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.

OsSPLs 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.

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.

Jasmonic Acid Plays a Pivotal Role in Pollen Development and Fertility Regulation in Different Types of P(T)GMS Rice Lines.

Two-line hybrid rice systems represent a new technical approach to utilizing the advantages of rice hybrids. However, the mechanism underlying the male sterile-line fertility transition in rice remains unclear. Peiai 64S (PA64S) is a photoperiod- and thermo-sensitive genic male sterile (PTGMS) line in which male sterility manifests at an average temperature above 23.5 °C under long-day (LD) conditions. Nongken 58S (NK58S) is a LD-sensitive genic male sterile (PGMS) rice that is sterile under LD conditions (above 13.75 h-day). In contrast, D52S is a short-day (SD)-PGMS line that manifests male sterility under SD conditions (below 13.5 h-day). In this study, we obtained fertile and sterile plants from all three lines and performed transcriptome analyses on the anthers of the plants. Gene ontology (GO) analysis suggested that the differentially expressed genes identified were significantly enriched in common terms involved in the response to jasmonic acid (JA) and in JA biosynthesis. On the basis of the biochemical and molecular validation of dynamic, tissue-specific changes in JA, indole-3-acetic acid (IAA) levels, gibberellin (GA) levels, and JA biosynthetic enzyme activities and expression, we proposed that JA could play a pivotal role in viable pollen production through its initial upregulation, constant fluctuation and leaf-spikelet signaling under certain fertility-inducing conditions. Furthermore, we also sprayed methyl jasmonate (MEJA) and salicylhydroxamic acid (SHAM) on the plants, thereby achieving fertility reversal in the PGMS lines NK58S and D52S, with 12.91-63.53% pollen fertility changes. Through qPCR and enzyme activity analyses, we identified two key enzymes-allene oxide synthase (AOS) and allene oxide cyclase (AOC)-that were produced and upregulated by 20-500-fold in PGMS in response to spraying; the activities of these enzymes reversed pollen fertility by influencing the JA biosynthetic pathway. These results provide a new understanding of hormone interactions and networks in male-sterile rice based on the role of JA that will help us to better understand the potential regulatory mechanisms of fertility development in rice in the future.

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.

Lipid metabolism is involved in male fertility regulation of the photoperiod- and thermo sensitive genic male sterile rice line Peiai 64S.

Peiai 64S (PA64S) is a photoperiod- and thermo sensitive genic male sterile (PTGMS) rice line that has been widely applied in two-line hybrid rice breeding. The male fertility mechanism of PTGMS has always been the research focus. We obtained fertile PA64S (F) and sterile fertile PA64S (S) plants at 21℃ and 28℃, respectively. Here, we analyzed the development of anthers and pollen grains of PA64S (S) and found that the degradation of tapetum and sporopollenin accumulation of pollen exine was abnormal. The content of lipid components in PA64S (F) and PA64S (S) were different by LC-MS, among which sterols, (O-acyl) ω-hydroxy fatty acids, ceramide, and other lipid components were upregulated in PA64S (F). The results of transcriptome showed that many significantly different genes were enriched in the lipid metabolism pathways. Additionally, lipid synthesis and transport genes were downregulated in PA64S (S). In summary, the differences of the PA64S fertility under different temperatures were analyzed through multi-levels comparison. These results suggest that lipid synthesis and transport during PA64S anther development affects the lipid accumulation of pollen exine, and ultimately affected fertility. The differences in lipids content may also be a factor affecting PA64S pollen fertility.

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.

Gene mapping and transcriptome profiling of a practical photo-thermo-sensitive rice male sterile line with seedling-specific green-revertible albino leaf.

Abnormal environment weather can cause rice photoperiod-thermo-sensitive genic male sterile (PTGMS) lines fertile or partially fertile and thus cause the mixture of true hybrids with selfing seeds. Seedling-specific green-revertible albino leaf color mutant can be used to distinguish the real hybrids. Besides, it can also be used as an ideal material to research the development of chloroplast and biosynthesis of chlorophyll. The phenotype of leaf color mutants includes light green, yellowing, albino, green-revertible albino. Gene mutations affecting the synthesis and degradation of photosynthetic pigments, lycopene and heme, the differentiation and development of chloroplast, gibberellins (GAs) biosynthesis, can change the leaf color. We have created a PTGMS line with seedling-specific green-revertible albino leaf named W01S. The leaf phenotype, pollen sterility and fertility, agronomic traits, heredity, gene mapping and RNA-Seq of the differentially expressed genes between albino and green-revertible leaves were investigated. The results showed that W01S is a practical PTGMS line as Pei'ai 64S. The mutation of candidate gene Os03g0594100 (ent-isokaurene C2-hydroxylase-like) in W01S can be related to the biosynthesis of GAs, indole acetic acids, ethylene.

Jasmonic acid deficiency leads to scattered floret opening time in cytoplasmic male sterile rice Zhenshan 97A.

Cytoplasmic male sterile (CMS) rice has been widely used for hybrid rice seed production in China. However, CMS rice suffers from undesirable flowering habits including scattered floret opening time (FOT), which causes different FOTs among parental rice plants and greatly reduces hybrid rice seed production. Little is known about the mechanism of scattered FOT in CMS rice. Our results demonstrate that scattered FOT in CMS rice Zhenshan 97A (ZS97A) resulted from the lack of a driving force to open florets, which was directly caused by retarded lodicule expansion. Our results indicate that retarded lodicule expansion in ZS97A was caused by reduced water accumulation due to retarded accumulation of osmotic regulation substances (ORSs). Further, the retardation in accumulation of ORSs and water were caused by jasmonic acid (JA) deficiency, resulting from down-regulation of OsAOC expression. Applying JA restored scattered FOT in ZS97A by promoting ORS and water accumulation, and inducing the expansion of the lodicules. Taken together, JA deficiency inhibited lodicule expansion by retarding the accumulation of ORSs and water, leading to scattered FOT in CMS rice ZS97A.

Reduction of pyruvate orthophosphate dikinase activity is associated with high temperature-induced chalkiness in rice grains.

Global warming affects both rice (Oryza sativa) yields and grain quality. Rice chalkiness due to high temperature during grain filling would lower the grain quality. The biochemical and molecular mechanisms responsible for the increased occurrence of chalkiness under high temperature are not fully understood. Previous research suggested that cytosolic pyruvate orthophosphate dikinase (cyPPDK, EC 2.7.9.1) in rice modulates carbon metabolism. The objective of this study was to determine the relationship between cyPPDK and high temperature-induced chalkiness. High temperature treatments were applied during the grain filling of two rice cultivars (9311 and TXZ-25) which had different sensitivity of chalkiness to high temperature. Chalkiness was increased significantly under high temperature treatment, especially for TXZ-25. A shortened grain filling duration and a decreased grain weight in both cultivars were caused by high temperature treatment. A reduction in PPDK activities due to high temperature was observed during the middle and late grain filling periods, accompanied by down regulated cyPPDK mRNA and protein levels. The temperature effects on the developmental regulation of PPDK activity were confirmed at transcription, translation and post-translational levels. PPDK activities were insensitive to variation in PPDK levels, suggesting the rapid phosphorylation mechanism of this protein. The two varieties showed similar responses to the high temperature treatment in both PPDK activities and chalkiness. We concluded that high temperature-induced chalkiness was associated with the reduction of PPDK activity.

Characterization of a novel glutelin subunit OsGluBX by the experimental approach and molecular dynamics simulations.

Rice glutelin is a multi-subunit storage protein and has high nutritional value. However, many glutelin subunits are still not identified by experiment approach. In this study, a novel subunit (OsGluBX) was discovered by sequence alignment in the UniProtKB database. And then, the OsGluBX of rice from japonica cv. Nipponbare and indica cv. 9311 were cloned and expressed in Escherichia coli system and further identified by Western blotting. The total storage proteins were extracted from the grains of Nipponbare and 9311, and the native OsGluBX were identified. The novel OsGluBX subunit was classified into the subfamily B based on its high homology to the subfamily B members and their immunoblotting identification against the subfamily-specific antibody. Furthermore, two-dimensional electrophoresis analysis showed similarity and difference of the entire glutelin profiles between the two subspecies. Moreover, the atomic coordinate of the OsGluBX was constructed based on homology modeling approach and refined by molecular dynamics simulations. The spatial conformation of the OsGluBX protein was stable during the simulation, and the obvious hydrogen bonds were observed to maintain the integrity and stability of the ß-sheets region of the OsGluBX. Research into this novel OsGluBX subunit has improved our understanding of the glutelin family in rice.

Origin of Oryza sativa in China inferred by nucleotide polymorphisms of organelle DNA.

China is rich of germplasm resources of common wild rice (Oryza rufipogon Griff.) and Asian cultivated rice (O. sativa L.) which consists of two subspecies, indica and japonica. Previous studies have shown that China is one of the domestication centers of O. sativa. However, the geographic origin and the domestication times of O. sativa 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 O. rufipogon and it might be the primary domestication region of O. sativa. 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 O. rufipogon while japonica was domesticated from O. rufipogon which located in higher latitude. These results provided molecular evidences for the hypotheses of (i) Southern China is the origin center of O. sativa in China and (ii) the two subspecies of O. sativa were domesticated multiple times.

Domestication and geographic origin of Oryza sativa in China: insights from multilocus analysis of nucleotide variation of O. sativa and O. rufipogon.

Previous studies have indicated that China is one of the domestication centres of Asian cultivated rice (Oryza sativa), and common wild rice (O. rufipogon) is the progenitor of O. sativa. However, the number of domestication times and the geographic origin of Asian cultivated rice in China are still under debate. In this study, 100 accessions of Asian cultivated rice and 111 accessions of common wild rice in China were selected to examine the relationship between O. sativa and O. rufipogon and thereby infer the domestication and evolution of O. sativa in China through sequence analyses of six gene regions, trnC-ycf6 in chloroplast genomes, cox3 in mitochondrial genomes and ITS, Ehd1, Waxy, Hd1 in nuclear genomes. The results indicated that the two subspecies of O. sativa (indica and japonica) were domesticated independently from different populations of O. rufipogon with gene flow occurring later from japonica to indica; Southern China was the genetic diversity centre of O. rufipogon, and the Pearl River basin near the Tropic of Cancer was the domestication centre of O. sativa in China.

Different effects of night versus day high temperature on rice quality and accumulation profiling of rice grain proteins during grain filling.

High temperature has adverse effects on rice yield and quality. The different influences of night high temperature (NHT) and day high temperature (DHT) on rice quality and seed protein accumulation profiles during grain filling in indica rice '9311' were studied in this research. The treatment temperatures of the control, NHT, and DHT were 28°C/20°C, 27°C/35°C, and 35°C/27°C, respectively, and all the treatments were maintained for 20 days. The result of rice quality analysis indicated that compared with DHT, NHT exerted less effect on head rice rate and chalkiness, whereas greater effect on grain weight. Moreover, the dynamic accumulation change profiles of 61 protein spots, differentially accumulated and successfully identified under NHT and DHT conditions, were performed by proteomic approach. The results also showed that the different suppressed extent of accumulation amount of cyPPDKB might result in different grain chalkiness between NHT and DHT. Most identified isoforms of proteins, such as PPDK and pullulanase, displayed different accumulation change patterns between NHT and DHT. In addition, compared with DHT, NHT resulted in the unique accumulation patterns of stress and defense proteins. Taken together, the mechanisms of seed protein accumulation profiles induced by NHT and DHT during grain filling should be different in rice, and the potential molecular basis is discussed in this study.

Construction of a stable expression vector for Leifsonia xyli subsp. cynodontis and its application in studying the effect of the bacterium as an endophytic bacterium in rice.

To study the possibility of utilizing genetically engineered Leifsonia xyli subsp. cynodontis (Lxc) as an endophytic bacterium in rice, we constructed an Escherichia coli-Lxc shuttle vector, pLGUS, containing a beta-glucuronidase reporter gene, which was stable both in vitro and in vivo. Lxc grows and expresses the beta-glucuronidase reporter gene in all parts of rice, except for seed. A 2-year field study using three rice varieties from China showed that Lxc inoculation did not have a negative effect on the growth and yield of any of these varieties. Therefore, Lxc has the potential to be used as a benign endophyte for the expression of foreign genes in rice.

[Effect of temperature on the fertility restoration of different two-line hybrid rice].

On the basis of the fertility observation of hybrids from Pei'ai 64S and Nongken 58S crossed with an indica variety Nanjing11 and japonica marker line FL235, respectively, The plant growth chambers were employed to expose the F2 plant individuals to such different day mean temperature as 24 degrees C, 27 degrees C and 30 degrees C during natural long hour daylight from July to August in Wuhan (30 degrees 27' N), with a view to analyzing the difference in the temperature sensitivity of fertility between the two kinds of different fertility-restoring genes for two years running. The results showed that, whether it was under long daylight and high temperature or under long daylight and middle temperature, the mean natural seed-set percent of F1 was higher than 68.75%, suggesting that Nanjing11 could completely restore the fertility of Pei'ai 64S. And however, under natural high temperature condition, N58S x FL235 F1 could set seed naturally with 21.93%-26.75%, the mean natural seed set percent of F1 was 46.36%-48.38% in natural middle temperature condition, ability of FL235 to restore the fertility of N58S was affected by high temperature. Further analysis proved that temperature could not alter the inheritance mode of F2 but affect the extent of fertility genes expression in Nanjing11. On the other hand, the expression of fertility-genes of FL235 was sensitive to high temperature, whose the putative critical temperature was 27 degrees C, and high temperature influenced not only the genetic interactions but also the segregation modes in F2 generations.

Genetic analysis of the low critical sterility temperature point in photoperiod-thermo sensitive genic male sterile rice.

It has been a long haul but photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice has not been freely used in hybrid rice production because there are two perplexing problems corresponding to the critical sterility temperature point (CSTP): the uncertainty of the CSTP segregating pattern and the instability of CSTP for every originally useful line. N5088S, the most widely commercialized japonica-type PTGMS line in China, also saw that its CSTP variants have been isolated but with all other agronomic characteristics unchanged. In this report we analyzed the genetic basis of CSTP, by employing the iterated expectation and conditional maximization (IECM) algorithm on four tiller-splitting-formed sets of seven generations from N5088S and its CSTP-variant H5088S, each set treated with one temperature regime. The main results indicated that there are two dominant major genes and polygene, as well as their respective epistasis conditioning the CSTP in the 23.5 degrees C regime. Based on the results obtained, the strategy for breeding of PTGMS lines with stable low CSTP was outlined.