil compaction as the restricted air space causes this gaseous signal to accumulate around root tips. Ethylene inhibits root elongation and promotes radial expansion in compacted soil, but its mechanistic basis remains unclear. Here, we report that ethylene promotes abscisic acid (ABA) biosynthesis and cortical cell radial expansion. Rice mutants of ABA biosynthetic genes had attenuated cortical cell radial expansion in compacted soil, leading to better penetration. Soil compaction-induced ethylene also up-regulates the auxin biosynthesis gene OsYUC8 . Mutants lacking OsYUC8 are better able to penetrate compacted soil. The auxin influx transporter OsAUX1 is also required to mobilize auxin from the root tip to the elongation zone during a root compaction response. Moreover, osaux1 mutants penetrate compacted soil better than the wild-type roots and do not exhibit cortical cell radial expansion. We conclude that ethylene uses auxin and ABA as downstream signals to modify rice root cell elongation and radial expansion, causing root tips to swell and reducing their ability to penetrate compacted soil.

The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

Organ size is mainly determined by cell division and cell expansion. Several genetic factors regulating development of plant lateral organs have been characterised, but those involved in determining reproductive organ size and separation in rice (Oryza sativa) remain unknown. We have isolated the rice gene SMALL REPRODUCTIVE ORGANS (SRO) encoding a nucleus-localised C2 H2 zinc finger protein orthologous to Arabidopsis transcription factor SUPERMAN (SUP). Combined developmental, genetic, histological and transcriptomic analyses were used to determine the function of SRO in regulating reproductive organ size. SRO affects genes involved in cell division, cell expansion and phytohormone signalling in the rice flower. SRO is specifically expressed in the first stages of stamen filament development to regulate their correct formation and separation. In addition, SRO non-cell-autonomously regulates the size and functionality of male and female reproductive organs. The B-class MADS-box gene OsMADS16/SPW1 is epistatic to SRO, while SRO regulates reproductive organ specification and floral meristem determinacy synergistically with C-class genes OsMADS3 and OsMADS58. These findings provide insights into how an evolutionarily conserved transcription factor has a pivotal role in reproductive organ development in core eudicots and monocots, through partially conserved expression, function, and regulatory network.

E3 ubiquitin ligases have been more widely reported to mediate the degradation of target proteins. In our research, one SIAH type E3 ligase was identified, named RIP1, and there are five genes were found to be highly homologous to it, named RIP2 to RIP6 dividedly. Y2H and BiFC were carried out to prove the interaction between RIPs and OsFH5, moreover, homo- and hetero- oligomers can be formed among RIPs and they all can affect the localization of OsFH5. Degradation experiments showed that RIP1 is able to slow down the degradation rate of OsFH5, and also RIP5, RIP6. There was no obvious phenotype in both RIP1 overexpression and mutant transgenic lines, whereas in rips multi-knock out lines showed up similar defects to osfh5, such as, dwarf height; less smooth and rounder seeds; leaves width became wilder. Otherwise, rips also exhibit some special phenotypes, for instance, the angles between leaves and stems were appreciable enlarged; spiral crimped roots. In addition, microfilaments organization in rips appeared disordered than wild type. All above illustrate that RIPs influence morphology by interacting with OsFH5 and affecting its subcellular localization, stability or maybe involved in other pathways in rice.

Summary Panicle architecture is a key determinant of grain yield in cereals, but the mechanisms governing panicle morphogenesis and organ development remain elusive. Here, we have identified a quantitative trait locus (qPA1) associated with panicle architecture using chromosome segment substitution lines from parents Nipponbare and 9311. The panicle length, branch number and grain number of Nipponbare were significantly higher than CSSL‐9. Through map‐based cloning and complementation tests, we confirmed that qPA1 was identical to SD1 (Semi Dwarf1), which encodes a gibberellin 20‐oxidase enzyme participating in gibberellic acid (GA) biosynthesis. Transcript analysis revealed that SD1 was widely expressed during early panicle development. Analysis of sd1/osga20ox2 and gnp1/ osga20ox1 single and double mutants revealed that the two paralogous enzymes have non‐redundant functions during panicle development, likely due to differences in spatiotemporal expression; GNP1 expression under control of the SD1 promoter could rescue the sd1 phenotype. The DELLA protein SLR1, a component of the GA signalling pathway, accumulated more highly in sd1 plants. We have demonstrated that SLR1 physically interacts with the meristem identity class I KNOTTED1‐LIKE HOMEOBOX (KNOX) protein OSH1 to repress OSH1‐mediated activation of downstream genes related to panicle development, providing a mechanistic link between gibberellin and panicle architecture morphogenesis.

The spatiotemporal control of meristem identity is critical for determining inflorescence architecture, and thus yield, of cereal plants. However, the precise mechanisms underlying inflorescence and spikelet meristem determinacy in cereals are still largely unclear. We have generated loss-of-function and overexpression mutants of the paralogous OsMADS5 and OsMADS34 genes in rice (Oryza sativa), and analysed their panicle phenotypes. Using chromatin immunoprecipitation, electrophoretic mobility-shift and dual-luciferase assays, we have also identified RICE CENTRORADIALIS 4 (RCN4), a TFL1-like gene, as a direct downstream target of both OsMADS proteins, and have analysed RCN4 mutants. The osmads5 osmads34 mutant lines had significantly enhanced panicle branching with increased secondary, and even tertiary and quaternary, branches, compared to wild-type (WT) and osmads34 plants. The osmads34 mutant phenotype could largely be rescued by also knocking out RCN4. Moreover, transgenic panicles overexpressing RCN4 had significantly increased branching, and initiated development of c. 7× more spikelets than WT. Our results reveal a role for OsMADS5 in panicle development, and show that OsMADS5 and OsMADS34 play similar functions in limiting branching and promoting the transition to spikelet meristem identity, in part by repressing RCN4 expression. These findings provide new insights to better understand the molecular regulation of rice inflorescence architecture.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

Several QTLs and genes responsible for seed dormancy were detected and SNP candidates were shown to cause changes in seed germination. Seed dormancy is a key agricultural trait to prevent pre-harvest sprouting in crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum), and barley (Hordeum vulgare L.). However, our knowledge of seed dormancy is hampered by the complexities of studying a trait that changes over time after seed harvest, and is complicated by interactions between phytohormones, seed coat components and the environment. Here, we have conducted a genome-wide association study using a panel of 311 natural accessions of cultivated rice, examining a total of 519,158 single nucleotide polymorphisms (SNPs). Eight quantitative trait loci (QTLs) were found to associate with seed dormancy in the whole panel and five in the Japonica and Indica subpanel; expression of candidate genes within 100 kb of each QTL was examined in two published, germination-specific transcriptomic datasets. Ten candidate genes, differentially expressed within the first four days post-imbibition, were identified. Five of these genes had previously been associated with awn length, heading date, yield, and spikelet length phenotypes. Two candidates were validated using Quantitative Reverse Transcription (qRT)-PCR. In addition, previously identified genes involved in hormone signaling during germination were found to be differentially expressed between a japonica and an indica line; SNPs in the promoter of Os9BGlu33 were associated with germination index, with qRT-PCR validation. Collectively, our results are useful for future characterization of seed dormancy mechanism and crop improvement, and suggest haplotypes for further analysis that may be of use to boost PHS resistance in rice.

Flag leaf angle impacts the photosynthetic capacity of densely grown plants and is thus an important agronomic breeding trait for crop architecture and yield. The hormone auxin plays a key role in regulating this trait, yet the underlying molecular and cellular mechanisms remain unclear. Here, we report that two rice (Oryza sativa) auxin response factors (ARFs), OsARF6 and OsARF17, which are highly expressed in lamina joint tissues, control flag leaf angle in response to auxin. Loss-of-function double osarf6 osarf17 mutants displayed reduced secondary cell wall levels of lamina joint sclerenchymatous cells (Scs), resulting in an exaggerated flag leaf angle and decreased grain yield under dense planting conditions. Mechanical measurements indicated that the mutant lamina joint tissues were too weak to support the weight of the flag leaf blade, resembling the phenotype of the rice increased leaf angle1 (ila1) mutant. We demonstrate that OsARF6 and OsARF17 directly bind to the ILA1 promoter independently and synergistically to activate its expression. In addition, auxin-induced ILA1 expression was dependent on OsARF6 and OsARF17. Collectively, our study reveals a mechanism that integrates auxin signaling with the secondary cell wall composition to determine flag leaf angle, providing breeding targets in rice, and potentially other cereals, for this key trait., Two auxin response genes influence the secondary cell wall biosynthesis and the strength of lamina joints, thereby contributing to the adjustment of flag leaf angles.

Environmental conditions, such as photoperiod and temperature, can affect male fertility in plants. While this feature is heavily exploited in rice to generate male-sterile lines for hybrid breeding, the underlying molecular mechanisms remain largely unknown. In this study, we use a transcriptomics approach to identify key genes and regulatory networks affecting pollen maturation in rice anthers in response to different day lengths. A total of 11,726 differentially expressed genes (DEGs) were revealed, of which 177 were differentially expressed at six time points over a 24-h period. GO enrichment analysis revealed that genes at all time points were enriched in transport, carbohydrate, and lipid metabolic processes, and signaling pathways, particularly phytohormone signaling. In addition, co-expression network analysis revealed four modules strongly correlated with photoperiod. Within these four modules, 496 hub genes were identified with a high degree of connectivity to other photoperiod-sensitive DEGs, including two previously reported photoperiod- and temperature-sensitive genes affecting male fertility, Carbon Starved Anther and UDP-glucose pyrophosphorylase, respectively. This work provides a new understanding on photoperiod-sensitive pollen development in rice, and our gene expression data will provide a new, comprehensive resource to identify new environmentally sensitive genes regulating male fertility for use in crop improvement.

Research on rice (Oryza sativa) roots demands the automatic analysis of root architecture during image processing. It is challenging for a digital filter to identify the roots from the obscure and cluttered background. The original Frangi algorithm, presented by Alejandro F. Frangi in 1998, is a successful low-pass filter dedicated to blood vessel image enhancement. Considering the similarity between vessels and roots, the Frangi filter algorithm is applied to outline the roots. However, the original Frangi only enhances the tube-like primary roots but erases the lateral roots during filtering. In this paper, an improved Frangi filtering algorithm (IFFA), designed for plant roots, is proposed. Firstly, an automatic root phenotyping system is designed to fulfill the high-throughput root image acquisition. Secondly, multilevel image thresholding, connected components labeling, and width correction are used to optimize the output binary image. Thirdly, to enhance the local structure, the Gaussian filtering operator in the original Frangi is redesigned with a truncated Gaussian kernel, resulting in more discernible lateral roots. Compared to the original Frangi filter and commercially available software, IFFA is faster and more accurate, achieving a pixel accuracy of 97.48%. IFFA is an effective morphological filtering approach to enhance the roots of rice for segmentation and further biological research. It is convincing that IFFA is suitable for different 2-D plant root image processing and morphological analysis.

The mammalian BREAST CANCER 2 (BRCA2) gene is a tumor suppressor that plays a crucial role in DNA repair and homologous recombination (HR). Here, we report the identification and characterization of OsBRCA2, the rice orthologue of human BRCA2. Osbrca2 mutant plants exhibit normal vegetative growth but experience complete male and female sterility as a consequence of severe meiotic defects. Pairing, synapsis and recombination are impaired in osbrca2 male meiocytes, leading to chromosome entanglements and fragmentation. In the absence of OsBRCA2, localization to the meiotic chromosome axes of the strand-invasion proteins OsRAD51 and OsDMC1 is severely reduced and in vitro OsBRCA2 directly interacts with OsRAD51 and OsDMC1. These results indicate that OsBRCA2 is essential for facilitating the loading of OsRAD51 and OsDMC1 onto resected ends of programmed double-strand breaks (DSB) during meiosis to promote single-end invasions of homologous chromosomes and accurate recombination. In addition, treatment of osbrca2-1 seedlings with mitomycin C (MMC) led to hypersensitivity. As MMC is a genotoxic agent that creates DNA lesions in the somatic cells that can only be repaired by HR, these results suggest that OsBRCA2 has a conserved role in DSB repair and HR in rice.

Floral patterning is regulated by intricate networks of floral identity genes. The peculiar MADS32 subfamily genes, absent in eudicots but prevalent in monocots, control floral organ identity. However, how the MADS32 family genes interact with other floral homeotic genes during flower development is mostly unknown. We show here that the rice homeotic transcription factor OsMADS32 regulates floral patterning by interacting synergistically with E class protein OsMADS6 in a dosage-dependent manner. Furthermore, our results indicate important roles for OsMADS32 in defining stamen, pistil, and ovule development through physical and genetic interactions with OsMADS1, OsMADS58, and OsMADS13, and in specifying floral meristem identity with OsMADS6, OsMADS3, and OsMADS58, respectively. Our findings suggest that OsMADS32 is an important factor for floral meristem identity maintenance and that it integrates the action of other MADS-box homeotic proteins to sustain floral organ specification and development in rice. Given that OsMADS32 is an orphan gene and absent in eudicots, our data substantially expand our understanding of flower development in plants.