The rice LEC1-like transcription factor OsNF-YB9 interacts with SPK, an endosperm-specific sucrose synthase protein kinase, and functions in seed development.

LEAFY COTYLEDON1 (LEC1), a NUCLEAR FACTOR-Y (NF-Y) family member, plays a critical role in embryogenesis and seed development in Arabidopsis. Previous studies have shown that rice OsNF-YB9 and OsNF-YB7 are homologous to Arabidopsis LEC1. However, the functions of LEC1-like genes in rice remain unclear. Here we report that OsNF-YB9 and OsNF-YB7 display sub-functionalization in rice. We demonstrate that OsNF-YB7 is expressed mainly in the embryo, whereas OsNF-YB9 is preferentially expressed in the developing endosperm. Heterologous expression of either OsNF-YB9 or OsNF-YB7 in Arabidopsis lec1-1 was able to complement the lec1-1 defects. We failed to generate osnf-yb7 homozygous mutants due to lethality caused by OsNF-YB7 defects. Loss of OsNF-YB9 function caused abnormal seed development: seeds were longer, narrower and thinner and exhibited a higher chalkiness ratio. Furthermore, the expression of genes related to starch synthesis was deregulated in osnf-yb9. OsNF-YB9 could interact with SPK, a sucrose synthase protein kinase that is predominantly expressed in rice endosperm. Knockout of SPK resulted in chalky seeds similar to those observed in the osnf-yb9 mutants. Ectopic expression of OsNF-YB9 in both rice and Arabidopsis resulted in unhealthy plants with small seeds. Taken together, these results suggest a critical role for OsNF-YB9 in rice seed development.

OsYUC11-mediated auxin biosynthesis is essential for endosperm development of rice.

Auxin is a phytohormone essential for plant development. However, our understanding of auxin-regulated endosperm development remains limited. Here, we described rice YUCCA (YUC) flavin-containing monooxygenase encoding gene OsYUC11 as a key contributor to auxin biosynthesis in rice (Oryza sativa) endosperm. Grain filling or storage product accumulation was halted by mutation of OsYUC11, but the deficiencies could be recovered by the exogenous application of auxin. A rice transcription factor (TF) yeast library was screened, and 41 TFs that potentially bind to the OsYUC11 promoter were identified, of which OsNF-YB1, a member of the nuclear factor Y family, is predominantly expressed in the endosperm. Both osyuc11 and osnf-yb1 mutants exhibited reduced seed size and increased chalkiness, accompanied by a reduction in indole-3-acetic acid biosynthesis. OsNF-YB1 can bind the OsYUC11 promoter to induce gene expression in vivo. We also found that OsYUC11 was a dynamically imprinted gene that predominantly expressed the paternal allele in the endosperm up to 10 d after fertilization (DAF) but then became a non-imprinted gene at 15 DAF. A functional maternal allele of OsYUC11 was able to recover the paternal defects of this gene. Overall, the findings indicate that OsYUC11-mediated auxin biosynthesis is essential for endosperm development in rice.

Five Rice Seed-Specific NF-YC Genes Redundantly Regulate Grain Quality and Seed Germination via Interfering Gibberellin Pathway.

NF-YCs are important transcription factors with diverse functions in the plant kingdoms including seed development. NF-YC8, 9, 10, 11 and 12 are close homologs with similar seed-specific expression patterns. Despite the fact that some of the NF-YCs are functionally known; their biological roles have not been systematically explored yet, given the potential functional redundancy. In this study, we generated pentuple mutant pnfyc of NF-YC8-12 and revealed their functions in the regulation of grain quality and seed germination. pnfyc grains displayed significantly more chalkiness with abnormal starch granule packaging. pnfyc seed germination and post-germination growth are much slower than the wild-type NIP, largely owing to the GA-deficiency as exogenous GA was able to fully recover the germination phenotype. The RNA-seq experiment identified a total of 469 differentially expressed genes, and several GA-, ABA- and grain quality control-related genes might be transcriptionally regulated by the five NF-YCs, as revealed by qRT-PCR analysis. The results demonstrated the redundant functions of NF-YC8-12 in regulating GA pathways that underpin rice grain quality and seed germination, and shed a novel light on the functions of the seed-specific NF-YCs.

Functional divergence of two duplicated Fertilization Independent Endosperm genes in rice with respect to seed development.

Fertilization Independent Endosperm (FIE) is an essential member of Polycomb Repressive Complex 2 (PRC2) that plays important roles in the developmental regulation of plants. OsFIE1 and OsFIE2 are two FIE homologs in the rice genome. Here, we showed that OsFIE1 probably duplicated from OsFIE2 after the origin of the tribe Oryzeae, but has a specific expression pattern and methylation landscape. During evolution, OsFIE1 underwent a less intensive purifying selection than did OsFIE2. The mutant osfie1 produced smaller seeds and displayed reduced dormancy, indicating that OsFIE1 predominantly functions in late seed development. Ectopic expression of OsFIE1, but not OsFIE2, was deleterious to vegetative growth in a dose-dependent manner. The newly evolved N-terminal tail of OsFIE1 was probably not the cause of the adverse effects on vegetative growth. The CRISPR/Cas9-derived mutant osfie2 exhibited impaired cellularization of the endosperm, which suggested that OsFIE2 is indispensable for early seed development as a positive regulator of cellularization. Autonomous endosperm was observed in both OsFIE2+- and osfie1/OsFIE2+- but at a very low frequency. Although OsFIE1-PRC2 exhibited H3K27me3 methyltransferase ability in plants, OsFIE1-PRC2 is likely to be less important for development in rice than is OsFIE2-PRC2. Our findings revealed the functional divergence of OsFIE1 and OsFIE2 and shed light on their distinct evolution following duplication.

OsbZIP76 interacts with OsNF-YBs and regulates endosperm cellularization in rice (Oryza sativa).

Following double fertilization, plant endosperm nuclei undergo syncytial divisions, followed by synchronous cellularization. Cellularization is a key event during endosperm development, but our understanding of its regulation is limited to Arabidopsis. In this study we show that OsbZIP76 regulates cellularization in rice (Oryza sativa). Activation of OsbZIP76 coincided with the initiation of cellularization, and its knockdown or knockout mutants exhibited precocious cellularization. Genes involved in endosperm development or starch biosynthesis were prematurely activated in the osbzip76 caryopsis. As a putative transcription factor, OsbZIP76 alone lacked transcriptional activation activity; however, it interacted with the nuclear factor Y (NF-Y) family transcription factors OsNF-YB9 and OsNF-YB1 in yeast and in planta. OsbZIP76 and OsNF-YB9 were predominantly expressed in the endosperm and the proteins colocalized. Seeds of osnf-yb1 and osbzip76 mutants showed reduced size and reduced apparent amylose content. The parent-of-origin-dependent expression of OsbZIP76 is variable in different rice accessions. In summary, OsbZIP76 is an endosperm-expressed imprinted gene that regulates endosperm development in rice.

The Arabidopsis anaphase-promoting complex/cyclosome subunit 8 is required for male meiosis.

Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation.

A group of nuclear factor Y transcription factors are sub-functionalized during endosperm development in monocots.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that consists of three subunits, NF-YA, NF-YB, and NF-YC. Gene functions of NF-Ys during endosperm development are not well understood. In this study, we identified eight rice NF-Y-encoding genes, namely OsNF-YA8, OsNF-YB1,9, and OsNF-YC8,9,10,11,12, that are predominantly expressed in the endosperm. Interestingly, the close homologs of these OsNF-Ys are present only in monocot species and are also preferentially expressed in the endosperm, suggesting that they have roles in the regulation of endosperm development. A systemic analysis of interactions between rice endosperm-preferential NF-Ys in yeast revealed that OsNF-YBs and OsNF-YCs could interact with each other. We also found that the endosperm-preferential OsNF-YBs and OsNF-YCs could interact with some ethylene response factors (ERFs) of rice. Unlike OsNF-YC8,9,10, the members of OsNF-YB1,9 or OsNF-YC 11,12 showed no transcriptional activation when present alone. However, they displayed functional activity while in dimer form. In addition, OsNF-YB1-knockout lines showed significant changes in seed morphology, further confirming its role in endosperm development. Our findings provide evidence that a group of phylogenetically conserved NF-Ys is probably differentiated in monocots to regulate endosperm development.

Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation.

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation.

Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice.

Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

The DYT1-interacting proteins bHLH010, bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome.

The anther is the male reproductive organ of flowering plants, and the Arabidopsis bHLH transcription factors encoded by DYSFUNCTIONAL TAPETUM1 (DYT1) and ABORTED MICROSPORE (AMS) are required for control of the complex transcriptional networks regulating anther development. Knowledge of the mechanisms by which the bHLH proteins affect this diverse gene expression is quite limited. We examine here three recently duplicated Arabidopsis bHLH genes, bHLH010, bHLH089 and bHLH091, using evolutionary, genetic, morphological and transcriptomic approaches, and uncover their redundant functions in anther development. These three genes are relatively highly expressed in the tapetum of the Arabidopsis anther; single mutants at each of the bHLH010, bHLH089 and bHLH091 loci are developmentally normal, but the various double and triple combinations progressively exhibit increasingly defective anther phenotypes (abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development), indicating their redundant functions in male fertility. Further transcriptomic and molecular analyses suggest that these three proteins act slightly later than DYT1, and also form protein complexes with DYT1, subsequently affecting the correct expression of many DYT1 target genes in the anther development transcriptional network. This study demonstrated that bHLH010, bHLH089 and bHLH091 together are important for the normal transcriptome of the developing Arabidopsis anther, possibly by forming a feed-forward loop with DYT1.

Genetic interactions between diverged alleles of Early heading date 1 (Ehd1) and Heading date 3a (Hd3a)/ RICE FLOWERING LOCUS T1 (RFT1) control differential heading and contribute to regional adaptation in rice (Oryza sativa).

Initiation of flowering, also called heading, in rice (Oryza sativa) is determined by the florigens encoded by Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1). Early heading date 1 (Ehd1) regulates Hd3a and RFT1. However, different rice varieties have diverged alleles of Ehd1 and Hd3a/RFT1 and their genetic interactions remain largely unclear. Here we generated three segregating populations for different combinations of diverged Ehd1 and Hd3a/RFT1 alleles, and analyzed their genetic interactions between these alleles. We demonstrated that, in an ehd1 mutant background, Hd3a was silenced, but RFT1 was expressed (although at lower levels than in plants with a functional Ehd1) under short-day (SD) and long-day (LD) conditions. We identified a nonfunctional RFT1 allele (rft1); the lines carrying homozygous ehd1 and Hd3a/rft1 failed to induce the floral transition under SD and LD conditions. Like Hd3a, RFT1 also interacted with 14-3-3 proteins, the florigen receptors, but a nonfunctional RFT1 with a crucial E105K mutation failed to interact with 14-3-3 proteins. Furthermore, analyses of sequence variation and geographic distribution suggested that functional RFT1 alleles were selected during rice adaptation to high-latitude regions. Our results demonstrate the important roles of RFT1 in rice flowering and regional adaptation.

The ATP-binding cassette transporter OsABCG15 is required for anther development and pollen fertility in rice.

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map-based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.

Hybrid male sterility in rice controlled by interaction between divergent alleles of two adjacent genes.

Sterility is common in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice (Oryza sativa). Although multiple loci for plant hybrid sterility have been identified, it remains unknown how alleles of the loci interact at the molecular level. Here we show that a locus for indica-japonica hybrid male sterility, Sa, comprises two adjacent genes, SaM and SaF, encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. Most indica cultivars contain a haplotype SaM(+)SaF(+), whereas all japonica cultivars have SaM(-)SaF(-) that diverged by nucleotide variations in wild rice. Male semi-sterility in this heterozygous complex locus is caused by abortion of pollen carrying SaM(-). This allele-specific gamete elimination results from a selective interaction of SaF(+) with SaM(-), a truncated protein, but not with SaM(+) because of the presence of an inhibitory domain, although SaM(+) is required for this male sterility. Lack of any one of the three alleles in recombinant plants does not produce male sterility. We propose a two-gene/three-component interaction model for this hybrid male sterility system. The findings have implications for overcoming male sterility in inter-subspecific hybrid rice breeding.

The maternally expressed polycomb group gene OsEMF2a is essential for endosperm cellularization and imprinting in rice.

Cellularization is a key event in endosperm development. Polycomb group (PcG) genes, such as Fertilization-Independent Seed 2 (FIS2), are vital for the syncytium-to-cellularization transition in Arabidopsis plants. In this study, we found that OsEMF2a, a rice homolog of the Arabidopsis PcG gene Embryonic Flower2 (EMF2), plays a role similar to that of FIS2 in regard to seed development, although there is limited sequence similarity between the genes. Delayed cellularization was observed in osemf2a, associated with an unusual activation of type I MADS-box genes. The cell cycle was persistently activated in osemf2a caryopses, which was likely caused by cytokinin overproduction. However, the overaccumulation of auxin was not found to be associated with the delayed cellularization. As OsEMF2a is a maternally expressed gene in the endosperm, a paternally inherited functional allele was unable to recover the maternal defects of OsEMF2a. Many imprinted rice genes were deregulated in the defective hybrid seeds of osemf2a (♀)/9311 (♂) (m9). The paternal expression bias of some paternally expressed genes was disrupted in m9 due to either the activation of maternal alleles or the repression of paternal alleles. These findings suggest that OsEMF2a-PRC2-mediated H3K27me3 is necessary for endosperm cellularization and genomic imprinting in rice.