The WD40 domain-containing protein Ehd5 positively regulates flowering in rice (Oryza sativa).

Heading date (flowering time), which greatly influences regional and seasonal adaptability in rice (Oryza sativa), is regulated by many genes in different photoperiod pathways. Here, we characterized a heading date gene, Early heading date 5 (Ehd5), using a modified bulked segregant analysis method. The ehd5 mutant showed late flowering under both short-day and long-day conditions, as well as reduced yield, compared to the wild type. Ehd5, which encodes a WD40 domain-containing protein, is induced by light and follows a circadian rhythm expression pattern. Transcriptome analysis revealed that Ehd5 acts upstream of the flowering genes Early heading date 1 (Ehd1), RICE FLOWERING LOCUS T 1 (RFT1), and Heading date 3a (Hd3a). Functional analysis showed that Ehd5 directly interacts with Rice outermost cell-specific gene 4 (Roc4) and Grain number, plant height, and heading date 8 (Ghd8), which might affect the formation of Ghd7-Ghd8 complexes, resulting in increased expression of Ehd1, Hd3a, and RFT1. In a nutshell, these results demonstrate that Ehd5 functions as a positive regulator of rice flowering and provide insight into the molecular mechanisms underlying heading date.

Armadillo repeat only protein GS10 negatively regulates brassinosteroid signaling to control rice grain size.

Grain yield and grain quality are major determinants in modern breeding controlled by many quantitative traits loci (QTLs) in rice (Oryza sativa). However, the mechanisms underlying grain shape and quality are poorly understood. Here, we characterize a QTL for grain size and grain quality via map-based cloning from wild rice (W1943), GS10 (Grain Size on Chromosome 10), which encodes a protein with 6 tandem armadillo repeats. The null mutant gs10 shows slender and narrow grains with altered cell size, which has a pleiotropic effect on other agronomical traits. Functional analysis reveals that GS10 interacts with TUD1 (Taihu Dwarf1) and is epistatic to OsGSK2 (glycogen synthase kinase 2) through regulating grain shape and lamina joint inclination, indicating it is negatively involved in brassinosteroid (BR) signaling. Pyramiding gs10 and the grain size gene GW5 into cultivar GLA4 substantially improved grain shape and appearance quality. Natural variation analysis revealed that gs10 from the wild rice Oryza rufipogon W1943 is a rare allele across the rice population. Collectively, these findings advance our understanding of the underlying mechanism of grain shape and provide the beneficial allele of gs10 for future rice breeding and genetic improvement.

A retrotransposon insertion in MUTL-HOMOLOG 1 affects wild rice seed set and cultivated rice crossover rate.

Wild rice (Oryza rufipogon) has a lower panicle seed setting rate (PSSR) and gamete fertility than domesticated rice (Oryza sativa), but the genetic mechanisms of this phenomenon remain unknown. Here, we cloned a null allele of OsMLH1, an ortholog of MutL-homolog 1 to yeast and mammals, from wild rice O. rufipogon W1943 and revealed a 5.4-kb retrotransposon insertion in OsMLH1 is responsible for the low PSSR in wild rice. In contrast to the wild-type, a near isogenic line NIL-mlh1 exhibits defective crossover (CO) formation during meiosis, resulting in reduced pollen viability, partial embryo lethality, and low PSSR. Except for the mutant of mismatch repair gene postmeiotic segregation 1 (Ospms1), all other MutL mutants from O. sativa indica subspecies displayed male and female semi-sterility similar to NIL-mlh1, but less severe than those from O. sativa japonica subspecies. MLH1 and MLH3 did not contribute in an additive fashion to fertility. Two types of MutL heterodimers, MLH1-PMS1 and MLH1-MLH3, were identified in rice, but only the latter functions in promoting meiotic CO formation. Compared to japonica varieties, indica cultivars had greater numbers of CO events per meiosis. Our results suggest that low fertility in wild rice may be caused by different gene defects, and indica and japonica subspecies have substantially different CO rates responsible for the discrepancy between the fertility of mlh1 and mlh3 mutants.

Developmental toxicity of glyphosate on embryo-larval zebrafish (Danio rerio).

Glyphosate (GLY) induces developmental toxicity in fish, but research on the toxicity mechanism is limited. In this study, zebrafish embryos were exposed for 120 hpf to 0.7, 7, and 35 mg L-1 GLY. The results show that GLY treatment induced developmental toxicity in the fish, including premature hatching, reduced heartbeats, pericardial and yolk sac oedema, swim bladder deficiency, and shortened body length, which was possibly due to a significantly decreased triiodothyronine (T3)/thyroxine (T4) ratio and the abnormal expression patterns of hypothalamic-pituitary-thyroid (HPT) (crh, tshß, tr α, tr ß, and t tr ) and growth hormone/insulin-like growth factor (GH/IGF) axis-related genes (gh, ghrα, ghrß, igf1, igf1rα, and igf1rß) in larvae exposed to GLY. In addition, GLY exposure altered the levels of SOD and CAT, increased ROS, promoted malondialdehyde (MDA) content, and significantly altered the levels of endoplasmic reticulum (ER) stress signalling pathway factors (perk, eif2α, gadd34, atf4, ire1α, xbp1, atf6, hspa5, and chop), suggesting that GLY treatment induced oxidative injury and ER stress in the larvae. Further research showed that treatment with a higher concentration of GLY upregulated the levels of iNOS, IL-1ß, and TNF-α while inhibiting the expression of IL-10 and TGF-ß, suggesting that GLY causes an inflammatory reaction in the larvae. In addition, we also found that apoptosis was induced in the larvae, which was determined by acridine orange staining and abnormal expression of p53, caspase-3, -8, and -9. Taken together, our results demonstrate that GLY exposure altered the T3/T4 ratio, disturbed the expression patterns of HPT and GH/IGF axis-related genes, and induced oxidative and ER stress, inflammatory reactions, and apoptosis in the zebrafish larvae. This investigation contributes to improved understanding of the developmental toxicity mechanism of GLY in fish.

The PLATZ Transcription Factor GL6 Affects Grain Length and Number in Rice.

Grain size is one of the key determinants of grain yield. Although a number of genes that control grain size in rice (Oryza sativa) have been identified, the overall regulatory networks behind this process remain poorly understood. Here, we report the map-based cloning and functional characterization of the quantitative trait locus GL6, which encodes a plant-specific plant AT-rich sequence- and zinc-binding transcription factor that regulates rice grain length and spikelet number. GL6 positively controls grain length by promoting cell proliferation in young panicles and grains. The null gl6 mutant possesses short grains, whereas overexpression of GL6 results in large grains and decreased grain number per panicle. We demonstrate that GL6 participates in RNA polymerase III transcription machinery by interacting with RNA polymerase III subunit C53 and transcription factor class C1 to regulate the expression of genes involved in rice grain development. Our findings reveal a further player involved in the regulation of rice grain size that may be exploited in future rice breeding.

An-1 encodes a basic helix-loop-helix protein that regulates awn development, grain size, and grain number in rice.

Long awns are important for seed dispersal in wild rice (Oryza rufipogon), but are absent in cultivated rice (Oryza sativa). The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown. We report here the molecular cloning of a major quantitative trait locus, An-1, which regulates long awn formation in O. rufipogon. An-1 encodes a basic helix-loop-helix protein, which regulates cell division. The nearly-isogenic line (NIL-An-1) carrying a wild allele An-1 in the genetic background of the awnless indica Guangluai4 produces long awns and longer grains, but significantly fewer grains per panicle compared with Guangluai4. Transgenic studies confirmed that An-1 positively regulates awn elongation, but negatively regulates grain number per panicle. Genetic variations in the An-1 locus were found to be associated with awn loss in cultivated rice. Population genetic analysis of wild and cultivated rice showed a significant reduction in nucleotide diversity of the An-1 locus in rice cultivars, suggesting that the An-1 locus was a major target for artificial selection. Thus, we propose that awn loss was favored and strongly selected by humans, as genetic variations at the An-1 locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice.

Genetic control of seed shattering in rice by the APETALA2 transcription factor shattering abortion1.

Seed shattering is an important agricultural trait in crop domestication. SH4 (for grain shattering quantitative trait locus on chromosome 4) and qSH1 (for quantitative trait locus of seed shattering on chromosome 1) genes have been identified as required for reduced seed shattering during rice (Oryza sativa) domestication. However, the regulatory pathways of seed shattering in rice remain unknown. Here, we identified a seed shattering abortion1 (shat1) mutant in a wild rice introgression line. The SHAT1 gene, which encodes an APETALA2 transcription factor, is required for seed shattering through specifying abscission zone (AZ) development in rice. Genetic analyses revealed that the expression of SHAT1 in AZ was positively regulated by the trihelix transcription factor SH4. We also identified a frameshift mutant of SH4 that completely eliminated AZs and showed nonshattering. Our results suggest a genetic model in which the persistent and concentrated expression of active SHAT1 and SH4 in the AZ during early spikelet developmental stages is required for conferring AZ identification. qSH1 functioned downstream of SHAT1 and SH4, through maintaining SHAT1 and SH4 expression in AZ, thus promoting AZ differentiation.

The rice OsLTP6 gene promoter directs anther-specific expression by a combination of positive and negative regulatory elements.

Characterization of tissue-specific plant gene promoters will benefit genetic improvement in crops. Here, we isolated a novel rice anther-specific plant lipid transfer protein (OsLTP6) gene through high through-put expressional profiling. The promoter of OsLTP6 was introduced to the upstream of the uidA gene, which encodes ß-glucuronidase (GUS), and transformed into rice plants for functional analysis. Histochemical and fluorometric GUS assay showed that GUS was specifically expressed in the anthers and pollens in OsLTP6 promoter::uidA transgenic plants. Transverse section of the rice anther further indicated that the OsLTP6 promoter directed the reporter gene specifically expressed in anther tapetum. To identify regulatory elements within OsLTP6 promoter region, four progressive deletions of the OsLTP6 promoter were constructed. The results indicated that the OsLTP6 promoter achieved anther-specific expression through a combination of positive and negative regulatory elements. A 26-bp motif upstream of TATA box was a key transcriptional activator for OsLTP6 gene. CAAT box and GTGA box were the putative motifs to increase the transcription level to full expression. Two negative regulatory elements were also found in two distinct regions within this promoter. They repressed the expression in leaf and stem, respectively. These results revealed the regulating complexity of anther-specific expression.

Genetic control of a transition from black to straw-white seed hull in rice domestication.

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.

Negative impacts of microcystin-LR and glyphosate on zebrafish intestine: Linked with gut microbiota and microRNAs?

Microcystin-LR (MC-LR) and glyphosate (GLY) have been classified as a Group 2B and Group 2A carcinogens for humans, respectively, and frequently found in aquatic ecosystems. However, data on the potential hazard of MC-LR and GLY exposure to the fish gut are relatively scarce. In the current study, a subacute toxicity test of zebrafish exposed to MC-LR (35 µg L-1) and GLY (3.5 mg L-1), either alone or in combination was performed for 21 d. The results showed that MC-LR or/and GLY treatment reduced the mRNA levels of tight junction genes (claudin-5, occludin, and zonula occludens-1) and altered the levels of diamine oxidase and D-lactic, indicating increased intestinal permeability in zebrafish. Furthermore, MC-LR and/or GLY treatment remarkably increased the levels of intestinal IL-1ß and IL-8 but decreased the levels of IL-10 and TGF-ß, indicating that MC-LR and/or GLY exposure induced an inflammatory response in the fish gut. MC-LR and/or GLY exposure also activated superoxide dismutase and catalase, generally upregulated the levels of p53, bax, bcl-2, caspase-3, and caspase-9, downregulated the levels of caspase-8 and caused notable histological injury in the fish gut. Moreover, MC-LR and/or GLY exposure also significantly altered the microbial community in the zebrafish gut and the expression of miRNAs (miR-146a, miR-155, miR-16, miR-21, and miR-223). Chronic exposure to MC-LR and/or GLY can induce intestinal damage in zebrafish, and this study is the first to demonstrate an altered gut microbiome and miRNAs in the zebrafish gut after MC-LR and GLY exposure.

Dissecting a heterotic gene through GradedPool-Seq mapping informs a rice-improvement strategy.

Hybrid rice breeding for exploiting hybrid vigor, heterosis, has greatly increased grain yield. However, the heterosis-related genes associated with rice grain production remain largely unknown, partly because comprehensive mapping of heterosis-related traits is still labor-intensive and time-consuming. Here, we present a quantitative trait locus (QTL) mapping method, GradedPool-Seq, for rapidly mapping QTLs by whole-genome sequencing of graded-pool samples from F2 progeny via bulked-segregant analysis. We implement this method and map-based cloning to dissect the heterotic QTL GW3p6 from the female line. We then generate the near isogenic line NIL-FH676::GW3p6 by introgressing the GW3p6 allele from the female line Guangzhan63-4S into the male inbred line Fuhui676. The NIL-FH676::GW3p6 exhibits grain yield highly increased compared to Fuhui676. This study demonstrates that it may be possible to achieve a high level of grain production in inbred rice lines without the need to construct hybrids.

The mechanical properties and in vitro biodegradation and biocompatibility of UV-treated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

Strong mechanical properties and controllable biodegradability, together with biocompatibility, are the important requirement for the development of medical implant materials. In this study, an ultraviolet (UV) radiation method was developed to achieve controlled degradation for bacterial biopolyester poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) which has a low biodegradation rate that limits its application for many implant applications required quick degradation. When UV radiation was applied directly to PHBHHx powder, significant molecular weight (Mw) losses were observed with the powder, Mw reduction depended on the UV radiation time. At the same time, a broad PHBHHx Mw distribution was the result of inhomogeneous radiation. Interestingly, this inhomogeneous radiation helped maintain the mechanical properties of films made of the UV-radiated powder. In comparison, the PHBHHx films subjected to direct UV radiation became very brittle although their degradation was faster than that of the PHBHHx powders subjected to direct UV radiation. After 15 weeks of degradation in simulated body fluid (SBF), films prepared from 8 and 16h UV-treated PHBHHx powders maintained 92% and 87% of their original weights, respectively, while the untreated PHBHHx films lost only 1% of its weight. Significant increases in growth of fibroblast L929 were observed on films prepared from UV-radiated powders. This improved biocompatibility could be attributed to increasing hydrophilic functional groups generated by increasing polar groups C-O and CO. In general, UV-treated PHBHHx powder had a broad Mw distribution, which contributed to fast degradation due to dissolution of low Mw polymer fragments, and strong mechanical property due to high Mw polymer chains. Combined with its improved biocompatibility, PHBHHx is one more step close to become a biomedical implant material.

OsSPL13 controls grain size in cultivated rice.

Although genetic diversity has a cardinal role in domestication, abundant natural allelic variations across the rice genome that cause agronomically important differences between diverse varieties have not been fully explored. Here we implement an approach integrating genome-wide association testing with functional analysis on grain size in a diverse rice population. We report that a major quantitative trait locus, GLW7, encoding the plant-specific transcription factor OsSPL13, positively regulates cell size in the grain hull, resulting in enhanced rice grain length and yield. We determine that a tandem-repeat sequence in the 5' UTR of OsSPL13 alters its expression by affecting transcription and translation and that high expression of OsSPL13 is associated with large grains in tropical japonica rice. Further analysis indicates that the large-grain allele of GLW7 in tropical japonica rice was introgressed from indica varieties under artificial selection. Our study demonstrates that new genes can be effectively identified on the basis of genome-wide association data.

An-2 Encodes a Cytokinin Synthesis Enzyme that Regulates Awn Length and Grain Production in Rice.

A wide range of morphological and physiological traits have changed between cultivated rice Oryza sativa and wild rice Oryza rufipogon under domestication. Here, we report cloning of the An-2 gene, encoding the Lonely Guy Like protein 6 (OsLOGL6), which catalyzes the final step of cytokinin synthesis in O. rufipogon. The near-isogenic line harboring a wild allele of An-2 in the genetic background of the awnless indica Guangluai 4 shows that An-2 promotes awn elongation by enhancing cell division, but decreases grain production by reducing grains per panicle and tillers per plant. We reveal that a genetic variation in the An-2 locus has a large impact on reducing awn length and increasing tiller and grain numbers in domesticated rice. Analysis of gene expression patterns suggests that An-1 regulates the formation of awn primordial, and An-2 promotes awn elongation. Nucleotide diversity of the An-2 locus in cultivated rice was found to be significantly reduced compared with that of wild rice, suggesting that the An-2 locus was subjected to artificial selection. We therefore propose that the selection of genetic variation in An-2 was due to reduced awn length and increased grain yield in cultivated rice.

Collection and comparative analysis of 1888 full-length cDNAs from wild rice Oryza rufipogon Griff. W1943.

A huge amount of cDNA and EST resources have been developed for cultivated rice species Oryza sativa; however, only few cDNA resources are available for wild rice species. In this study, we isolated and completely sequenced 1888 putative full-length cDNA (FLcDNA) clones from wild rice Oryza rufipogon Griff. W1943 for comparative analysis between wild and cultivated rice species. Two cDNA libraries were constructed from 3-week-old leaf samples under either normal or cold-treated conditions. Homology searching of these cDNA sequences revealed that >96.8% of the wild rice cDNAs were matched to the cultivated rice O. sativa ssp. japonica cv. Nipponbare genome sequence. However, <22% of them were fully matched to the cv. Nipponbare genome sequence. The comparative analysis showed that O. rufipogon W1943 had greater similarity to O. sativa ssp. japonica than to ssp. indica cultivars. In addition, 17 novel rice cDNAs were identified, and 41 putative tissue-specific expression genes were defined through searching the rice massively parallel signature-sequencing database. In conclusion, these FLcDNA clones are a resource for further function verification and could be broadly utilized in rice biological studies.

A collection of 10,096 indica rice full-length cDNAs reveals highly expressed sequence divergence between Oryza sativa indica and japonica subspecies.

Relatively few indica rice full-length cDNAs were available to aid in the annotation of rice genes. The data presented here described the sequencing and analysis of 10,096 full-length cDNAs from Oryza sativa subspecies indica Guangluai 4. Of them, 9,029 matched rice genomic sequences in publicly-available databases, and 1,200 were identified as new rice genes. Comparison with the knowledge-based Oryza Molecular Biological Encyclopedia japonica cDNA collection indicated that 3,316 (41.6%) of the 7,965 indica-japonica cDNA pairs showed no distinct variations at protein level (2,117 indica-japonica cDNA pairs showed fully identical and 1,199 indica-japonica cDNA pairs showed no frame shift). Moreover, 3,645 (45.8%) of the indica-japonica pairs showed substantial differences at the protein level due to single nucleotide polymorphisms (SNPs), insertions or deletions, and sequence-segment variations between indica and japonica subspecies. Further experimental verifications using PCR screening and quantitative reverse transcriptional PCR revealed unique transcripts for indica subspecies. Comparative analysis also showed that most of rice genes were evolved under purifying selection. These variations might distinguish the phenotypic changes of the two cultivated rice subspecies indica and japonica. Analysis of these cDNAs extends known rice genes and identifies new ones in rice.