Fluorescent cytoskeletal markers reveal associations between the actin and microtubule cytoskeleton in rice cells.

Rice (Oryza sativa) is one of our main food crops, feeding ∼3.5 billion people worldwide. An increasing number of studies note the importance of the cytoskeleton, including actin filaments and microtubules, in rice development and environmental responses. Yet, reliable in vivo cytoskeleton markers are lacking in rice, which limits our knowledge of cytoskeletal functions in living cells. Therefore, we generated bright fluorescent marker lines of the actin and microtubule cytoskeletons in rice, suitable for live-cell imaging in a wide variety of rice tissues. Using these lines, we show that actin bundles and microtubules engage and co-function during pollen grain development, how the cytoskeletal components are coordinated during root cell development, and that the actin cytoskeleton is robust and facilitates microtubule responses during salt stress. Hence, we conclude that our cytoskeletal marker lines, highlighted by our findings of cytoskeletal associations and dynamics, will substantially further future investigations in rice biology.

DWARF TILLER1 regulates apical-basal pattern formation and proper orientation of rice embryos.

Body axis establishment is one of the earliest patterning events in plant embryogenesis. Asymmetric zygote division is critical for apical-basal axis formation in Arabidopsis (Arabidopsis thaliana). However, how the orientation of the cell division plane is regulated and its relation to apical-basal axis establishment and proper position of embryos in grasses remain poorly understood. By characterizing mutants of 3 rice (Oryza sativa) WUSCHEL HOMEOBOX9 (WOX9) genes, whose paralogs in Arabidopsis play essential roles in zygotic asymmetric cell division and cell fate determination, we found 2 kinds of independent embryonic defects: topsy-turvy embryos, in which apical-basal axis twists from being parallel to the longitudinal axis of the seed to being perpendicular; and organ-less embryos. In contrast to their Arabidopsis orthologs, OsWOX9s displayed dynamic distribution during embryo development. Both DWT1/OsWOX9A and DWL2/WOX9C play major roles in the apical-basal axis formation and initiation of stem cells. In addition, DWT1 has a distinct function in regulating the first few embryonic cell divisions to ensure the correct orientation of the embryo in the ovary. In summary, DWT1 acts in 2 steps during rice embryo pattern formation: the initial zygotic division, and with DWL2 to establish the main body axes and stem cell fate 2 to 3 d after pollination.

A leucine-rich-repeat receptor-like kinase regulates pollen aperture formation in rice.

Apertures in pollen grains exhibit species-specific patterns and provide an ideal model for studying cell-surface patterning. Pollen apertures are critical for cereal crop fertility, and while DEFECTIVE IN APERTURE FORMATION1 (OsDAF1) and INAPERTURATE POLLEN1 (OsINP1) have been documented to participate in pollen aperture formation in rice (Oryza sativa), the molecular transduction pathway regulating aperture formation is largely unknown. Here, we report that a leucine-rich-repeat receptor-like kinase (LRR-RLK), AM1, plays a key role in rice pollen aperture formation. Mutations of OsAM1 lead to complete sterility due to disappearance of the pollen aperture and failure in pollen tube germination. OsAM1 encodes a LRR-RLK that belongs to the STRUBBELIG-receptor family. Similar to other reported aperture regulators, OsAM1 assembles to future aperture sites on tetrads after meiosis to regulate aperture formation. The extracellular and intracellular domain of OsAM1 interacts with OsINP1 and OsDAF1, respectively. However, despite their interaction and the absence of aperture formation in osam1 pollen grains, OsINP1 and OsDAF1 localize to future aperture sites at the tetrad stage. Mutation of OsINP1, however, disrupts normal localization of OsAM1, indicating that OsAM1 acts downstream of OsINP1. Our findings reveal the role of a LRR-RLK protein in pollen aperture formation and shed light on the regulatory network of pollen aperture formation.

mRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

Ethylene inhibits rice root elongation in compacted soil via ABA- and auxin-mediated mechanisms.

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.

m6A demethylase OsALKBH5 is required for double-strand break formation and repair by affecting mRNA stability in rice meiosis.

N6-methyladenosine (m6A) RNA modification is the most prevalent messenger RNA (mRNA) modification in eukaryotes and plays critical roles in the regulation of gene expression. m6A is a reversible RNA modification that is deposited by methyltransferases (writers) and removed by demethylases (erasers). The function of m6A erasers in plants is highly diversified and their roles in cereal crops, especially in reproductive development essential for crop yield, are largely unknown. Here, we demonstrate that rice OsALKBH5 acts as an m6A demethylase required for the normal progression of male meiosis. OsALKBH5 is a nucleo-cytoplasmic protein, highly enriched in rice anthers during meiosis, that associates with P-bodies and exon junction complexes, suggesting that it is involved in regulating mRNA processing and abundance. Mutations of OsALKBH5 cause reduced double-strand break (DSB) formation, severe defects in DSB repair, and delayed meiotic progression, leading to complete male sterility. Transcriptome analysis and m6A profiling indicate that OsALKBH5-mediated m6A demethylation stabilizes the mRNA level of multiple meiotic genes directly or indirectly, including several genes that regulate DSB formation and repair. Our study reveals the indispensable role of m6A metabolism in post-transcriptional regulation of meiotic progression in rice.

A promoter polymorphism defines distinct roles in anther development for Col-0 and Ler-0 alleles of Arabidopsis ACYL-COA BINDING PROTEIN3.

Acyl-CoA-Binding Proteins (ACBPs) bind acyl-CoA esters and function in lipid metabolism. Although acbp3-1, the ACBP3 mutant in Arabidopsis thaliana ecotype Col-0, displays normal floral development, the acbp3-2 mutant from ecotype Ler-0 characterized herein exhibits defective adaxial anther lobes and improper sporocyte formation. To understand these differences and identify the role of ERECTA in ACBP3 function, the acbp3 mutants and acbp3-erecta (er) lines were analyzed by microscopy for anther morphology and high-performance liquid chromatography for lipid composition. Defects in Landsberg anther development were related to the ERECTA-mediated pathway because the progenies of acbp3-2 × La-0 and acbp3-1 × er-1 in Col-0 showed normal anthers, contrasting to that of acbp3-2 in Ler-0. Polymorphism in the regulatory region of ACBP3 enabled its function in anther development in Ler-0 but not Col-0 which harbored an AT-repeat insertion. ACBP3 expression and anther development in acbp3-2 were restored using ACBP3pro (Ler)::ACBP3 not ACBP3pro (Col)::ACBP3. SPOROCYTELESS (SPL), a sporocyte formation regulator activated ACBP3 transcription in Ler-0 but not Col-0. For anther development, the ERECTA-related role of ACBP3 is required in Ler-0, but not Col-0. The disrupted promoter regulatory region for SPL binding in Col-0 eliminates the role of ACBP3 in anther development.

Rice HEAT SHOCK PROTEIN60-3B maintains male fertility under high temperature by starch granule biogenesis.

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.

AUXIN RESPONSE FACTORS 6 and 17 control the flag leaf angle in rice by regulating secondary cell wall biosynthesis of lamina joints.

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.

EPAD1 Orthologs Play a Conserved Role in Pollen Exine Patterning.

The pollen wall protects pollen during dispersal and is critical for pollination recognition. In the Poaceae family, the pollen exine stereostructure exhibits a high degree of conservation with similar patterns across species. However, there remains controversy regarding the conservation of key factors involved in its formation among various Poaceae species. EPAD1, as a gene specific to the Poaceae family, and its orthologous genes play a conserved role in pollen wall formation in wheat and rice. However, they do not appear to have significant functions in maize. To further confirm the conserved function of EPAD1 in Poaceae, we performed an analysis on four EPAD1 orthologs from two distinct sub-clades within the Poaceae family. The two functional redundant barley EPAD1 genes (HvEPAD1 and HvEPAD2) from the BOP clade, along with the single copy of sorghum (SbEPAD1) and millet (SiEPAD1) from the PACMAD clade were examined. The CRISPR-Cas9-generated mutants all exhibited defects in pollen wall formation, consistent with previous findings on EPAD1 in rice and wheat. Interestingly, in barley, hvepad2 single mutant also showed apical spikelets abortion, aligning with a decreased expression level of HvEPAD1 and HvEPAD2 from the apical to the bottom of the spike. Our finding provides evidence that EPAD1 orthologs contribute to Poaceae specific pollen exine pattern formation via maintaining primexine integrity despite potential variations in copy numbers across different species.

THERMOSENSITIVE BARREN PANICLE (TAP) is required for rice panicle and spikelet development at high ambient temperature.

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.

The regulatory role of CARBON STARVED ANTHER-mediated photoperiod-dependent male fertility 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.

Multiomics analysis reveals a link between Brassica-specific miR1885 and rapeseed tolerance to low temperature.

Brassica crops include various edible vegetable and plant oil crops, and their production is limited by low temperature beyond their tolerant capability. The key regulators of low-temperature resistance in Brassica remain largely unexplored. To identify posttranscriptional regulators of plant response to low temperature, we performed small RNA profiling, and found that 16 known miRNAs responded to cold treatment in Brassica rapa. The cold response of seven of those miRNAs were further confirmed by qRT-PCR and/or northern blot analyses. In parallel, a genome-wide association study of 220 accessions of Brassica napus identified four candidate MIRNA genes, all of which were cold-responsive, at the loci associated with low-temperature resistance. Specifically, these large-scale data analyses revealed a link between miR1885 and the plant response to low temperature in both B. rapa and B. napus. Using 5' rapid amplification of cDNA ends approach, we validated that miR1885 can cleave its putative target gene transcripts, Bn.TIR.A09 and Bn.TNL.A03, in B. napus. Furthermore, overexpression of miR1885 in Semiwinter type B. napus decreased the mRNA abundance of Bn.TIR.A09 and Bn.TNL.A03 and resulted in increased sensitivity to low temperature. Knocking down of miR1885 in Spring type B. napus led to increased mRNA abundance of its targets and improved rapeseed tolerance to low temperature. Together, our results suggested that the loci of miR1885 and its targets could be potential candidates for the molecular breeding of low temperature-tolerant Spring type Brassica crops.

Combined genome-wide association study and epistasis analysis reveal multifaceted genetic architectures of plant height in Asian cultivated rice.

Plant height (PH) in rice (Oryza sativa) is an important trait for its adaptation and agricultural performance. Discovery of the semi-dwarf1 (SD1) mutation initiated the Green Revolution, boosting rice yield and fitness, but the underlying genetic regulation of PH in rice remains largely unknown. Here, we performed genome-wide association study (GWAS) and identified 12 non-repetitive QTL/genes regulating PH variation in 619 Asian cultivated rice accessions. One of these was an SD1 structural variant, not normally detected in standard GWAS analyses. Given the strong effect of SD1 on PH, we also divided 619 accessions into subgroups harbouring distinct SD1 haplotypes, and found a further 85 QTL/genes for PH, revealing genetic heterogeneity that may be missed by analysing a broad, diverse population. Moreover, we uncovered two epistatic interaction networks of PH-associated QTL/genes in the japonica (Geng)-dominant SD1NIP subgroup. In one of them, the hub QTL/gene qphSN1.4/GAMYB interacted with qphSN3.1/OsINO80, qphSN3.4/HD16/EL1, qphSN6.2/LOC_Os06g11130, and qphSN10.2/MADS56. Sequence variations in GAMYB and MADS56 were associated with their expression levels and PH variations, and MADS56 was shown to physically interact with MADS57 to coregulate expression of gibberellin (GA) metabolic genes OsGA2ox3 and Elongated Uppermost Internode1 (EUI1). Our study uncovered the multifaceted genetic architectures of rice PH, and provided novel and abundant genetic resources for breeding semi-dwarf rice and new candidates for further mechanistic studies on regulation of PH in rice.

Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

Grass-Specific EPAD1 Is Essential for Pollen Exine Patterning in Rice.

The highly variable and species-specific pollen surface patterns are formed by sporopollenin accumulation. The template for sporopollenin deposition and polymerization is the primexine that appears on the tetrad surface, but the mechanism(s) by which primexine guides exine patterning remain elusive. Here, we report that the Poaceae-specific EXINE PATTERN DESIGNER 1 (EPAD1), which encodes a nonspecific lipid transfer protein, is required for primexine integrity and pollen exine patterning in rice (Oryza sativa). Disruption of EPAD1 leads to abnormal exine pattern and complete male sterility, although sporopollenin biosynthesis is unaffected. EPAD1 is specifically expressed in male meiocytes, indicating that reproductive cells exert genetic control over exine patterning. EPAD1 possesses an N-terminal signal peptide and three redundant glycosylphosphatidylinositol (GPI)-anchor sites at its C terminus, segments required for its function and localization to the microspore plasma membrane. In vitro assays indicate that EPAD1 can bind phospholipids. We propose that plasma membrane lipids bound by EPAD1 may be involved in recruiting and arranging regulatory proteins in the primexine to drive correct exine deposition. Our results demonstrate that EPAD1 is a meiocyte-derived determinant that controls primexine patterning in rice, and its orthologs may play a conserved role in the formation of grass-specific exine pattern elements.

Identification and validation of new MADS-box homologous genes in 3010 rice pan-genome.

KEY MESSAGE: Identification and validation of ten new MADS-box homologous genes in 3010 rice pan-genome for rice breeding. The functional genome is significant for rice breeding. MADS-box genes encode transcription factors that are indispensable for rice growth and development. The reported 15,362 novel genes in the rice pan-genome (RPAN) of Asian cultivated rice accessions provided a useful gene reservoir for the identification of more MADS-box candidates to overcome the limitation for the usage of only 75 MADS-box genes identified in Nipponbare for rice breeding. Here, we report the identification and validation of ten MADS-box homologous genes in RPAN. Origin and identity analysis indicated that they are originated from different wild rice accessions and structure of motif analysis revealed high variations in their amino acid sequences. Phylogenetic results with 277 MADS-box genes in 41 species showed that all these ten MADS-box homologous genes belong to type I (SRF-like, M-type). Gene expression analysis confirmed the existence of these ten MADS-box genes in IRIS_313-10,394, all of them were expressed in flower tissues, and six of them were highly expressed during seed development. Altogether, we identified and validated experimentally, for the first time, ten novel MADS-box genes in RPAN, which provides new genetic sources for rice improvement.

Function of the pseudo phosphotransfer proteins has diverged between rice and Arabidopsis.

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.

Wheat AGAMOUS LIKE 6 transcription factors function in stamen development by regulating the expression of Ta APETALA3.

Previous studies have revealed the functions of rice and maize AGAMOUS LIKE 6 (AGL6) genes OsMADS6 and ZAG3, respectively, in floral development; however, the functions of three wheat (Triticum aestivum) AGL6 genes are still unclear. Here, we report the main functions of wheat AGL6 homoeologous genes in stamen development. In RNAi plants, stamens showed abnormality in number and morphology, and a tendency to transform into carpels. Consistently, the expression of the B-class gene TaAPETALA3 (AP3) and the auxin-responsive gene TaMGH3 was downregulated, whereas the wheat ortholog of the rice carpel identity gene DROOPING LEAF was ectopically expressed in RNAi stamens. TaAGL6 proteins bind to the promoter of TaAP3 directly. Yeast one-hybrid and transient expression assays further showed that TaAGL6 positively regulates the expression of TaAP3 in vivo. Wheat AGL6 transcription factors interact with TaAP3, TaAGAMOUS and TaMADS13. Our findings indicate that TaAGL6 transcription factors play an essential role in stamen development through transcriptional regulation of TaAP3 and other related genes. We propose a model to illustrate the function and probable mechanism of this regulation. This study extends our understanding of AGL6 genes.

Temperature-sensitive male sterility in rice determined by the roles of AGO1d in reproductive phasiRNA biogenesis and function.

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.