Photo-thermosensitive Genic Male Sterile Line Research Articles

Superoxide dismutase promotes early flowering in Triticum aestivum L.

Superoxide dismutase (SOD) is a first-line-defense antioxidant enzyme that plays a crucial role in scavenging reactive oxygen species (ROS) to maintain homeostasis in plants. SOD catalyzes the conversion of superoxide (O2-) into oxygen (O2) and hydrogen peroxide (H2O2), and besides its role in stress resistance, SOD also impacts plant growth and development. Here, we cloned and characterized a TaCSOD gene from the wheat photo-thermosensitive genic male sterile line BS366. Phylogenetic and motif analyses identified TaCSOD as a Cu/Zn-dependent SOD due to the presence of conserved Cu2+ and Zn2+ binding sites. Overexpression of TaCSOD enhanced drought and salt tolerance in both Arabidopsis thaliana and yeast. In addition, seed germination rate, primary root length, and fresh weight of the transgenic plants were higher than those of the wild-type under drought- and salt-stressed conditions. The Arabidopsis TaCSOD overexpression lines also exhibited an early flowering phenotype, with fewer leaves and shorter flowering period. Nitroblue tetrazolium (NBT) and 3, 3-diaminobenzidine (DAB) staining, along with transcriptome analysis, demonstrated that TaCSOD regulates ROS homeostasis and flowering time through carbohydrate signaling, aging, vernalization, and gibberellic acid pathways. Our study provides valuable insights into the functions of SOD genes in regulating flowering through the regulation of ROS homeostasis in plants.

Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.)

The Photothermosensitive Genic-Male-Sterile (PTGMS) line, Y58S, an indica rice variety, combines high-quality and high-light-efficiency use, disease and stress resistance, and excellent plant type and mating force. Y58S is widely used to assemble two-line hybrid rice varieties, especially super hybrids. The Wx gene is the main effector gene for controlling amylose synthesis, which determines the amylose content (AC) of rice grains. By editing this gene, a glutinous line with a low AC can be obtained. In this study, the CRISPR/Cas9 system was used to mediate the editing of the Wx gene, which caused ultra-low AC mutations that produced a PTGMS glutinous rice strain with excellent waxiness. The results showed that 18 positively transformed plants were obtained from the T0 generation, with a mutation rate of 64.29%, of which six were homozygous mutant plants, indicating that the gene-editing target had a higher targeting efficiency and a higher homozygosity mutation rate. Compared to the wild type, the AC of the mutants was significantly lower. Through molecular marker detection and screening of T1 and T2 generations, five homozygous T-DNA-free mutant strains were identified that were consistent with Y58S in fertility and other agronomic traits except for AC. Among these, the AC of the W-1-B-5 homozygous mutant, the glutinous PTGMS line wx-Y58S, was as low as 0.6%. Our research revealed that the Wx gene of excellent PTGMS rice can be edited to generate a new waxy PTGMS line using the CRISPR/Cas9 system. This study provided a simple and effective strategy for breeding high-yield, high-quality, and glutinous two-line hybrid rice, and provided excellent sterile lines for their large-scale application. Once put into use, waxy hybrid rice will greatly improve the yield of glutinous rice and increase social benefits.

Genome-Wide Identification of lncRNAs Involved in Fertility Transition in the Photo-Thermosensitive Genic Male Sterile Rice Line Wuxiang S.

Long non-coding RNAs (lncRNAs) act as universal regulators of various biological processes, but no genome-wide screening of lncRNAs involved in the fertility transition of the photo-thermosensitive genic male sterile (PTGMS) rice line has been reported. Here, we performed strand-specific RNA sequencing at three developmental stages of a novel PTGMS line Wuxiang S (WXS). A total of 3,948 lncRNAs were identified; 622 of these were detected as differentially expressed lncRNAs (DE-lncRNAs) between male-sterile WXS (WXS-S) and male-fertile WXS (WXS-F). A large proportion of lncRNAs differentially expressed at the stage of pollen mother cells meiosis, suggested that it may be the most critical stage for fertility transition of WXS. Furthermore, functional annotation of the cis- and trans- targets of DE-lncRNAs showed that 150 targets corresponding to 141 DE-lncRNAs were identified as involved in anther and pollen development. Moreover, computational analysis predicted 97 lncRNAs as precursors for 72 miRNAs, and 94 DE-lncRNAs as potential endogenous target mimics (eTMs) for 150 miRNAs. Finally, using the dual luciferase reporter assays, we demonstrated that two lncRNAs act as eTMs to regulate the expression of the SPL and GRF genes by competing for the shared osa-miR156 and osa-miR396, respectively. These genomic characteristics, differential expression, and interaction of lncRNAs with miRNAs and mRNAs contribute to our understanding of the roles of lncRNAs during the fertility transition in PTGMS rice lines.

Uncovering ceRNA integrated networks that associate with fertility in a photoperiod and temperature sensitive male sterile wheat line

The pollen fertility of photoperiod/temperature sensitive genic male sterile (P/TGMS) wheat is controlled by light and/or temperature. Circular RNA (circRNA) and long non-coding RNA (lncRNA) are known to participate in the development of anthers in plants, but their impact on male sterility in the P/TGMS line is not well understood. In this study, we carried out high-throughput sequencing to investigate the differential expression of lncRNAs and circRNAs and their biological functions in anthers of photo-thermosensitive genic male sterile (PTGMS) wheat line BS366-42L during the transition phase of male fertility under four different photoperiod and temperature treatments. Eight lncRNAs, 40 mRNAs and three circRNAs were screened out and thought as essential candidates that closely related to male sterility. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential functions of differentially expressed RNAs. The results indicated that carbohydrate-related metabolism was important for male sterility in the wheat PTGMS line BS366-42L. lncRNA/circRNA-mRNA-miRNA (ceRNA) integrate networks were constructed to reflect their complex inner association with male sterility. Our study provides a systematic perspective on the potential function of RNAs in male fertility in PTGMS lines of wheat.

Jasmonates alleviate spikelet‐opening impairment caused by high temperature stress during anthesis of photo‐thermo‐sensitive genic male sterile rice lines

AbstractHybrids produced by photo‐thermo‐sensitive genic male sterile (PTSGMS) rice (Oryza sativa L.) lines crossed with restorer lines have huge heterosis. However, PTSGMS rice lines show serious spikelet‐opening impairment as high temperature (HT) stress occurs during anthesis. Its physiological mechanism remains unclear. Jasmonates (JAs) are novel phytohormones and play important roles in responses to biotic and abiotic stresses. This study investigated whether and how JAs mediate the effect of HT stress during anthesis on spikelet‐opening of PTSGMS rice lines. Three PTSGMS rice lines were pot‐grown and subjected to HT stress during anthesis. The concentrations of jasmonic acid (JA), methyl jasmonate (MeJA), hydrogen peroxide (H2O2), ascorbic acid (AsA), and soluble sugars and activities of catalase and α‐amylase in the lodicules that locate at the base inside the lemma and function spikelet‐opening were determined. The results showed that increases or decreases in JA and MeJA concentrations in the lodicules under HT stress varied with PTSGMS lines. A PTSGMS line with higher JA and MeJA concentrations under HT stress exhibited a higher opened‐spikelet rate and also showed a higher fertilized‐spikelet rate and a higher seed yield when the line was crossed with a restorer line. Both JA and MeJA concentrations were very significantly (p = .01) and positively correlated with AsA concentrations and activities of catalase and α‐amylase, whereas were negatively correlated with H2O2 concentration. Increases in concentrations of JAs in lodicules by applying JA or MeJA under HT stress during anthesis significantly increased activities of catalase and α‐amylase and concentrations of AsA and soluble sugars, and significantly decreased H2O2 concentration in the lodicules, and, accordingly, increased opened‐spikelet rate. The results suggest that JAs play an important role in alleviating spikelet‐opening impairment caused by HT stress during anthesis through enhancing antioxidant ability and osmotic regulation in the lodicules of PTSGMS rice lines.

Physiological mechanism underlying the effect of high temperature during anthesis on spikelet-opening of photo-thermo-sensitive genic male sterile rice lines

Decrease in the grain yield resulted from a low percentage of opened spikelets under high temperature (HT) during anthesis is a serious problem in the seed production of photo-thermo-sensitive genic male sterile (PTGMS) rice (Oryza sativa L.) lines, and the mechanism is little understood. Elucidating the physiological mechanism underlying the effect of HT during anthesis on spikelet-opening of PTGMS lines would have great significance in exploring the effective way to mitigate the adverse effect of HT. In this study, two PTGMS lines and one restorer line of rice were used and were subjected to normal temperature (NT) and HT treatments. The results showed that, compared with NT, HT significantly decreased the percentage of opened spikelets, fertilization percentage and seed-setting by significantly increasing the percentage of wrapped spikelets and reducing the spikelet-opening angle, length of spikelet-opening time. The HT significantly decreased the contents of soluble sugars, jasmonic acid (JA) and methyl jasmonate (MeJA) in the lodicules before and at glume-opening, which were significantly correlated with and accounts for the low percentage of opened spikelets under HT for rice, especially for the PTGMS lines.

Improvement of rice blast and brown planthopper resistance of PTGMS line C815S in two-line hybrid rice through marker-assisted selection

Two-line hybrid rice breeding, which plays an important role in ensuring food security in China, is one of important approaches to utilize the heterosis in rice. Breeding of photo-thermo-sensitive genic male sterile (PTGMS) lines of two-line hybrid rice with high resistance to diseases and pests is vital for enhancing rice yield stability, reducing environmental pollution, and increasing rice production efficiency. To breed PTGMS rice with resistance to both rice blast and brown planthopper (BPH), four broad-spectrum blast resistance genes Pi9, Pi47, Pi48, and Pi49 and two BPH resistance genes Bph14 and Bph15 were introgressed into a PTGMS line C815S through backcrossing and gene pyramiding coupled with molecular marker-assisted selection (MAS) in this study. Finally, several improved PTGMS lines of C815S which pyramided 1–3 homozygous rice blast resistance genes and two BPH resistance genes were developed. Evaluation of rice blast resistance showed that the improved PTGMS lines were resistant, while C815S was susceptible or only moderately resistant. Assessment of the BPH resistance showed that C815S was highly susceptible, while the improved lines were resistant. The improved lines had similar major agronomic traits and temperature of fertility transition to C815S, and several traits were even better. The newly developed PTGMS lines can be used for breeding of superior two-line hybrid rice or advanced PTGMS lines.

Genomic identification and characterization of MYC family genes in wheat (Triticum aestivum L.)

BackgroundMYC transcriptional factors are members of the bHLH (basic helix-loop-helix) superfamily, and play important roles in plant growth and development. Recent studies have revealed that some MYCs are involved in the crosstalk between Jasmonic acid regulatory pathway and light signaling in Arabidopsis, but such kinds of studies are rare in wheat, especially in photo-thermo-sensitive genic male sterile (PTGMS) wheat line.Results27 non-redundant MYC gene copies, which belonged to 11 TaMYC genes, were identified in the whole genome of wheat (Chinese Spring). These gene copies were distributed on 13 different chromosomes, respectively. Based on the results of phylogenetic analysis, 27 TaMYC gene copies were clustered into group I, group III, and group IV. The identified TaMYC genes copies contained different numbers of light, stress, and hormone-responsive regulatory elements in their 1500 base pair promoter regions. Besides, we found that TaMYC3 was expressed highly in stem, TaMYC5 and TaMYC9 were expressed specially in glume, and the rest of TaMYC genes were expressed in all tissues (root, stem, leaf, pistil, stamen, and glume) of the PTGMS line BS366. Moreover, we found that TaMYC3, TaMYC7, TaMYC9, and TaMYC10 were highly sensitive to methyl jasmonate (MeJA), and other TaMYC genes responded at different levels. Furthermore, we confirmed the expression profiles of TaMYC family members under different light quality and plant hormone stimuli, and abiotic stresses. Finally, we predicted the wheat microRNAs that could interact with TaMYC family members, and built up a network to show their integrative relationships.ConclusionsThis study analyzed the size and composition of the MYC gene family in wheat, and investigated stress-responsive and light quality induced expression profiles of each TaMYC gene in the PTGMS wheat line BS366. In conclusion, we obtained lots of important information of TaMYC family, and the results of this study was supposed to contribute novel insights and gene and microRNA resources for wheat breeding, especially for the improvement of PTGMS wheat lines.

Circular RNA profiling of the rice photo-thermosensitive genic male sterile line Wuxiang S reveals circRNA involved in the fertility transition

BackgroundCircular RNAs (circRNAs) are known to play an important role in the regulation of gene expression in eukaryotes. Photo-thermosensitive genic male sterile (PTGMS) is a very important germplasm resource in two-line hybrid rice breeding. Although many circRNAs have been identified in rice (Oryza sativa L.), little is known about the biological roles of circRNAs in the fertility transition of the PTGMS rice line.ResultsIn the present study, RNA-sequencing libraries were constructed from the young panicles of the Wuxiang S sterile line rice (WXS (S)) and its fertile line rice (WXS (F)) at three development stages with three biological replicates. A total of 9994 circRNAs were obtained in WXS rice based on high-throughput strand-specific RNA sequencing and bioinformatic approaches, of which 5305 were known circRNAs and 4689 were novel in rice. And 14 of 16 randomly selected circRNAs were experimentally validated with divergent primers. Our results showed that 186 circRNAs were significantly differentially expressed in WXS (F) compared with WXS (S), of which 97, 87 and 60 circRNAs were differentially expressed at the pollen mother cell (PMC) formation stage (P2), the meiosis stage (P3) and the microspore formation stage (P4), respectively. Fertility specific expression patterns of eight circRNAs were analysis by qRT-PCR. Gene ontology (GO) and KEGG pathway analysis of the parental genes of differentially expressed circRNAs (DECs) revealed that they mainly participated in various biological processes such as development, response to stimulation, hormonal regulation, and reproduction. Furthermore, 15 DECs were found to act as putative miRNA sponges to involved in fertility transition in PTGMS rice line.ConclusionIn the present study, the abundance and characteristics of circRNAs were investigated in the PTGMS rice line using bioinformatic approaches. Moreover, the expression patterns of circRNAs were different between WXS (F) and WXS (S). Our findings primarily revealed that circRNAs might be endogenous noncoding regulators of flower and pollen development, and were involved in the fertility transition in the PTGMS rice line, and guide the production and application of two-line hybrid rice.

Uncovering Male Fertility Transition Responsive miRNA in a Wheat Photo-Thermosensitive Genic Male Sterile Line by Deep Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) are endogenous small RNAs which play important negative regulatory roles at both the transcriptional and post-transcriptional levels in plants. Wheat is the most commonly cultivated plant species worldwide. In this study, RNA-seq analysis was used to examine the expression profiles of miRNA in the spikelets of photo-thermosenisitive genic male sterile (PTGMS) wheat line BS366 during male fertility transition. Through mapping on their corresponding precursors, 917–7,762 novel miRNAs were found in six libraries. Six novel miRNAs were selected for examination of their secondary structures and confirmation by stem-loop RT-PCR. In a differential expression analysis, 20, 22, and 58 known miRNAs exhibited significant differential expression between developmental stages 1 (secondary sporogenous cells had formed), 2 (all cells layers were present and mitosis had ceased), and 3 (meiotic division stage), respectively, of fertile and sterile plants. Some of these differential expressed miRNAs, such as tae-miR156, tae-miR164, tae-miR171, and tae-miR172, were shown to be associated with their targets. These targets were previously reported to be related to pollen development and/or male sterility, indicating that these miRNAs and their targets may be involved in the regulation of male fertility transition in the PTGMS wheat line BS366. Furthermore, target genes of miRNA cleavage sites were validated by degradome sequencing. In this study, a possible signal model for the miRNA-mediated signaling pathway during the process of male fertility transition in the PTGMS wheat line BS366 was developed. This study provides a new perspective for understanding the roles of miRNAs in male fertility in PTGMS lines of wheat.

The research progress and prospects of two-line hybrid rice in China

Two-line hybrid rice is new technology for exploiting rice heterosis established based on the discoveries of photo- thermo-sensitive genic male sterile (PTGMS) rice in China. The male fertility alteration of PTGMS line was controlled by recessive nuclear genes in sporophyte and induced by day-length and temperature during young panicle differentiation stage. Compared with three-line hybrid rice technology, it has three advantages: (1) The sterility is controlled by recessive nuclear genes. Any genotype with good combining ability can be used as a male parent. The frequency of obtaining heterotic hybrids in testcrosses is higher than in three-line system. (2) It simplifies the process of hybrid breeding program and shortens the breeding period for rapidly developing new hybrid rice. (3) It provides more kinds of sterile lines for diversifying hybrid rice. More than 300 PTGMS lines and over 900 two-line hybrid rice hybrids were developed and certificated in past three decades, and released in twenty provinces of China. The area covered under two-line hybrid rice was 5.5 million hectares yearly on the average, occupying 35% of total area of hybrid rice planting. Developing PTGMS lines with resistances to multiple-diseases and insect pests, short growth duration and lodging tolerance, exploiting indica-japonica heterosis for increasing yield, and ensuring safe and stable two-line hybrid rice seed production, will be the tasks for further maintaining and expanding two-line hybrid rice in future.

Small RNA Profiles of the Rice PTGMS Line Wuxiang S Reveal miRNAs Involved in Fertility Transition.

MicroRNAs (miRNAs) play key roles in the regulation of plant growth and developmental processes. In this study, RNA-seq was used to examine the expression profiles of miRNAs in a novel, photo-thermosensitive genic male sterile (PTGMS) rice line, Wuxiang S (WXS), during fertility transition. A total of 497 known miRNAs and 273 novel miRNAs were identified. In a differential expression analysis, 26 miRNAs exhibited significant differential expression between WXS (Sterile, S) and WXS (Fertile, F). Some of these miRNAs were validated by quantitative real-time PCR. Among these miRNAs, 11 showed decreased expression levels, and 15 showed increased expression levels in WXS (S) compared to WXS (F). Some of these miRNAs, such as osa-miR156a-j, osa-miR164d, and osa-miR528, were shown to be negatively correlated with their targets. These targets have previously been reported to be related to pollen development and male sterility, suggesting that these miRNAs may be involved in the regulation of pollen development in the rice PTGMS line WXS. Furthermore, miRNA-mediated editing events were also observed. In this study, a possible model for the control of signaling pathways during the process of fertility transition in the rice PTGMS line WXS by miRNAs was developed. These findings contribute to our understanding of the roles of miRNAs during anther development in PTGMS lines in rice.

Comparative Analysis of Anther Transcriptome Profiles of Two Different Rice Male Sterile Lines Genotypes under Cold Stress.

Rice is highly sensitive to cold stress during reproductive developmental stages, and little is known about the mechanisms of cold responses in rice anther. Using the HiSeq™ 2000 sequencing platform, the anther transcriptome of photo thermo sensitive genic male sterile lines (PTGMS) rice Y58S and P64S (Pei’ai64S) were analyzed at the fertility sensitive stage under cold stress. Approximately 243 million clean reads were obtained from four libraries and aligned against the oryza indica genome and 1497 and 5652 differentially expressed genes (DEGs) were identified in P64S and Y58S, respectively. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for these DEGs. Functional classification of DEGs was also carried out. The DEGs common to both genotypes were mainly involved in signal transduction, metabolism, transport, and transcriptional regulation. Most of the DEGs were unique for each comparison group. We observed that there were more differentially expressed MYB (Myeloblastosis) and zinc finger family transcription factors and signal transduction components such as calmodulin/calcium dependent protein kinases in the Y58S comparison group. It was also found that ribosome-related DEGs may play key roles in cold stress signal transduction. These results presented here would be particularly useful for further studies on investigating the molecular mechanisms of rice responses to cold stress.

The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-<i>Waxy</i> gene

Transgenic photo-thermo sensitive genic male sterility Oryza sativa L. cv. “261S” plants with the anti-Waxy gene were successfully obtained using an Agrobacterium tumefaciens-mediated co-transformation method. Marker-free homozygous transgenic lines with the anti-Waxy gene were obtained. The setting seed rates of the transgenic plants via self-pollination or via crossing with the restorer line WX99075 rice and the 1000-grain weight of the transgenic plants and the F2 hybrid seeds obtained by crossing the transgenic or non-transgenic plants with the restorer line WX99075 rice, and the number of panicles of the transgenic plants and yields of the F2 hybrid rice, were analysed. Quality indexes of the transgenic plants and of the F2 hybrid seeds were analysed. Our researches results indicate that hybrid female and hybrid descendant edibility could be improved via the introduction of the anti-Waxy gene, but the grain yields of the reserve seeds via self-pollination of the transgenic photo-thermo sensitive genic sterile lines and of the hybrid rice were not affected.

Breeding of Zi-Biao S line, the indica photothermosensitive genic male sterile line with recessive purple leaf marker

Breeding of Zi-Biao S line, the indica photothermosensitive genic male sterile line with recessive purple leaf marker