Heat shock-inducible clonal analysis reveals the stepwise establishment of cell fate in the rice stem.

The stem, consisting of nodes and internodes, is the shoot axis, which supports aboveground organs and connects them to roots. In contrast to other organs, developmental processes of the stem remain elusive, especially those initiating nodes and internodes. By introducing an intron into the Cre recombinase gene, we established a heat shock-inducible clonal analysis system in a single binary vector and applied it to the stem in the flag leaf phytomer of rice (Oryza sativa). With detailed characterizations of stem structure and development, we show that cell fate acquisition for each domain of the stem occurs stepwise. Cell fate for a single phytomer was established in the shoot apical meristem (SAM) by one plastochron before leaf initiation. Cells destined for the foot (nonelongating domain at the stem base) also started emerging before leaf initiation. Cell fate acquisition for the node began just before leaf initiation at the flank of the SAM, separating cell lineages for leaves and stems. Subsequently, cell fates for the axillary bud were established in early leaf primordia. Finally, cells committed to the internode emerged from, at most, a few cell tiers of the 12- to 25-cell stage stem epidermis. Thus, internode cell fate is established last during stem development. This study provides the groundwork to unveil underlying molecular mechanisms in stem development and a valuable tool for clonal analysis, which can be applied to various species.

A stepwise route to domesticate rice by controlling seed shattering and panicle shape.

Asian rice (Oryza sativa L.) is consumed by more than half of the world's population. Despite its global importance, the process of early rice domestication remains unclear. During domestication, wild rice (Oryza rufipogon Griff.) acquired non-seed-shattering behavior, allowing humans to increase grain yield. Previous studies argued that a reduction in seed shattering triggered by the sh4 mutation led to increased yield during rice domestication, but our experiments using wild introgression lines show that the domesticated sh4 allele alone is insufficient for shattering loss in O. rufipogon. The interruption of abscission layer formation requires both sh4 and qSH3 mutations, demonstrating that the selection of shattering loss in wild rice was not as simple as previously suggested. Here we identified a causal single-nucleotide polymorphism at qSH3 within the seed-shattering gene OsSh1, which is conserved in indica and japonica subspecies but absent in the circum-aus group of rice. Through harvest experiments, we further demonstrated that seed shattering alone did not significantly impact yield; rather, yield increases were observed with closed panicle formation controlled by SPR3 and further augmented by nonshattering, conferred by integration of sh4 and qSH3 alleles. Complementary manipulation of panicle shape and seed shattering results in a mechanically stable panicle structure. We propose a stepwise route for the earliest phase of rice domestication, wherein selection of visible SPR3-controlled closed panicle morphology was instrumental in the sequential recruitment of sh4 and qSH3, which together led to the loss of shattering.

Rice GLUCAN SYNTHASE-LIKE5 promotes anther callose deposition to maintain meiosis initiation and progression.

Callose is a plant cell wall polysaccharide whose deposition is spatiotemporally regulated in various developmental processes and environmental stress responses. The appearance of callose in premeiotic anthers is a prominent histological hallmark for the onset of meiosis in flowering plants; however, the biological role of callose in meiosis remains unknown. Here, we show that rice (Oryza sativa) GLUCAN SYNTHASE LIKE5 (OsGSL5), a callose synthase, localizes on the plasma membrane of pollen mother cells (PMCs) and is responsible for biogenesis of callose in anther locules through premeiotic and meiotic stages. In Osgsl5 mutant anthers mostly lacking callose deposition, aberrant PMCs accompanied by aggregated, unpaired, or multivalent chromosomes were frequently observed and, furthermore, a considerable number of mutant PMCs had untimely progress into meiosis compared to that of wild-type PMCs. Immunostaining of meiosis-specific protein HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS2 in premeiotic PMCs revealed precocious meiosis entry in Osgsl5 anthers. These findings provide insights into the function of callose in controlling the timing of male meiosis initiation and progression, in addition to roles in microsporogenesis, in flowering plants.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

Widespread and transgenerational retrotransposon activation in inter- and intraspecies recombinant inbred populations of Lotus japonicus.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes, including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a 'shock' that stimulates genome reorganization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained unclear. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the RILs investigated. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as an unexpectedly common event in plant reproduction.

Correction: EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

[This corrects the article DOI: 10.1371/journal.pgen.1007238.].

RNA-Binding Protein MAC5A Is Required for Gibberellin-Regulated Stamen Development.

The development of floral organs is coordinated by an elaborate network of homeotic genes, and gibberellin (GA) signaling is involved in floral organ development; however, the underlying molecular mechanisms remain elusive. In the present study, we found that MOS4-ASSOCIATED COMPLEX 5A (MAC5A), which is a protein containing an RNA-binding motif, was involved in the development of sepals, petals, and stamens; either the loss or gain of MAC5A function resulted in stamen malformation and a reduced seed set. The exogenous application of GA considerably exacerbated the defects in mac5a null mutants, including fewer stamens and male sterility. MAC5A was predominantly expressed in pollen grains and stamens, and overexpression of MAC5A affected the expression of homeotic genes such as APETALA1 (AP1), AP2, and AGAMOUS (AG). MAC5A may interact with RABBIT EARS (RBE), a repressor of AG expression in Arabidopsis flowers. The petal defect in rbe null mutants was at least partly rescued in mac5a rbe double mutants. These findings suggest that MAC5A is a novel factor that is required for the normal development of stamens and depends on the GA signaling pathway.

EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.

Collection, preservation and distribution of Oryza genetic resources by the National Bioresource Project RICE (NBRP-RICE).

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.

KNOTTED1 Cofactors, BLH12 and BLH14, Regulate Internode Patterning and Vein Anastomosis in Maize.

Monocot stems lack the vascular cambium and instead have characteristic structures in which intercalary meristems generate internodes and veins remain separate and scattered. However, developmental processes of these unique structures have been poorly described. BELL1-like homeobox (BLH) transcription factors (TFs) are known to heterodimerize with KNOTTED1-like homeobox TFs to play crucial roles in shoot meristem maintenance, but their functions are elusive in monocots. We found that maize (Zea mays) BLH12 and BLH14 have redundant but important roles in stem development. BLH12/14 interact with KNOTTED1 (KN1) in vivo and accumulate in overlapping domains in shoot meristems, young stems, and provascular bundles. Similar to kn1 loss-of-function mutants, blh12 blh14 (blh12/14) double mutants fail to maintain axillary meristems. Unique to blh12/14 is an abnormal tassel branching and precocious internode differentiation that results in dwarfism and reduced veins in stems. Micro-computed tomography observation of vascular networks revealed that blh12/14 double mutants had reduced vein number due to fewer intermediate veins in leaves and precocious anastomosis in young stems. Based on these results, we propose two functions of BLH12/14 during stem development: (1) maintaining intercalary meristems that accumulate KN1 and prevent precocious internode differentiation and (2) preventing precocious anastomosis of provascular bundles in young stems to ensure the production of sufficient independent veins.

A wide reprogramming of histone H3 modifications during male meiosis I in rice is dependent on the Argonaute protein MEL1.

The roles of epigenetic mechanisms, including small-RNA-mediated silencing, in plant meiosis largely remain unclear, despite their importance in plant reproduction. This study unveiled that rice chromosomes are reprogrammed during the premeiosis-to-meiosis transition in pollen mother cells (PMCs). This large-scale meiotic chromosome reprogramming (LMR) continued throughout meiosis I, during which time H3K9 dimethylation (H3K9me2) was increased, and H3K9 acetylation and H3S10 phosphorylation were broadly decreased, with an accompanying immunostaining pattern shift of RNA polymerase II. LMR was dependent on the rice Argonaute protein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which is specifically expressed in germ cells prior to meiosis, because LMR was severely diminished in mel1 mutant anthers. Pivotal meiotic events, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes, were also disrupted in this mutant. Interestingly, and as opposed to the LMR loss in most chromosomal regions, aberrant meiotic protein loading and hypermethylation of H3K9 emerged on the nucleolar organizing region in the mel1 PMCs. These results suggest that MEL1 plays important roles in epigenetic LMR to promote faithful homologous recombination and synapsis during rice meiosis.

Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs.

Small RNAs that interact with Argonaute (AGO) proteins play central roles in RNA-mediated silencing. MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice AGO, has specific functions in the development of pre-meiotic germ cells and the progression of meiosis. Here, we show that MEL1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21-nucleotide phased small interfering RNAs (phasiRNAs) that bear a 5'-terminal cytosine. Most phasiRNAs are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non-coding RNAs (lincRNAs) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNAs are processed via DICER-LIKE4 (DCL4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118-dependent and the DCL4-dependent pathways are both required for biogenesis of 21-nt phasiRNAs associated with germline-specific MEL1 AGO in rice, and over 700 lincRNAs are key factors for induction of this biogenesis during reproductive-specific stages.

Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

PCR-based INDEL markers co-dominant between Oryza sativa, japonica cultivars and closely-related wild Oryza species.

Wild relatives genetically close to cultivars are precious genetic resources for plant breeding. Oryza rufipogon, O. barthii, O. glumaepatula, O. meridionalis and O. longistaminata are such wild species, and are also categorized as AA genome species based on their structural similarities. Chromosome segment substitution lines (CSSLs) are a powerful resource in breeding and genetics, and numerous rice CSSLs have been produced. This study aimed to develop DNA markers for evaluation of CSSLs directly by PCR and subsequent gel electrophoresis. We confirmed that up to 155 of 188 markers developed for detection of japonica-indica INDELs could also detect INDELs between rice cultivars and wild AA-species accessions. Percentages of applicable markers were higher in O. rufipogon accessions (61.7 to 85.6%), and lower in accessions of other four AA species (39.8 to 51.4%). These markers were distributed throughout the rice chromosomes, and will be useful for genotyping of CSSLs and other genetic resources derived from crosses between rice cultivars and closely related wild species.

Evaluation of genetic variation among wild rice populations in Cambodia.

A total of 448 samples in five natural populations of wild rice (Oryza rufipogon) were collected in Cambodia. They were examined using 12 SSR and two chloroplast markers to evaluate the degree of variation among populations and the genetic structure within populations. In the two annual populations, the number of plants with homozygous alleles at all 12 SSR loci were high (66.3% and 79.5%), suggesting that these plants propagate mainly through self-pollination. In the three perennial populations, no individuals had all homozygous genotypes, but redundant genotypes resulted from clonal propagation were observed. Percentages of the redundant genotypes were highly varied (3.6%, 29.2% and 86.0%). This may be due to the different stable levels of environmental conditions. As for chloroplast genome, most of the wild plants showed the same chloroplast types as most Indica-type cultivars have. However, plants with different chloroplast types were maintained, even in the same population. In tropical Asian countries, many wild rice populations were observed under similar ecological conditions examined in this study. Therefore, the present results concerning population structure will be important to further elucidate genetic features of wild rice, and will also give strong clues to utilize and conserve wild natural genetic resources.

Collapsed abnormal pollen1 gene encoding the Arabinokinase-like protein is involved in pollen development in rice.

We isolated a pollen-defective mutant, collapsed abnormal pollen1 (cap1), from Tos17 insertional mutant lines of rice (Oryza sativa). The cap1 heterozygous plant produced equal numbers of normal and collapsed abnormal grains. The abnormal pollen grains lacked almost all cytoplasmic materials, nuclei, and intine cell walls and did not germinate. Genetic analysis of crosses revealed that the cap1 mutation did not affect female reproduction or vegetative growth. CAP1 encodes a protein consisting of 996 amino acids that showed high similarity to Arabidopsis (Arabidopsis thaliana) l-arabinokinase, which catalyzes the conversion of l-arabinose to l-arabinose 1-phosphate. A wild-type genomic DNA segment containing CAP1 restored mutants to normal pollen grains. During rice pollen development, CAP1 was preferentially expressed in anthers at the bicellular pollen stage, and the effects of the cap1 mutation were mainly detected at this stage. Based on the metabolic pathway of l-arabinose, cap1 pollen phenotype may have been caused by toxic accumulation of l-arabinose or by inhibition of cell wall metabolism due to the lack of UDP-l-arabinose derived from l-arabinose 1-phosphate. The expression pattern of CAP1 was very similar to that of another Arabidopsis homolog that showed 71% amino acid identity with CAP1. Our results suggested that CAP1 and related genes are critical for pollen development in both monocotyledonous and dicotyledonous plants.

A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L.).

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.

YABBY and diverged KNOX1 genes shape nodes and internodes in the stem.

Plant stems comprise nodes and internodes that specialize in solute exchange and elongation. However, their boundaries are not well defined, and how these basic units arise remains elusive. In rice with clear nodes and internodes, we found that one subclade of class I knotted1-like homeobox (KNOX1) genes for shoot meristem indeterminacy restricts node differentiation and allows internode formation by repressing YABBY genes for leaf development and genes from another node-specific KNOX1 subclade. YABBYs promote nodal vascular differentiation and limit stem elongation. YABBY and node-specific KNOX1 genes specify the pulvinus, which further elaborates the nodal structure for gravitropism. Notably, this KNOX1 subclade organization is specific to seed plants. We propose that nodes and internodes are distinct domains specified by YABBY-KNOX1 cross-regulation that diverged in early seed plants.

Development of INDEL markers to discriminate all genome types rapidly in the genus Oryza.

The wild Oryza species are rich in genetic diversity and are good resources for modern breeding of rice varieties. The reliable ex situ conservation of various genetic resources supports both basic and applied rice research. For this purpose, we developed PCR-based and co-dominant insertion/deletion (INDEL) markers which enable the discrimination of the genome types or species in the genus Oryza. First, 12,107 INDEL candidate sequences were found in the BAC end sequences for 12 Oryza species available in public databases. Next, we designed PCR primers for INDEL-flanking sequences to match the characteristics of each INDEL, based on an assessment of their likelihood to give rise to a single or few PCR products in all 102 wild accessions, covering most Oryza genome types. Then, we selected 22 INDEL markers to discriminate all genome types in the genus Oryza. A phylogenetic tree of 102 wild accessions and two cultivars according to amplicon polymorphisms for the 22 INDEL markers corresponded well to those in previous studies, indicating that the INDEL markers developed in this study were a useful tool to improve the reliability of identification of wild Oryza species in the germplasm stocks.

Genetic Regulation of Mitosis-Meiosis Fate Decision in Plants: Is Callose an Oversighted Polysaccharide in These Processes?

Timely progression of the meiotic cell cycle and synchronized establishment of male meiosis in anthers are key to ascertaining plant fertility. With the discovery of novel regulators of the plant cell cycle, the mechanisms underlying meiosis initiation and progression appear to be more complex than previously thought, requiring the conjunctive action of cyclins, cyclin-dependent kinases, transcription factors, protein-protein interactions, and several signaling components. Broadly, cell cycle regulators can be classified into two categories in plants based on the nature of their mutational effects: (1) those that completely arrest cell cycle progression; and (2) those that affect the timing (delay or accelerate) or synchrony of cell cycle progression but somehow complete the division process. Especially the latter effects reflect evasion or obstruction of major steps in the meiosis but have sometimes been overlooked due to their subtle phenotypes. In addition to meiotic regulators, very few signaling compounds have been discovered in plants to date. In this review, we discuss the current state of knowledge about genetic mechanisms to enter the meiotic processes, referred to as the mitosis-meiosis fate decision, as well as the importance of callose (ß-1,3 glucan), which has been unsung for a long time in male meiosis in plants.