HEI10 was first described in human as a RING domain-containing protein that regulates cell cycle and cell invasion. Mice HEI10(mei4) mutant displays no obvious defect other than meiotic failure from an absence of chiasmata. In this study, we characterize rice HEI10 by map-based cloning and explore its function during meiotic recombination. In the rice hei10 mutant, chiasma frequency is markedly reduced, and those remaining chiasmata exhibit a random distribution among cells, suggesting possible involvement of HEI10 in the formation of interference-sensitive crossovers (COs). However, mutation of HEI10 does not affect early recombination events and synaptonemal complex (SC) formation. HEI10 protein displays a highly dynamic localization on the meiotic chromosomes. It initially appears as distinct foci and co-localizes with MER3. Thereafter, HEI10 signals elongate along the chromosomes and finally restrict to prominent foci that specially localize to chiasma sites. The linear HEI10 signals always localize on ZEP1 signals, indicating that HEI10 extends along the chromosome in the wake of synapsis. Together our results suggest that HEI10 is the homolog of budding yeast Zip3 and Caenorhabditis elegans ZHP-3, and may specifically promote class I CO formation through modification of various meiotic components.

Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), zeta-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene beta-cyclase (beta-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.

Meiotic cytokinesis influences the fertility and ploidy of gametes. However, limited information is available on the genetic control of meiotic cytokinesis in plants. Here, we identified a rice mutant with low male fertility, defective callose in meiosis 1 (dcm1). The pollen grains of dcm1 are proved to be defective in exine formation. Meiotic cytokinesis is disrupted in dcm1, resulting in disordered spindle orientation during meiosis II and formation of pollen grains with varied size and DNA content. We demonstrated that meiotic cytokinesis defect in dcm1 is caused by prematurely dissolution of callosic plates. Furthermore, peripheral callose surrounding the dcm1 pollen mother cells (PMCs) also disappeared untimely around pachytene. The DCM1 protein contains five tandem CCCH motifs and interacts with nuclear poly (A) binding proteins (PABNs) in nuclear speckles. The expression profiles of genes related to callose synthesis and degradation are significantly modified in dcm1. Together, we propose that DCM1 plays an essential role in male meiotic cytokinesis by preserving callose from prematurely dissolution in rice.

Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.

The widespread agricultural problem of pre-harvest sprouting (PHS) could potentially be overcome by improving seed dormancy. Here, we report that miR156, an important grain yield regulator, also controls seed dormancy in rice. We found that mutations in one MIR156 subfamily enhance seed dormancy and suppress PHS with negligible effects on shoot architecture and grain size, whereas mutations in another MIR156 subfamily modify shoot architecture and increase grain size but have minimal effects on seed dormancy. Mechanistically, mir156 mutations enhance seed dormancy by suppressing the gibberellin (GA) pathway through de-represssion of the miR156 target gene Ideal Plant Architecture 1 (IPA1), which directly regulates multiple genes in the GA pathway. These results provide an effective method to suppress PHS without compromising productivity, and will facilitate breeding elite crop varieties with ideal plant architectures.

The repair of SPO11-dependent double-strand breaks (DSBs) by homologous recombination (HR) ensures the correct segregation of homologous chromosomes. In yeast and human, RAD17 is involved in DNA damage checkpoint control and DSB repair. However, little is known about its function in plants. In this study, we characterized the RAD17 homolog in rice. In Osrad17 pollen mother cells (PMCs), associations between non-homologous chromosomes and chromosome fragmentation were constantly observed. These aberrant chromosome associations were dependent on the formation of programmed DSBs. OsRAD17 interacts with OsRAD1 and the meiotic phenotype of Osrad1 Osrad17 is indistinguishable from the two single mutants which have similar phenotypes, manifesting they could act in the same pathway. OsZIP4, OsMSH5 and OsMER3 are members of ZMM proteins in rice that are required for crossover formation. We found that homologous pairing and synapsis, which was roughly unaffected in Oszip4 and Osrad17 single mutant, was severely disturbed in the Oszip4 Osrad17 double mutant. Similar phenotypes were observed in the Osmsh5 Osrad17 and Osmer3 Osrad1 double mutants, suggesting the cooperation between the checkpoint proteins and ZMM proteins in assuring accurate HR in rice.

The widespread introduction of semi-dwarf1 (sd1), also known as the 'Green Revolution' gene, has dramatically increased rice yield. However, the extensive use of limited sources of dwarf genes may cause 'bottleneck' effects in breeding new rice varieties. Alternative dwarf germplasms are quite urgent for rice breeding. Here, we characterized a new allele of the rice Slr1-d mutant, Slr1-d6, which reduced plant height by 37%, a much milder allele for dwarfism. Slr-d6 was still responsive to gibberellin (GA) to a reduced extent. The mutation site in Slr1-d6 was less conserved in the TVHYNP domain, leading to the specific semi-dominant dwarf phenotype. Expression of SLR1 and five key GA biosynthetic genes was disturbed in Slr1-d6, and the interaction between Slr1-d6 and GID1 was decreased. In the genetic background of cultivar 9311 with sd1 eliminated, Slr1-d6 homozygous plants were ~70 cm tall. Moreover, Slr1-d6 heterozygous plants were equivalent in height to the standard sd1 semi-dwarf 9311, but with a 25% yield increase, showing its potential application in hybrid rice breeding.

Sexual reproduction is essential for the life cycle of most angiosperms. However, pseudovivipary is an important reproductive strategy in some grasses. In this mode of reproduction, asexual propagules are produced in place of sexual reproductive structures. However, the molecular mechanism of pseudovivipary still remains a mystery. In this work, we found three naturally occurring mutants in rice, namely, phoenix (pho), degenerative palea (dep), and abnormal floral organs (afo). Genetic analysis of them indicated that the stable pseudovivipary mutant pho was a double mutant containing both a Mendelian mutation in DEP and a non-Mendelian mutation in AFO. Further map-based cloning and microarray analysis revealed that dep mutant was caused by a genetic alteration in OsMADS15 while afo was caused by an epigenetic mutation in OsMADS1. Thus, OsMADS1 and OsMADS15 are both required to ensure sexual reproduction in rice and mutations of them lead to the switch of reproductive habit from sexual to asexual in rice. For the first time, our results reveal two regulators for sexual and asexual reproduction modes in flowering plants. In addition, our findings also make it possible to manipulate the reproductive strategy of plants, at least in rice.

The flag leaf and grain belong to the source and sink, respectively, of cereals, and both have a bearing on final yield. Premature leaf senescence significantly reduces the photosynthetic rate and severely lowers crop yield. Cytokinins play important roles in leaf senescence and determine grain number. Here, we characterized the roles of the rice (Oryza sativa L.) cytokinin oxidase/dehydrogenase OsCKX11 in delaying leaf senescence, increasing grain number, and coordinately regulating source and sink. OsCKX11 was predominantly expressed in the roots, leaves, and panicles and was strongly induced by abscisic acid and leaf senescence. Recombinant OsCKX11 protein catalysed the degradation of various types of cytokinins but showed preference for trans-zeatin and cis-zeatin. Cytokinin levels were significantly increased in the flag leaves of osckx11 mutant compared to those of the wild type (WT). In the osckx11 mutant, the ABA-biosynthesizing genes were down-regulated and the ABA-degrading genes were up-regulated, thereby reducing the ABA levels relative to the WT. Thus, OsCKX11 functions antagonistically between cytokinins and ABA in leaf senescence. Moreover, osckx11 presented with significantly increased branch, tiller, and grain number compared with the WT. Collectively, our findings reveal that OsCKX11 simultaneously regulates photosynthesis and grain number, which may provide new insights into leaf senescence and crop molecular breeding.

Abnormal temperature caused by global climate change threatens the rice production. Defense signaling network for chilling has been uncovered in plants. However, less is known about repairing DNA damage produced from overwhelmed defense and its evolution during domestication. Here, we genetically identified a major QTL, COLD11, using the data-merging genome-wide association study based on an algorithm combining polarized data from two subspecies, indica and japonica, into one system. Rice loss-of-function mutations of COLD11 caused reduced chilling tolerance. Genome evolution analysis of representative rice germplasms suggested that numbers of GCG sequence repeats in the first exon of COLD11 were subjected to strong domestication selection during the northern expansion of rice planting. The repeat numbers affected the biochemical activity of DNA repair protein COLD11/RAD51A1 in renovating DNA damage under chilling stress. Our findings highlight a potential way to finely manipulate key genes in rice genome and effectively improve chilling tolerance through molecular designing.

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

Grain weight and quality are always determined by grain filling. Plant microRNAs have drawn attention as key targets for regulation of grain size and yield. However, the mechanisms that underlie grain size regulation remain largely unclear because of the complex networks that control this trait. Our earlier studies demonstrated that suppressed expression of miR167 (STTM/MIM167) substantially increased grain weight. In a field test, the yield increased up to 12.90%-21.94% because of a significantly enhanced grain filling rate. Here, biochemical and genetic analyses revealed the regulatory effects of miR159 on miR167 expression. Further analysis indicated that OsARF12 is the major mediator by which miR167 regulates rice grain filling. Overexpression of OsARF12 produced grain weight and grain filling phenotypes resembling those of STTM/MIM167 plants. Upon in-depth analysis, we found that OsARF12 activates OsCDKF;2 expression by directly binding to the TGTCGG motif in its promoter region. Flow cytometry analysis of young panicles from OsARF12-overexpressing plants and examination of cell number in cdkf;2 mutants verified that OsARF12 positively regulates grain filling and grain size by targeting OsCDKF;2. Moreover, RNA sequencing results suggested that the miR167-OsARF12 module is involved in the cell development process and hormone pathways. OsARF12-overexpressing plants and cdkf;2 mutants exhibited enhanced and reduced sensitivity to exogenous auxin and brassinosteroid (BR) treatment, confirming that targeting of OsCDKF;2 by OsARF12 mediates auxin and BR signaling. Our results reveal that the miR167-OsARF12 module works downstream of miR159 to regulate rice grain filling and grain size via OsCDKF;2 by controlling cell division and mediating auxin and BR signals.

The shape of grass leaves possesses great value in both agronomy and developmental biology research. Leaf rolling is one of the important traits in rice (Oryza sativa L.) breeding. MYB transcription factors are one of the largest gene families and have important roles in plant development, metabolism and stress responses. However, little is known about their functions in rice.

During meiosis, the paired homologous chromosomes are tightly held together by the synaptonemal complex (SC). This complex consists of two parallel axial/lateral elements (AEs/LEs) and one central element. Here, we observed that PAIR3 localized to the chromosome core during prophase I and associated with both unsynapsed AEs and synapsed LEs. Analyses of the severe pair3 mutant demonstrated that PAIR3 was essential for bouquet formation, homologous pairing and normal recombination, and SC assembly. In addition, we showed that although PAIR3 was not required for the initial recruitment of PAIR2, it was required for the proper association of PAIR2 with chromosomes. Dual immunostaining revealed that PAIR3 highly colocalized with REC8. Moreover, studies using a rec8 mutant indicated that PAIR3 localized to chromosomes in a REC8-dependent manner.

Recent completion of rice genome sequencing has revealed that more than 40% of its genome consists of repetitive sequences, and most of them are related to inactive transposable elements. In the present study, a transposable element, nDaiZ0, which is induced by tissue culture with high frequency, was identified by sequence analysis of an allelic line of the golden hull and internode 2 (gh2) mutant, which was integrated into the forth exon of GH2. The 528-bp nDaiZ0 has 14-bp terminal inverted repeats (TIRs), and generates an 8-bp duplication of its target sites (TSD) during its mobilization. nDaiZs are non-autonomous transposons and have no coding capacity. Bioinformatics analysis and southern blot hybridization showed that at least 16 copies of nDaiZ elements exist in the japonica cultivar Nipponbare genome and 11 copies in the indica cultivar 93-11 genome. During tissue culture, only one copy, nDaiZ9, located on chromosome 5 in the genome of Nipponbare can be activated with its transposable frequency reaching 30%. However, nDaiZ9 was not present in the 93-11 genome. The larger elements, DaiZs, were further identified by database searching using nDaiZ0 as a query because they share similar TIRs and subterminal sequences. DaiZ can also generate an 8-bp TSD. DaiZ elements contain a conserved region with a high similarity to the hAT dimerization motif, suggesting that the nDaiZ-DaiZ transposon system probably belongs to the hAT superfamily of class II transposons. Phylogenetic analysis indicated that it is a new type of plant hAT-like transposon. Although nDaiZ is activated by tissue culture, the high transposable frequency indicates that it could become a useful gene tagging system for rice functional genomic studies. In addition, the mechanism of the high transposable ability of nDaiZ9 is discussed.

The leptotene-zygotene transition is a major step in meiotic progression during which pairing between homologous chromosomes is initiated and double strand breaks occur. OsAM1, a homologue of maize AM1 and Arabidopsis SWI1, encodes a protein with a coiled-coil domain in its central region that is required for the leptotene-zygotene transition during rice meiosis. To gain more insight into the role of OsAM1 in rice meiosis and identify additional meiosis-specific genes, we characterized the transcriptomes of young panicles of Osam1 mutant and wild-type rice plants using RNA-Seq combined with bioinformatic and statistical analyses. As a result, a total of 25,750 and 28,455 genes were expressed in young panicles of wild-type and Osam1 mutant plants, respectively, and 4,400 differentially expressed genes (DEGs; log2 Ratio ≥ 1, FDR ≤ 0.05) were identified. Of these DEGs, four known rice meiosis-specific genes were detected, and 22 new putative meiosis-related genes were found by mapping these DEGs to reference biological pathways in the KEGG database. We identified eight additional well-conserved OsAM1-responsive rice meiotic genes by comparing our RNA-Seq data with known meiotic genes in Arabidopsis and fission yeast.

Potato (Solanum tuberosum) is the most consumed non-cereal food crop. Most commercial potato cultivars are autotetraploids with highly heterozygous genomes, severely hampering genetic analyses and improvement. By leveraging the state-of-the-art sequencing technologies and polyploid graph binning, we achieved a chromosome-scale, haplotype-resolved genome assembly of a cultivated potato, Cooperation-88 (C88). Intra-haplotype comparative analyses revealed extensive sequence and expression differences in this tetraploid genome. We identified haplotype-specific pericentromeres on chromosomes, suggesting a distinct evolutionary trajectory of potato homologous centromeres. Furthermore, we detected double reduction events that are unevenly distributed on haplotypes in 1021 of 1034 selfing progeny, a feature of autopolyploid inheritance. By distinguishing maternal and paternal haplotype sets in C88, we simulated the origin of heterosis in cultivated tetraploid with a survey of 3110 tetra-allelic loci with deleterious mutations, which were masked in the heterozygous condition by two parents. This study provides insights into the genomic architecture of autopolyploids and will guide their breeding.

Synaptonemal complex (SC) assembly and homologous recombination, the most critical events during prophase I, are the prerequisite for faithful meiotic chromosome segregation. However, the underlying regulatory mechanism remains largely unknown. Here, we reveal that a functional RING finger E3 ubiquitin ligase, DESYNAPSIS1 (DSNP1), plays significant roles in SC assembly and homologous recombination during rice meiosis. In the dsnp1 mutant, homologous synapsis is discontinuous and aberrant SC-like polycomplexes occur independent of coaligned homologous chromosomes. Accompanying the decreased foci of HEI10, ZIP4, and MER3 on meiotic chromosomes, the number of crossovers (COs) decreases dramatically in dsnp1 meiocytes. Furthermore, the absence of central elements largely restores the localization of non-ZEP1 ZMM proteins and the number of COs in the dsnp1 background. Collectively, DSNP1 stabilizes the canonical tripartite SC structure along paired homologous chromosomes and further promotes the formation of COs.

Rice (Oryza sativa) OsMADS58 is a C-class MADS box protein, and characterization of a transposon insertion mutant osmads58 suggested that OsMADS58 plays a role in stamen development. However, as no null mutation has been obtained, its role has remained unclear. Here, we report that the CRISPR knockout mutant osmads58 exhibits complex altered phenotypes, including anomalous diploid germ cells, aberrant meiosis, and delayed tapetum degeneration. This CRISPR mutant line exhibited stronger changes in expression of OsMADS58 target genes compared with the osmads58 dSpm (transposon insertion) line, along with changes in multiple pathways related to early stamen development. Notably, transcriptional regulatory circuits in young panicles covering the stamen at stages 4-6 were substantially altered in the CRISPR line compared to the dSpm line. These findings strongly suggest that the pleiotropic effects of OsMADS58 on stamen development derive from a potential role in stabilizing gene regulatory circuits during early stamen development. Thus, this work opens new avenues for viewing and deciphering the regulatory mechanisms of early stamen development from a network perspective.

Grain size and plant architecture are critical factors determining crop productivity. Here, we performed gene editing of the MIR396 gene family in rice and found that MIR396e and MIR396f are two important regulators of grain size and plant architecture. mir396ef mutations can increase grain yield by increasing grain size. In addition, mir396ef mutations resulted in an altered plant architecture, with lengthened leaves but shortened internodes, especially the uppermost internode. Our research suggests that mir396ef mutations promote leaf elongation by increasing the level of a gibberellin (GA) precursor, mevalonic acid, which subsequently promotes GA biosynthesis. However, internode elongation in mir396ef mutants appears to be suppressed via reduced CYP96B4 expression but not via the GA pathway. This research provides candidate gene-editing targets to breed elite rice varieties.