Jiulin Wang

Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight

Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci (QTLs). We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

D14–SCF D3 -dependent degradation of D53 regulates strigolactone signalling

Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.

Brassinosteroids Regulate Grain Filling in Rice

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO2 assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.

A Chlorophyll-Deficient Rice Mutant with Impaired Chlorophyllide Esterification in Chlorophyll Biosynthesis

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.

Ehd4 Encodes a Novel and Oryza-Genus-Specific Regulator of Photoperiodic Flowering in Rice

Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the “florigen” genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice.

Association of functional nucleotide polymorphisms at DTH2 with the northward expansion of rice cultivation in Asia

Flowering time (i.e., heading date in crops) is an important ecological trait that determines growing seasons and regional adaptability of plants to specific natural environments. Rice (Oryza sativa L.) is a short-day plant that originated in the tropics. Increasing evidence suggests that the northward expansion of cultivated rice was accompanied by human selection of the heading date under noninductive long-day (LD) conditions. We report here the molecular cloning and characterization of DTH2 (for Days to heading on chromosome 2), a minor-effect quantitative trait locus that promotes heading under LD conditions. We show that DTH2 encodes a CONSTANS-like protein that promotes heading by inducing the florigen genes Heading date 3a and RICE FLOWERING LOCUS T 1, and it acts independently of the known floral integrators Heading date 1 and Early heading date 1. Moreover, association analysis and transgenic experiments identified two functional nucleotide polymorphisms in DTH2 that correlated with early heading and increased reproductive fitness under natural LD conditions in northern Asia. Our combined population genetics and network analyses suggest that DTH2 likely represents a target of human selection for adaptation to LD conditions during rice domestication and/or improvement, demonstrating an important role of minor-effect quantitative trait loci in crop adaptation and breeding.

Pollen Semi-Sterility1 Encodes a Kinesin-1–Like Protein Important for Male Meiosis, Anther Dehiscence, and Fertility in Rice

This work describes Pollen Semi-Sterility1, a kinesin-1 like protein that is involved in regulating male meiosis in rice; in pss1 mutants, chromosome segregation is perturbed, causing reduced pollen viability and partial sterility. In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1–like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1–green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.

Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax

Epicuticular wax in plants limits non-stomatal water loss, inhibits postgenital organ fusion, protects plants against damage from UV radiation and imposes a physical barrier against pathogen infection. Here, we give a detailed description of the genetic, physiological and morphological consequences of a mutation in the rice gene WSL2, based on a comparison between the wild-type and an EMS mutant. The mutant’s leaf cuticle membrane is thicker and less organized than that of the wild type, and its total wax content is diminished by ~80%. The mutant is also more sensitive to drought stress. WSL2 was isolated by positional cloning, and was shown to encode a homologue of the Arabidopsis thaliana genes CER3/WAX2/YRE/FLP1 and the maize gene GL1. It is expressed throughout the plant, except in the root. A transient assay carried out in both A. thaliana and rice protoplasts showed that the gene product is deposited in the endoplasmic reticulum. An analysis of the overall composition of the wax revealed that the mutant produces a substantially reduced quantity of C22–C32 fatty acids, which suggests that the function of WSL2 is associated with the elongation of very long-chain fatty acids.

Identification and Characterization of an Epi-Allele of FIE1 Reveals a Regulatory Linkage between Two Epigenetic Marks in Rice

This work identifies an epi-allele of rice Fertilization Independent Endosperm1 (FIE1) with DNA hypomethylation, reduced H3 Lys 9 dimethylation, increased H3 Lys 4 trimethylation, ectopic FIE1 expression, and loss imprinting, plus altered H3 Lys 27 trimethylation and perturbed expression of hundreds of genes. This suggests a regulatory link among these epigenetic marks. DNA methylation and histone H3 Lys 9 dimethylation (H3K9me2) are important epigenetic repression marks for silencing transposons in heterochromatin and for regulating gene expression. However, the mechanistic relationship to other repressive marks, such as histone H3 Lys 27 trimethylation (H3K27me3) is unclear. FERTILIZATION-INDEPENDENT ENDOSPERM1 (FIE1) encodes an Esc-like core component of the Polycomb repressive complex 2, which is involved in H3K27me3-mediated gene repression. Here, we identify a gain-of-function epi-allele (Epi-df) of rice (Oryza sativa) FIE1; this allele causes a dwarf stature and various floral defects that are inherited in a dominant fashion. We found that Epi-df has no changes in nucleotide sequence but is hypomethylated in the 5′ region of FIE1 and has reduced H3K9me2 and increased H3K4me3. In Epi-df, FIE1 was ectopically expressed and its imprinting was disrupted. FIE1 interacted with rice Enhancer of Zeste homologs, consistent with its role in H3K27me3 repression. Ectopic expression of FIE1 in Epi-df resulted in alteration of H3K27me3 levels in hundreds of genes. In summary, this work identifies an epi-allele involved in H3K27me3-mediated gene repression that itself is highly regulated by DNA methylation and histone H3K9me2, thereby shedding light on the link between DNA methylation and histone methylation, the two important epigenetic marks regulating rice development.

A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants

Disruption of the OsClpP6 gene causes a virescent-yellow leaf phenotype, demonstrating an important role of caseinolytic proteases in regulating chloroplast biogenesis and leaf development in rice. The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.