Combined analysis of transcriptome and metabolome reveals that sugar, lipid, and phenylpropane metabolism are essential for male fertility in temperature-induced male sterile rice

Photoperiod- and thermosensitive genic male sterility (PTGMS) rice is a vital germplasm resource consisting of two-line hybrid rice in which light and temperature strictly control their fertility changes. Variable environmental conditions present huge risks to the two-lines hybrid seed production. Explaining the regulatory mechanism of male fertility in rice PTGMS lines is an essential prerequisite to ensuring food security production. A group of near-isogenic lines (NILs) of a rice PTGMS line unique to this research group was used for this study. These lines have the same genetic background and regulate male fertility by responding to different temperature changes. Transcriptomic analysis revealed that 315 upregulated genes and 391 regulated genes regulated male fertility in response to temperature changes, and differentially expressed genes (DEGs) were mainly characterized in enrichment analysis as having roles in the metabolic pathways of sugar, lipid and phenylpropanoid. Electron microscopy analysis revealed that a lack of starch accumulation in sterile pollen grains induced by high temperature, with an abnormal exine development and a lack of inner pollen grains. Defective processes for sporopollenin synthesis, sporopollenin transport and pollen wall formation in sterile anthers were verified using qPCR. Targeted metabolomics analysis revealed that most lipids (phospholipids, sphingolipids and fatty acids) and flavonoids (flavones and flavanones) were upregulated in fertile anthers and involved in pollen wall development and male fertility formation, while lignin G units and C-type lignin were the major contributors to pollen wall development. The coding genes for trehalose 6-phosphate phosphatase, beta-1,3-glucanase, phospholipase D and 4-coumarate-CoA ligase are considered essential regulators in the process of male fertility formation. In conclusion, our results indicated that the expression of critical genes and accumulation of metabolites in the metabolism of sugar, lipid, and phenylpropanoid are essential for male fertility formation. The results provide new insights for addressing the negative effects of environmental variation on two-line hybrid rice production

Cia Zeaxanthin Biosynthesis, OsZEP and OsVDE Regulate Striped Leaves Occurring in Response to Deep Transplanting of Rice

The rice leaf color mutant B03S was previously generated from the photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice line Efeng 1S, of which male sterility manifests by photoperiod and temperature but exhibits mainly temperature-sensitive characteristics. After these plants were deeply transplanted, the new leaves manifested typical zebra stripe patterns. Here, B03S was subjected to deep and shallow transplanting, shading with soil and aluminum foil, and control conditions in situ to determine the cause of the striped-leaf trait. The direct cause of striped leaves is the base of the leaf sheath being under darkness during deep transplanting, of which the critical shading range reached or exceeds 4 cm above the base. Moreover, typical striped leaves were analyzed based on the targeted metabolome method by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) combined with transcriptome and real-time quantitative PCR (qPCR)-based verification to clarify the metabolic pathways and transcriptional regulation involved. Carotenoids enter the xanthophyll cycle, and the metabolites that differentially accumulate in the striped leaves include zeaxanthin and its derivatives for photooxidative stress protection, driven by the upregulated expression of OsZEP. These findings improve the understanding of the physiological and metabolic mechanisms underlying the leaf color mutation in rice plants, enrich the theoretical foundation of the nonuniform leaf color phenomenon widely found in nature and highlight key advancements concerning rice production involving the transplanting of seedlings or direct broadcasting of seeds

Research and Progress on the Mechanism of Iron Transfer and Accumulation in Rice Grains

Iron (Fe) is one of the most important micronutrients for organisms. Currently, Fe deficiency is a growing nutritional problem and is becoming a serious threat to human health worldwide. A method that could help alleviate this “hidden hunger” is increasing the bioavailable Fe concentrations in edible tissues of major food crops. Therefore, understanding the molecular mechanisms of Fe accumulation in different crop tissues will help to develop crops with higher Fe nutritional values. Biofortification significantly increases the concentration of Fe in crops. This paper considers the important food crop of rice (Oryza sativa L.) as an example and highlights recent research advances on the molecular mechanisms of Fe uptake and allogeneic uptake in different tissues of rice. In addition, different approaches to the biofortification of Fe nutrition in rice and their outcomes are described and discussed. To address the problems that occur during the development and application of improving nutritional Fe in rice, technical strategies and long-term solutions are also proposed as a reference for the future improvement of staple food nutrition with micronutrients

Integrated Analysis of Small RNA, Transcriptome, and Degradome Sequencing Reveals the MiR156, MiR5488 and MiR399 Are Involved in the Regulation of Male Sterility in PTGMS Rice

A photoperiod- and thermo-sensitive genic male sterile (PTGMS) line is the basic material for two-hybrid rice and is an important genetic breeding resource. Peiai64S (PA64S) is an important germplasm resource of PTGMS rice, and it has been applied to two-line hybrid rice systems in China. Pollen fertility in PA64S is regulated by the temperature and photoperiod, but the mechanism of the fertility transition is unclear. In this study, we obtained the male fertile plant PA64S(F) and the male sterile plant PA64S(S) by controlling different temperatures under long light conditions and used the male fertile and sterile plants to investigate the role of microRNAs (miRNAs) in regulating male fertility in rice. We performed the small RNA library sequencing of anthers from PA64S(S) and PA64S(F). A total of 196 miRNAs were identified—166 known miRNAs among 27 miRNA families and 30 novel miRNAs. In the transcriptome analysis, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes revealed significant enrichment in the synthesis and metabolism of fatty acids and some secondary metabolism pathways such as fatty acid metabolism and phenylalanine metabolism. With a comprehensive analysis of miRNA, transcriptome, and degradome sequencing, we identified that 13 pairs of miRNA/target genes regulated male fertility in rice by responding to temperature change, among which the miR156, miR5488, and miR399 affect the male fertility of PA64S by influencing SPLs, the lignin synthesis of anther walls, and the flavonoid metabolism pathway. The results provide a new understanding of PTGMS rice, which will help us better understand the potential regulatory mechanisms of male sterility in the future

<i>OsSPLs</i> Regulate Male Fertility in Response to Different Temperatures by Flavonoid Biosynthesis and Tapetum PCD in PTGMS Rice

Photoperiod and thermo-sensitive genic male sterile (PTGMS) rice is an important resource for two line hybrid rice production. The SQUAMOSA–promoter binding, such as the (SPL) gene family, encode the plant specific transcription factors that regulate development and defense responses in plants. However, the reports about SPLs participating in male fertility regulation are limited. Here, we identified 19 OsSPL family members and investigated their involvement in the fertility regulation of the PTGMS rice lines, PA2364S and PA2864S, with different fertility transition temperatures. The results demonstrated that OsSPL2, OsSPL4, OsSPL16 and OsSPL17 affect male fertility in response to temperature changes through the MiR156-SPL module. WGCNA (weighted gene co-expression network analysis) revealed that CHI and APX1 were co-expressed with OsSPL17. Targeted metabolite and flavonoid biosynthetic gene expression analysis revealed that OsSPL17 regulates the expression of flavonoid biosynthesis genes CHI, and the up regulation of flavanones (eriodictvol and naringenin) and flavones (apigenin and luteolin) content contributed to plant fertility. Meanwhile, OsSPL17 negatively regulates APX1 to affect APX (ascorbate peroxidase) activity, thereby regulating ROS (reactive oxygen species) content in the tapetum, controlling the PCD (programmed cell death) process and regulating male fertility in rice. Overall, this report highlights the potential role of OsSPL for the regulation of male fertility in rice and provides a new insight for the further understanding of fertility molecular mechanisms in PTGMS rice

Salinity Inhibits Rice Seed Germination by Reducing α-Amylase Activity via Decreased Bioactive Gibberellin Content

Seed germination plays important roles in the establishment of seedlings and their subsequent growth; however, seed germination is inhibited by salinity, and the inhibitory mechanism remains elusive. Our results indicate that NaCl treatment inhibits rice seed germination by decreasing the contents of bioactive gibberellins (GAs), such as GA1 and GA4, and that this inhibition can be rescued by exogenous bioactive GA application. To explore the mechanism of bioactive GA deficiency, the effect of NaCl on GA metabolic gene expression was investigated, revealing that expression of both GA biosynthetic genes and GA-inactivated genes was up-regulated by NaCl treatment. These results suggest that NaCl-induced bioactive GA deficiency is caused by up-regulated expression of GA-inactivated genes, and the up-regulated expression of GA biosynthetic genes might be a consequence of negative feedback regulation of the bioactive GA deficiency. Moreover, we provide evidence that NaCl-induced bioactive GA deficiency inhibits rice seed germination by decreasing α-amylase activity via down-regulation of α-amylase gene expression. Additionally, exogenous bioactive GA rescues NaCl-inhibited seed germination by enhancing α-amylase activity. Thus, NaCl treatment reduces bioactive GA content through promotion of bioactive GA inactivation, which in turn inhibits rice seed germination by decreasing α-amylase activity via down-regulation of α-amylase gene expression

Table_1.DOC

<p>Seed germination plays important roles in the establishment of seedlings and their subsequent growth; however, seed germination is inhibited by salinity, and the inhibitory mechanism remains elusive. Our results indicate that NaCl treatment inhibits rice seed germination by decreasing the contents of bioactive gibberellins (GAs), such as GA₁ and GA_4, and that this inhibition can be rescued by exogenous bioactive GA application. To explore the mechanism of bioactive GA deficiency, the effect of NaCl on GA metabolic gene expression was investigated, revealing that expression of both GA biosynthetic genes and GA-inactivated genes was up-regulated by NaCl treatment. These results suggest that NaCl-induced bioactive GA deficiency is caused by up-regulated expression of GA-inactivated genes, and the up-regulated expression of GA biosynthetic genes might be a consequence of negative feedback regulation of the bioactive GA deficiency. Moreover, we provide evidence that NaCl-induced bioactive GA deficiency inhibits rice seed germination by decreasing α-amylase activity via down-regulation of α-amylase gene expression. Additionally, exogenous bioactive GA rescues NaCl-inhibited seed germination by enhancing α-amylase activity. Thus, NaCl treatment reduces bioactive GA content through promotion of bioactive GA inactivation, which in turn inhibits rice seed germination by decreasing α-amylase activity via down-regulation of α-amylase gene expression.</p

Origin of <em>Oryza sativa</em> in China Inferred by Nucleotide Polymorphisms of Organelle DNA

<div><p>China is rich of germplasm resources of common wild rice (<em>Oryza rufipogon</em> Griff.) and Asian cultivated rice (<em>O. sativa</em> L.) which consists of two subspecies, indica and japonica. Previous studies have shown that China is one of the domestication centers of <em>O</em>. <em>sativa</em>. However, the geographic origin and the domestication times of <em>O</em>. <em>sativa</em> in China are still under debate. To settle these disputes, six chloroplast loci and four mitochondrial loci were selected to examine the relationships between 50 accessions of Asian cultivated rice and 119 accessions of common wild rice from China based on DNA sequence analysis in the present study. The results indicated that Southern China is the genetic diversity center of <em>O</em>. <em>rufipogon</em> and it might be the primary domestication region of <em>O</em>. <em>sativa</em>. Molecular dating suggested that the two subspecies had diverged 0.1 million years ago, much earlier than the beginning of rice domestication. Genetic differentiations and phylogeography analyses indicated that indica was domesticated from tropical <em>O</em>. <em>rufipogon</em> while japonica was domesticated from <em>O</em>. <em>rufipogon</em> which located in higher latitude. These results provided molecular evidences for the hypotheses of (i) Southern China is the origin center of <em>O</em>. <em>sativa</em> in China and (ii) the two subspecies of <em>O</em>. <em>sativa</em> were domesticated multiple times.</p> </div

A map showing the sampled populations of <i>O</i>.

<p><b><i>rufipogon</i></b><b> and the distribution of haplotypes.</b> Detailed information of the samples is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049546#pone.0049546.s005" target="_blank">Table S1</a>. Phylogenetic relationship of the haplotype based on the NJ analysis is indicated below the map. Pie charts show the proportions of the haplotypes within each population. Haplotypes are indicated by different colors. The Tropical of Cancer is indicated by the green dotted line. Codes: CL, Chaling; DX, Dongxiang; JH, Jinghong; JY, Jiangyong; YJ, Yuanjiang; ZP, Zhangpu;</p

Longitude and latitude of the <i>O</i>.

<p><b><i>rufipogon</i></b><b> from H1, H2 and H3.</b> Accessions from H2 and H3 are indicated by circles and accessions from H1 are indicated by triangles. The horizontal ordinate origins from 23.5° (N) which is the Tropical of Cancer located in.</p