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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.

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.

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.

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

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 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.

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.

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.

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.

Background In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice (Oryza sativa). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice. Results Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana. In subcellular localization analysis of the four RopGEFs through tobacco (Nicotiana benthamiana) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes. Conclusions In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.

•Uniform growth of the main shoot and tillers significantly influences rice plant architecture and grain yield. The WUSCHEL‐related homeobox transcription factor DWT1 is a key regulator of this important agronomic trait, disruption of which causes enhanced main shoot dominance and tiller dwarfism by an unknown mechanism. •Here, we have used yeast‐two‐hybrid screening to identify OsPIP5K1, a member of the rice phosphatidylinositol‐4‐phosphate 5‐kinase family, as a protein that interacts with DWT1. Cytological analyses confirmed that DWT1 induces accumulation of OsPIP5K1 and its product PI(4,5)P2, a phosphoinositide secondary messenger, in nuclear bodies. •Mutation of OsPIP5K1 compounds the dwarf dwt1 phenotype but abolishes the main shoot dominance. Conversely, overexpression of OsPIP5K1 partially rescues dwt1 developmental defects. Furthermore, we showed that DWL2, the homologue of DWT1, is also able to interact with OsPIP5K1 and shares partial functional redundancy with DWT1 in controlling rice uniformity. •Overall, our data suggest that nuclear localised OsPIP5K1 acts with DWT1 and/or DWL2 to coordinate the uniform growth of rice shoots, likely to be through nuclear phosphoinositide signals, and provides insights into the regulation of rice uniformity via a largely unexplored plant nuclear signalling pathway.

Summary Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3‐LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA‐sequencing analyses revealed that 1958 genes were up‐regulated and 2096 genes were down‐regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.

SEPALLATA (SEP)-like genes, which encode a subfamily of MADS-box transcription factors, are essential for specifying floral organ and meristem identity in angiosperms. Rice (Oryza sativa) has five SEP-like genes with partial redundancy and overlapping expression domains, yet their functions and evolutionary conservation are only partially known. Here, we describe the biological role of one of the SEP genes of rice, OsMADS5, in redundantly controlling spikelet morphogenesis. OsMADS5 belongs to the conserved LOFSEP subgroup along with OsMADS1 and OsMADS34OsMADS5 was expressed strongly across a broad range of reproductive stages and tissues. No obvious phenotype was observed in the osmads5 single mutants when compared with the wild type, which was largely due to the functional redundancy among the three LOFSEP genes. Genetic and molecular analyses demonstrated that OsMADS1, OsMADS5, and OsMADS34 together regulate floral meristem determinacy and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes. Experiments conducted in yeast also suggested that OsMADS1, OsMADS5, and OsMADS34 form protein-protein interactions with other MADS-box floral homeotic members, which seems to be a typical, conserved feature of plant SEP proteins.

Plants employ dynamic molecular networks to control development in response to environmental changes, yet the underlying mechanisms are largely unknown. Here we report the identification of two rice leucine-rich repeat receptor-like kinases, Thermo-Sensitive Genic Male Sterile 10 (TMS10) and its close homolog TMS10-Like (TMS10L), which redundantly function in the maintenance of the tapetal cell layer and microspore/pollen viability under normal temperature conditions with TMS10 playing an essential role in higher temperatures (namely, 28 °C). tms10 displays male sterility under high temperatures but male fertility under low temperatures, and the tms10 tms10l double mutant shows complete male sterility under both high and low temperatures. Biochemical and genetic assays indicate that the kinase activity conferred by the intracellular domain of TMS10 is essential for tapetal degeneration and male fertility under high temperatures. Furthermore, indica or japonica rice varieties that contain mutations in TMS10, created by genetic crosses or genome editing, also exhibit thermo-sensitive genic male sterility. These findings demonstrate that TMS10 and TMS10L act as a key switch in postmeiotic tapetal development and pollen development by buffering environmental temperature changes, providing insights into the molecular mechanisms by which plants develop phenotypic plasticity via genotype-environment temperature interaction. TMS10 may be used as a genetic resource for the development of hybrid seed production systems in crops.

Summary Angiosperm male reproductive organs (anthers and pollen grains) have complex and interesting morphological features, but mechanisms that underlie their patterning are poorly understood. Here we report the isolation and characterization of a male sterile mutant of No Pollen 1 (NP1) in rice (Oryza sativa). The np1-4 mutant exhibited smaller anthers with a smooth cuticle surface, abnormal Ubisch bodies, and aborted pollen grains covered with irregular exine. Wild-type exine has two continuous layers; but np1-4 exine showed a discontinuous structure with large granules of varying size. Chemical analysis revealed reduction in most of the cutin monomers in np1-4 anthers, and less cuticular wax. Map-based cloning suggested that NP1 encodes a putative glucose-methanol-choline oxidoreductase; and expression analyses found NP1 preferentially expressed in the tapetal layer from stage 8 to stage 10 of anther development. Additionally, the expression of several genes involved in biosynthesis and in the transport of lipid monomers of sporopollenin and cutin was decreased in np1-4 mutant anthers. Taken together, these observations suggest that NP1 is required for anther cuticle formation, and for patterning of Ubisch bodies and the exine. We propose that products of NP1 are likely important metabolites in the development of Ubisch bodies and pollen exine, necessary for polymerization, assembly, or both.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

Rice flower development determines grain yield. Here we reveal the genetic interactions between the rice meristem maintenance gene FON4 and six floral homeotic genes in flower development., The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice.

The aperture on the pollen surface provides an exit for the emerging pollen tube. Apertures exhibit huge morphological variation across plant species-grasses, including rice, possess a complex aperture consisting of an annulus and an operculum-but little is known about how this species-specific cell-surface pattern forms. Here, we report a lectin receptor-like kinase in Oryza sativa, OsDAF1, which is essential for annulus formation and thus for fertility. OsDAF1 is evenly distributed in early microsporocytes but localizes to the distal pre-aperture site at the tetrad stage. We further reveal that the rice orthologue of a key aperture factor in Arabidopsis, OsINP1, has conserved and diversified roles in rice aperture formation. Disruption of OsINP1 prevents formation of the aperture, precluding pollen-tube germination. Furthermore, our results demonstrate that OsINP1 is required for polarization of OsDAF1 via direct protein interaction, suggesting that OsINP1 has an additional role in the formation of annulus that is absent in Arabidopsis. Our study reveals the importance of the aperture for rice grain yield and reveals mechanisms controlling pollen aperture development in cereal species.

In most eukaryotes, a set of conserved proteins that are collectively termed ZMM proteins (named for molecular zipper 1 [ZIP1], ZIP2, ZIP3, and ZIP4, MutS homologue 4 [MSH4] and MSH5, meiotic recombination 3, and sporulation 16 [SPO16] in yeast [Saccharomyces cerevisiae]) are essential for the formation of the majority of meiotic crossovers (COs). Recent reports indicated that ZIP2 acts together with SPO16 and ZIP4 to control CO formation through recognizing and stabilizing early recombination intermediates in budding yeast. However, whether this mechanism is conserved in plants is not clear. Here, we characterized the functions of SHORTAGE OF CHIASMATA 1 (OsSHOC1; ZIP2 ortholog) and PARTING DANCERS (OsPTD; SPO16 ortholog) and their interactions with other ZMM proteins in rice (Oryza sativa). We demonstrated that disruption of OsSHOC1 caused a reduction of CO numbers to ∼83% of wild-type CO numbers, whereas synapsis and early meiotic recombination steps were not affected. Furthermore, OsSHOC1 interacts with OsPTD, which is responsible for the same set of CO formations as OsSHOC1. In addition, OsSHOC1 and OsPTD are required for the normal loading of other ZMM proteins, and conversely, the localizations of OsSHOC1 and OsPTD were also affected by the absence of OsZIP4 and human enhancer of invasion 10 in rice (OsHEI10). OsSHOC1 interacts with OsZIP4 and OsMSH5, and OsPTD interacts with OsHEI10. Furthermore, bimolecular fluorescence complementation and yeast-three hybrid assays demonstrated that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 were able to form various combinations of heterotrimers. Moreover, statistical and genetic analysis indicated that OsSHOC1 and OsPTD are epistatic to OsHEI10 and OsZIP4 in meiotic CO formation. Taken together, we propose that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 form multiple protein complexes that have conserved functions in promoting class I CO formation.

Light and gravity are two key determinants in orientating plant stems for proper growth and development. The organization and dynamics of the actin cytoskeleton are essential for cell biology and critically regulated by actin-binding proteins. However, the role of actin cytoskeleton in shoot negative gravitropism remains controversial. In this work, we report that the actin-binding protein Rice Morphology Determinant (RMD) promotes reorganization of the actin cytoskeleton in rice (Oryza sativa) shoots. The changes in actin organization are associated with the ability of the rice shoots to respond to negative gravitropism. Here, light-grown rmd mutant shoots exhibited agravitropic phenotypes. By contrast, etiolated rmd shoots displayed normal negative shoot gravitropism. Furthermore, we show that RMD maintains an actin configuration that promotes statolith mobility in gravisensing endodermal cells, and for proper auxin distribution in light-grown, but not dark-grown, shoots. RMD gene expression is diurnally controlled and directly repressed by the phytochrome-interacting factor-like protein OsPIL16. Consequently, overexpression of OsPIL16 led to gravisensing and actin patterning defects that phenocopied the rmd mutant. Our findings outline a mechanism that links light signaling and gravity perception for straight shoot growth in rice.

Previous studies have revealed the functions of rice and maize

Background During pollen wall formation in flowering plants, a conserved metabolon consisting of acyl-CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide α-pyrone reductase (TKPR), is required for sporopollenin synthesis. Despite this, the precise function of each of these components in different species remains unclear. Results In this study, we characterized the function of OsTKPR1, a rice orthologue of Arabidopsis TKPR1. Loss of function of OsTKPR1 delayed tapetum degradation, reduced the levels of anther cuticular lipids, and impaired Ubisch body and pollen exine formation, resulting in complete male sterility. In addition, the phenylpropanoid pathway in mutant anthers was remarkably altered. Localization studies suggest that OsTKPR1 accumulates in the endoplasmic reticulum, while specific accumulation of OsTKPR1 mRNA in the anther tapetum and microspores is consistent with its function in anther and pollen wall development. Conclusions Our results show that OsTKPR1 is indispensable for anther cuticle development and pollen wall formation in rice, providing new insights into the biochemical mechanisms of the conserved sporopollenin metabolon in flowering plants. Electronic supplementary material The online version of this article (10.1186/s12870-019-1711-4) contains supplementary material, which is available to authorized users.

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression.

Two Arabidopsis ABC transporters, ABCG1 and ABCG16, are expressed in the tapetal layer, specifically after postmeiotic microspore release, and play important roles in pollen surface development. The male gametophytic cells of terrestrial plants, the pollen grains, travel far before fertilization, and thus require strong protective layers, which take the form of a pollen coat and a pollen wall. The protective surface structures are generated by the tapetum, the tissue surrounding the developing gametophytes. Many ABC transporters, including Arabidopsis thaliana ABCG1 and ABCG16, have been shown to play essential roles in the development of such protective layers. However, the details of the mechanism of their function remain to be clarified. In this study, we show that ABCG1 and ABCG16 are localized at the plasma membrane of tapetal cells, specifically after postmeiotic microspore release, and play critical roles in the postmeiotic stages of male gametophyte development. Consistent with this stage-specific expression, the abcg1 abcg16 double knockout mutant exhibited defects in pollen development after postmeiotic microspore release; their microspores lacked intact nexine and intine layers, exhibited defects in pollen mitosis I, displayed ectopic deposits of arabinogalactan proteins, failed to complete cytokinesis, and lacked sperm cells. Interestingly, the double mutant exhibited abnormalities in the internal structures of tapetal cells, too; the storage organelles of tapetal cells, tapetosomes and elaioplasts, were morphologically altered. Thus, this work reveals that the lack of ABCG1 and ABCG16 at the tapetal cell membrane causes a broad range of defects in pollen, as well as in tapetal cells themselves. Furthermore, these results suggest that normal pollen surface development is necessary for normal development of the pollen cytoplasm.

Root angle has a major impact on acquisition of nutrients like phosphate that accumulate in topsoil and in many species; low phosphate induces shallower root growth as an adaptive response. Identifying genes and mechanisms controlling root angle is therefore of paramount importance to plant breeding. Here we show that the actin-binding protein Rice Morphology Determinant (RMD) controls root growth angle by linking actin filaments and gravity-sensing organelles termed statoliths. RMD is upregulated in response to low external phosphate and mutants lacking of RMD have steeper crown root growth angles that are unresponsive to phosphate levels. RMD protein localizes to the surface of statoliths, and rmd mutants exhibit faster gravitropic response owing to more rapid statoliths movement. We conclude that adaptive changes to root angle in response to external phosphate availability are RMD dependent, providing a potential target for breeders., The orientation of plant roots responds to gravity and influences nutrient acquisition. Here the authors show that the formin RMD buffers movement of specialized gravity-sensing organelles and report enhanced RMD expression during phosphate deficiency that could alter root angle to improve phosphate uptake.

Aminoacyl-tRNA synthetases (aaRSs) have housekeeping roles in protein synthesis, but little is known about how these aaRSs are involved in organ development. Here, we report that a rice (Oryza sativa) glutamyl-tRNA synthetase (OsERS1) maintains proper somatic cell organization and limits the overproliferation of male germ cells during early anther development. The expression of OsERS1 is specifically detectable in meristematic layer 2-derived cells of the early anther, and osers1 anthers exhibit overproliferation and disorganization of layer 2-derived cells, producing fused lobes and extra germ cells in early anthers. The conserved biochemical function of OsERS1 in ligating glutamate to tRNAGlu is enhanced by its cofactor aaRS OsARC. Furthermore, metabolomics profiling revealed that OsERS1 is an important node for multiple metabolic pathways, indicated by the accumulation of amino acids and tricarboxylic acid cycle components in osers1 anthers. Notably, the anther defects of the osers1 mutant are causally associated with the abnormal accumulation of hydrogen peroxide, which can reconstitute the osers1 phenotype when applied to wild-type anthers. Collectively, these findings demonstrate how aaRSs affect male organ development in plants, likely through protein synthesis, metabolic homeostasis, and redox status.

Anther development, particularly around the time of meiosis, is extremely crucial for plant sexual reproduction. Meanwhile, cell-to-cell communication between somatic (especial tapetum) cells and meiocytes are important for both somatic anther development and meiosis. To investigate possible molecular mechanisms modulating protein activities during anther development, we applied high-resolution mass spectrometry-based proteomic and phosphoproteomic analyses for developing rice (Oryza sativa) anthers around the time of meiosis (RAM). In total, we identified 4984 proteins and 3203 phosphoproteins with 8973 unique phosphorylation sites (p-sites). Among those detected here, 1544 phosphoproteins are currently absent in the Plant Protein Phosphorylation DataBase (P3 DB), substantially enriching plant phosphorylation information. Mapman enrichment analysis showed that 'DNA repair','transcription regulation' and 'signaling' related proteins were overrepresented in the phosphorylated proteins. Ten genetically identified rice meiotic proteins were detected to be phosphorylated at a total of 25 p-sites; moreover more than 400 meiotically expressed proteins were revealed to be phosphorylated and their phosphorylation sites were precisely assigned. 163 putative secretory proteins, possibly functioning in cell-to-cell communication, are also phosphorylated. Furthermore, we showed that DNA synthesis, RNA splicing and RNA-directed DNA methylation pathways are extensively affected by phosphorylation. In addition, our data support 46 kinase-substrate pairs predicted by the rice Kinase-Protein Interaction Map, with SnRK1 substrates highly enriched. Taken together, our data revealed extensive protein phosphorylation during anther development, suggesting an important post-translational modification affecting protein activity.

During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, the molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins. We show that the physical and genetic interactions between OsMADS1 and OsMADS3 are essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regulating floral meristem determinacy and suppressing spikelet meristem reversion. We provided important genetic evidence to support the neofunctionalization of two rice C-class genes (OsMADS3 and OsMADS58) during flower development. Gene expression profiling and quantitative RT-PCR analyses further revealed that OsMADS1 affects the expression of many genes involved in floral identity and hormone signaling, and chromatin immunoprecipitation (ChIP)–PCR assay further demonstrated that OsMADS17 is a direct target gene of OsMADS1. Taken together, these results reveal that OsMADS1 has diversified regulatory functions in specifying rice floral organ and meristem identity, probably through its genetic and physical interactions with different floral homeotic regulators.

Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over-expression of the BR-synthesis gene D11 or a BR-signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non-transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop.

Grasses display highly diversified inflorescence architectures that differ in the arrangement of spikelets and flowers and determine cereal yields. However, the molecular basis underlying grass inflorescence morphogenesis remains largely unknown. Here we investigate the role of a functionally diversified SEPALLATA MADS-box transcription factor, OsMADS34, in regulating rice (Oryza sativa L.) inflorescence and spikelet development. Microarray analysis showed that, at the very early stages of inflorescence formation, dysfunction of OsMADS34 caused altered expression of 379 genes that are associated with protein modification and degradation, transcriptional regulation, signaling and metabolism activity. Genetic analysis revealed that OsMADS34 controls different aspects of inflorescence structure, branching and meristem activity synergistically with LAX PANICLE1 (LAX1) and FLORAL ORGAN NUMBER4 (FON4), as evidenced by the enhanced phenotypes of osmads34 lax1 and osmads34 fon4 compared with the single mutants. Additionally, double mutant between osmads34 and the sterile lemma defective mutant elongated empty glume (ele) displayed an enhanced phenotype, that is, longer and wider sterile lemmas that were converted into lemma/palea-like organs, suggesting that ELE and OsMADS34 synergistically control the sterile lemma development. OsMADS34 may act together with OsMADS15 in controlling sterile lemma development. Collectively, these findings provide insights into the regulatory function of OsMADS34 in rice inflorescence and spikelet development.

Loss of function mutation of rice OsACOS12 impairs lipid metabolism-mediated anther cuticle and pollen wall formation, and interferes with tapetum programmed cell death, leading to male sterility. Acyl-CoA Synthetase (ACOS) is one of the enzymes activating fatty acids for various metabolic functions in plants. Here, we show that OsACOS12, an orthologue of Arabidopsis ACOS5 in rice, is crucial for rice fertility. Similar to acos5, osaocs12 mutant had no mature pollen. But unlike acos5, osaocs12 produced defective anthers lacking cutin and Ubisch bodies on the epidermal and inner surfaces, respectively, and delayed programmed cell death (PCD)-induced tapetum degradation. Those phenotypic changes were evident at stage 10, during which OsACOS12 had its maximum expression in tapetal cells and microspores. Chemical analysis revealed that the levels of anther cuticular lipid components (wax and cutin monomers) were significantly reduced in osaocs12, while the expression levels of three known lipid biosynthetic genes were unchanged. Recombinant OsACOS12 enzyme was shown to catalyze the conversion of C18:1 fatty acid to C18:1 CoA in vitro. Phylogenetic analysis indicated that OsACOS12 is an ancient and conserved enzyme associated with the plant’s colonization to earth. Collectively, our study suggests that OsACOS12 is an ancient enzyme participating in a conserved metabolic pathway for diversified biochemical functions to secure male reproduction in plants.

The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:ω-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall.

AGAMOUS-LIKE6 (AGL6) genes play essential roles in flower development, but whether and how they work with floral organ identity genes remain less understood. Here, we describe interactions of the rice (Oryza sativa) AGL6 gene MADS6 with other rice floral homeotic genes in flower development. Genetic analyses revealed that MADS6 specifies the identity of the three inner whorls and floral meristem determinacy redundantly with SUPERWOMAN1/MADS16 (B-gene) or MADS3 (C-gene). MADS6 was shown to define carpel/ovule development and floral determinacy by interacting with MADS13 (D-gene) and control the palea and floral meristem identities together with the YABBY gene DROOPING LEAF. Expression analyses revealed that the transcript levels of six B-, C-, and E-class genes were reduced in mads6-1 at the early flower developmental stage, suggesting that MADS6 is a key regulator of early flower development. Moreover, MADS6 can directly bind to a putative regulatory motif on MADS58 (C-gene), and mads6-1 mads58 displayed phenotypes similar to that of mads6-1. These results suggest that MADS6 is a key player in specifying flower development via interacting with other floral homeotic genes in rice, thus providing new insights into the mechanism by which flower development is controlled.

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.

Grass plants develop unique floral patterns that determine grain production. However, the molecular mechanism underlying the specification of floral organ identities and meristem determinacy, including the interaction among floral homeotic genes, remains largely unknown in grasses. Here, we report the interactions of rice (Oryza sativa) floral homeotic genes, OsMADS3 (a C-class gene), OsMADS13 (a D-class gene), and DROOPING LEAF (DL), in specifying floral organ identities and floral meristem determinacy. The interaction among these genes was revealed through the analysis of double mutants. osmads13-3 osmads3-4 displayed a loss of floral meristem determinacy and generated abundant carpelloid structures containing severe defective ovules in the flower center, which were not detectable in the single mutant. In addition, in situ hybridization and yeast two-hybrid analyses revealed that OsMADS13 and OsMADS3 did not regulate each other’s transcription or interact at the protein level. This indicates that OsMADS3 plays a synergistic role with OsMADS13 in both ovule development and floral meristem termination. Strikingly, osmads3-4 dl-sup6 displayed a severe loss of floral meristem determinacy and produced supernumerary whorls of lodicule-like organs at the forth whorl, suggesting that OsMADS3 and DL synergistically terminate the floral meristem. Furthermore, the defects of osmads13-3 dl-sup6 flowers appeared identical to those of dl-sup6, and the OsMADS13 expression was undetectable in dl-sup6 flowers. These observations suggest that DL and OsMADS13 may function in the same pathway specifying the identity of carpel/ovule and floral meristem. Collectively, we propose a model to illustrate the role of OsMADS3, DL, and OsMADS13 in the specification of flower organ identity and meristem determinacy in rice.

Although AGAMOUS-LIKE6 (AGL6) MADS-box genes are ancient with wide distributions in gymnosperms and angiosperms, their functions remain poorly understood. Here, we show the biological role of the AGL6-like gene, OsMADS6, in specifying floral organ and meristem identities in rice (Oryza sativa L.). OsMADS6 was strongly expressed in the floral meristem at early stages. Subsequently, OsMADS6 transcripts were mainly detectable in paleas, lodicules, carpels and the integument of ovule, as well as in the receptacle. Compared to wild type plants, osmads6 mutants displayed altered palea identity, extra glume-like or mosaic organs, abnormal carpel development and loss of floral meristem determinacy. Strikingly, mutation of a SEPALLATA (SEP)-like gene, OsMADS1 (LHS1), enhanced the defect of osmads6 flowers, and no inner floral organs or glume-like structures were observed in whorls 2 and 3 of osmads1-z osmads6-1 flowers. Furthermore, the osmads1-z osmads6-1 double mutants developed severely indeterminate floral meristems. Our finding, therefore, suggests that the ancient OsMADS6 gene is able to specify "floral state" by determining floral organ and meristem identities in monocot crop rice together with OsMADS1.

As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR ( Tapetum Degeneration Retardation ) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC–MS (gas chromatography–mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr . Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total—154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr . In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development.

After meiosis, tapetal cells in the innermost anther wall layer undergo program cell death (PCD)-triggered degradation. This step is essential for microspore development and pollen wall maturation. We identified a key gene, Defective Tapetum Cell Death 1 (DTC1), that controls this degeneration by modulating the dynamics of reactive oxygen species (ROS) during rice male reproduction. Mutants defective in DTC1 exhibit phenotypes of an enlarged tapetum and middle layer with delayed degeneration, causing male sterility. The gene is preferentially expressed in the tapetal cells during early anther development. In dtc1 anthers, expression of genes encoding secretory proteases or lipid transporters is significantly reduced, while transcripts of PCD regulatory genes, e.g. UDT1, TDR1, and EAT1/DTD, are not altered. Moreover, levels of DTC1 transcripts are diminished in udt1, tdr, and eat1 anthers. These results suggest that DTC1 functions downstream of those transcription factor genes and upstream of the genes encoding secretory proteins. DTC1 protein interacts with OsMT2b, a ROS scavenger. Whereas wild-type plants accumulate large amounts of ROS in their anthers at Stage 9 of development, those levels remain low during all stages of development in dtc1 anthers. These findings indicate that DTC1 is a key regulator for tapetum PCD by inhibiting ROS-scavenging activity.

Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development.

The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in plant and animal genomes. They are known to play important roles in the specification of tissue types in animals. On the other hand, few plant bHLH proteins have been studied functionally. Recent completion of whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) allows genome-wide analysis and comparison of the bHLH family in flowering plants. We have identified 167 bHLH genes in the rice genome, and their phylogenetic analysis indicates that they form well-supported clades, which are defined as subfamilies. In addition, sequence analysis of potential DNA-binding activity, the sequence motifs outside the bHLH domain, and the conservation of intron/exon structural patterns further support the evolutionary relationships among these proteins. The genome distribution of rice bHLH genes strongly supports the hypothesis that genome-wide and tandem duplication contributed to the expansion of the bHLH gene family, consistent with the birth-and-death theory of gene family evolution. Bioinformatics analysis suggests that rice bHLH proteins can potentially participate in a variety of combinatorial interactions, endowing them with the capacity to regulate a multitude of transcriptional programs. In addition, similar expression patterns suggest functional conservation between some rice bHLH genes and their close Arabidopsis homologs.

To understand the expansion of multicopy microRNA (miRNA) families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miR160 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.