Summary: Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.

Significance: In recent years, phenotypic plasticity has received attention for improving plant adaptability to variable environments. For more than half a century, it has been known that rice and cereal plants develop different types of lateral roots (LRs), unlike the dicot model plant Arabidopsis . Despite the importance of plastic LR development under variable water conditions, the molecular mechanisms regulating LR types are unknown. Here, we report the regulatory mechanism of LR primordium size in rice, an important determinant of LR type. We identified two WUSCHEL -related homeobox (WOX) transcription factors that opposingly regulate LR primordium size. Our findings form the basis for improving root phenotypic plasticity for sustainable crop production under variable environments.

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.

The recent whole-genome sequencing of soybean (Glycine max) revealed that soybean experienced whole-genome duplications 59 million and 13 million years ago, and it has an octoploid-like genome in spite of its diploid nature. We analyzed a natural green-cotyledon mutant line, Tenshin-daiseitou. The physiological analysis revealed that Tenshin-daiseitou shows a non-functional stay-green phenotype in senescent leaves, which is similar to that of the mutant of Mendel’s green-cotyledon gene I, the ortholog of SGR in pea. The identification of gene mutations and genetic segregation analysis suggested that defects in GmSGR1 and GmSGR2 were responsible for the green-cotyledon/stay-green phenotype of Tenshin-daiseitou, which was confirmed by RNA interference (RNAi) transgenic soybean experiments using GmSGR genes. The characterized green-cotyledon double mutant d1d2 was found to have the same mutations, suggesting that GmSGR1 and GmSGR2 are D1 and D2. Among the examined d1d2 strains, the d1d2 strain K144a showed a lower Chl a/b ratio in mature seeds than other strains but not in senescent leaves, suggesting a seed-specific genetic factor of the Chl composition in K144a. Analysis of the soybean genome sequence revealed four genomic regions with microsynteny to the Arabidopsis SGR1 region, which included the GmSGR1 and GmSGR2 regions. The other two regions contained GmSGR3a/GmSGR3b and GmSGR4, respectively, which might be pseudogenes or genes with a function that is unrelated to Chl degradation during seed maturation and leaf senescence. These GmSGR genes were thought to be produced by the two whole-genome duplications, and they provide a good example of such whole-genome duplication events in the evolution of the soybean genome.

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T₀ plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T₁ generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

The Oryza officinalis complex is the largest species group in Oryza, with more than nine species from four continents, and is a tertiary gene pool that can be exploited in breeding programs for the improvement of cultivated rice. Most diploid and tetraploid members of this group have a C genome. Using a new reference C genome for the diploid species Oryza officinalis, and draft genomes for two other C genome diploid species O. eichingeri and O. rhizomatis, we examine the influence of transposable elements on genome structure and provide a detailed phylogeny and evolutionary history of the Oryza C genomes. The O. officinalis genome is 1.6 times larger than the A genome of cultivated O. sativa, mostly due to proliferation of Gypsy type long-terminal repeat (LTR) transposable elements, but overall syntenic relationships are maintained with other Oryza genomes (A, B and F). Draft genome assemblies of the two other C genome diploid species, O. eichingeri and O. rhizomatis, and short-read resequencing of a series of other C genome species and accessions reveal that after the divergence of the C genome progenitor, there was still a substantial degree of variation within the C genome species through proliferation and loss of both DNA and LTR transposable elements. We provide a detailed phylogeny and evolutionary history of the Oryza C genomes, and a genomic resource for the exploitation of the Oryza tertiary gene pool.