Comparative transcriptome and DNA methylation analysis in temperature-sensitive genic male sterile wheat BS366.

BACKGROUND: Known as the prerequisite component for the heterosis breeding system, the male sterile line determines the hybrid yield and seed purity. Therefore, a deep understanding of the mechanism and gene network that leads to male sterility is crucial. BS366, a temperature-sensitive genic male sterile (TGMS) line, is male sterile under cold conditions (12 °C with 12 h of daylight) but fertile under normal temperature (20 °C with 12 h of daylight). RESULTS: During meiosis, BS366 was defective in forming tetrads and dyads due to the abnormal cell plate. During pollen development, unusual vacuolated pollen that could not accumulate starch grains at the binucleate stage was also observed. Transcriptome analysis revealed that genes involved in the meiotic process, such as sister chromatid segregation and microtubule-based movement, were repressed, while genes involved in DNA and histone methylation were induced in BS366 under cold conditions. MethylRAD was used for reduced DNA methylation sequencing of BS366 spikes under both cold and control conditions. The differentially methylated sites (DMSs) located in the gene region were mainly involved in carbohydrate and fatty acid metabolism, lipid metabolism, and transport. Differentially expressed and methylated genes were mainly involved in cell division. CONCLUSIONS: These results indicated that the methylation of genes involved in carbon metabolism or fatty acid metabolism might contribute to male sterility in BS366 spikes, providing novel insight into the molecular mechanism of wheat male sterility.

Genome-wide identification and transcriptional characterization of DNA methyltransferases conferring temperature-sensitive male sterility in wheat.

BACKGROUND: DNA methyltransferase (DMT) genes contribute to plant stress responses and development by de novo establishment and subsequent maintenance of DNA methylation during replication. The photoperiod and/or temperature-sensitive genic male sterile (P/TGMS) lines play an important role in hybrid seed production of wheat. However, only a few studies have reported on the effect of DMT genes on temperature-sensitive male sterility of wheat. Although DMT genes have been investigated in some plant species, the identification and analysis of DMT genes in wheat (Triticum aestivum L.) based on genome-wide levels have not been reported. RESULTS: In this study, a detailed overview of phylogeny of 52 wheat DMT (TaDMT) genes was presented. Homoeolog retention for TaDMT genes was significantly above the average retention rate for whole-wheat genes, indicating the functional importance of many DMT homoeologs. We found that the strikingly high number of TaDMT genes resulted mainly from the significant expansion of the TaDRM subfamily. Intriguingly, all 5 paralogs belonged to the wheat DRM subfamily, and we speculated that tandem duplications might play a crucial role in the TaDRM subfamily expansion. Through the transcriptional analysis of TaDMT genes in a TGMS line BS366 and its hybrids with the other six fertile lines under sterile and fertile conditions, we concluded that TaCMT-D2, TaMET1-B1, and TaDRM-U6 might be involved in male sterility in BS366. Furthermore, a correlation analysis showed that TaMET1-B1 might negatively regulate the expression of TaRAFTIN1A, an important gene for pollen development, so we speculated regarding an epigenetic regulatory mechanism underlying the male sterility of BS366 via the interaction between TaMET1-B1 and TaRAFTIN1A. CONCLUSIONS: Our findings presented a detailed phylogenic overview of the DMT genes and could provide novel insights into the effects of DMT genes on TGMS wheat.

Transcriptome analysis of wheat seedling and spike tissues in the hybrid Jingmai 8 uncovered genes involved in heterosis.

MAIN CONCLUSION: Transcriptome analysis was carried out for wheat seedlings and spikes from hybrid Jingmai 8 and both inbred lines to unravel mechanisms underlying heterosis. Heterosis, known as one of the most successful strategies for increasing crop yield, has been widely exploited in plant breeding systems. Despite its great importance, the molecular mechanism underlying heterosis remains elusive. In the present study, RNA sequencing (RNA-seq) was performed on the seedling and spike tissues of the wheat (Triticum aestivum) hybrid Jingmai 8 (JM8) and its homozygous parents to unravel the underlying mechanisms of wheat heterosis. In total, 1686 and 2334 genes were identified as differentially expressed genes (DEGs) between the hybrid and the two inbred lines in seedling and spike tissues, respectively. Gene Ontology analysis revealed that DEGs from seedling tissues were significantly enriched in processes involved in photosynthesis and carbon fixation, and the majority of these DEGs expressed at a higher level in JM8 compared to both inbred lines. In addition, cell wall biogenesis and protein biosynthesis-related pathways were also significantly represented. These results confirmed that a combination of different pathways could contribute to heterosis. The DEGs between the hybrid and the two inbred progenitors from the spike tissues were significantly enriched in biological processes related to transcription, RNA biosynthesis and molecular function categories related to transcription factor activities. Furthermore, transcription factors such as NAC, ERF, and TIF-IIA were highly expressed in the hybrid JM8. These results may provide valuable insights into the molecular mechanisms underlying wheat heterosis.

ABI-like transcription factor gene TaABL1 from wheat improves multiple abiotic stress tolerances in transgenic plants.

The phytohormone abscisic acid (ABA) plays crucial roles in adaptive responses of plants to abiotic stresses. ABA-responsive element binding proteins (AREBs) are basic leucine zipper transcription factors that regulate the expression of downstream genes containing ABA-responsive elements (ABREs) in promoter regions. A novel ABI-like (ABA-insensitive) transcription factor gene, named TaABL1, containing a conserved basic leucine zipper (bZIP) domain was cloned from wheat. Southern blotting showed that three copies were present in the wheat genome. Phylogenetic analyses indicated that TaABL1 belonged to the AREB subfamily of the bZIP transcription factor family and was most closely related to ZmABI5 in maize and OsAREB2 in rice. Expression of TaABL1 was highly induced in wheat roots, stems, and leaves by ABA, drought, high salt, and low temperature stresses. TaABL1 was localized inside the nuclei of transformed wheat mesophyll protoplast. Overexpression of TaABL1 enhanced responses of transgenic plants to ABA and hastened stomatal closure under stress, thereby improving tolerance to multiple abiotic stresses. Furthermore, overexpression of TaABL1 upregulated or downregulated the expression of some stress-related genes controlling stomatal closure in transgenic plants under ABA and drought stress conditions, suggesting that TaABL1 might be a valuable genetic resource for transgenic molecular breeding.

Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway.

R2R3-MYB transcription factors play an important role in the synthesis of phenylpropanoid-derived compounds, which in turn provide salt tolerance in plant. In this study, we found that the expression of foxtail millet R2R3-MYB factor SiMYB16 can be induced by salt and drought. SiMYB16 is localized in the nucleus and acts as a transcriptional activator. Phylogenetic analysis indicates that SiMYB16 belongs to the R2R3-MYB transcription factor family subgroup 24. Transgenic rice expressing SiMYB16 (OX16) had a higher survival rate, lower malondialdehyde content, and heavier fresh weight compared with type (WT) under salt stress conditions. The transgenic plants also had a higher germination rate in salt treatment conditions and higher yield in the field compared with wild-type plants. Transcriptome analysis revealed that the up-regulated differential expression genes in the transgenic rice were mainly involved in phenylpropanoid biosynthesis, fatty acid elongation, phenylalanine metabolism, and flavonoid biosynthesis pathways. Quantitative real-time PCR analysis also showed that the genes encoding the major enzymes in the lignin and suberin biosynthesis pathways had higher expression level in SiMYB16 transgenic plants. Correspondingly, the content of flavonoid and lignin, and the activity of fatty acid synthase increased in SiMYB16 transgenic rice compared with wild-type plants under salt stress treatment. These results indicate that SiMYB16 gene can enhance plant salt tolerance by regulating the biosynthesis of lignin and suberin.

The soybean GmbZIP1 transcription factor enhances multiple abiotic stress tolerances in transgenic plants.

Abscisic acid (ABA)-responsive element binding proteins (AREBs) are basic domain/leucine zipper transcription factors that bind to the ABA-responsive element (ABRE) in the promoter regions of ABA-inducible genes in plants. A novel bZIP transcription factor gene, GmbZIP1, encoding 438 amino acids with a conserved bZIP domain composed of 60 amino acids was isolated from salt-tolerant soybean cv. Tiefeng 8. Southern blotting showed that only one copy was present in the soybean genome. Phylogenetic analyses showed that GmbZIP1 belonged to the AREB subfamily of the bZIP family and was most closely related to AtABF2 and OsTRAB1. The expression of GmbZIP1 was highly induced by ABA, drought, high salt and low temperature; and GmbZIP1 was expressed in soybean roots, stems and leaves under different stress conditions. GmbZIP1 was localized inside the nuclei of transformed onion epidermal cells. Overexpression of GmbZIP1 enhanced the responses of transgenic plants to ABA and triggered stomatal closure under stresses, potentially leading to improved tolerances to several abiotic stresses such as high salt, low temperature and drought in transgenic plants. Furthermore, overexpression of GmbZIP1 affected the expression of some ABA or stress-related genes involved in regulating stomatal closure in Arabidopsis under ABA, drought and high salt stress conditions. A few AREB elements were detected in the promoter region of those ABA or stress-related genes, suggesting that GmbZIP1 regulates the ABA response or stomatal closure mediated by those downstream genes in transgenic Arabidopsis. Moreover, GmbZIP1 was used to improve the drought tolerance trait of Chinese wheat varieties BS93. Functional analysis showed that overexpression of GmbZIP1 enhanced the drought tolerance of transgenic wheat, and transcripts of GmbZIP1 were detected in transgenic wheat using RT-PCR. In addition, GmbZIP1 overexpression did not result in growth retardation in all transgenic plants, suggesting that GmbZIP1 may be a valuable genetic resource for engineering stress tolerance of crops.

The G-Protein ß Subunit AGB1 Promotes Hypocotyl Elongation through Inhibiting Transcription Activation Function of BBX21 in Arabidopsis.

Hypocotyl development in Arabidopsis thaliana is regulated by light and endogenous hormonal cues, making it an ideal model to study the interplay between light and endogenous growth regulators. BBX21, a B-box (BBX)-like zinc-finger transcription factor, integrates light and abscisic acid signals to regulate hypocotyl elongation in Arabidopsis. Heterotrimeric G-proteins are pivotal regulators of plant development. The short hypocotyl phenotype of the G-protein ß-subunit (AGB1) mutant (agb1-2) has been previously identified, but the precise role of AGB1 in hypocotyl elongation remains enigmatic. Here, we show that AGB1 directly interacts with BBX21, and the short hypocotyl phenotype of agb1-2 is partially suppressed in agb1-2bbx21-1 double mutant. BBX21 functions in the downstream of AGB1 and overexpression of BBX21 in agb1-2 causes a more pronounced reduction in hypocotyl length, indicating that AGB1 plays an oppositional role in relation to BBX21 during hypocotyl development. Furthermore, we demonstrate that the C-terminal region of BBX21 is important for both its intracellular localization and its transcriptional activation activity that is inhibited by interaction with AGB1. ChIP assays showed that BBX21 specifically associates with its own promoter and with those of BBX22, HY5, and GA2ox1. which is not altered in agb1-2. These data suggest that the AGB1-BBX21 interaction only affects the transcriptional activation activity of BBX21 but has no effect on its DNA binding ability. Taken together, our data demonstrate that AGB1 positively promotes hypocotyl elongation through repressing BBX21 activity.

A cotton (Gossypium hirsutum) DRE-binding transcription factor gene, GhDREB, confers enhanced tolerance to drought, high salt, and freezing stresses in transgenic wheat.

A cotton (G. hirsutum L.) dehydration responsive element binding protein gene, GhDREB, which encodes a 153 amino acid protein containing a conserved AP2/EREBP domain, was isolated from the cDNA library of cotton cv. Simian 3 by a yeast one-hybrid system. RNA blot analysis showed that the GhDREB gene was induced in cotton seedlings by drought, high salt and cold stresses. An electrophoretic mobility shift assay (EMSA) indicated that the GhDREB protein bound specifically to the DRE core element (A/GCCGAC) in vitro. Two expression vectors containing the GhDREB gene with either of the Ubiqutin or rd29A promoters were constructed and transferred into wheat (Triticum aestivum L.) by bombardment. Fifty-eight Ubi::GhDREB and 17 rd29A::GhDREB T(0) plants of Yangmai (36 plants) and Lumai (39 plants) were identified by PCR analysis, respectively. Southern blot and RT-PCR analyses showed that two or three copies of the GhDREB were integrated into the Yangmai 10 genome and were expressed at the transcriptional level, and three or four copies were integrated into the Lumai 23 genome. Functional analysis indicated that the transgenic plants had improved tolerance to drought, high salt, and freezing stresses through accumulating higher levels of soluble sugar and chlorophyll in leaves after stress treatments. No phenotype differences were observed between transgenic plants and their non-transgenic controls. These results indicated that GhDREB might be useful in improving wheat stress tolerance through genetic engineering.