LcMYB4, an unknown function transcription factor gene from sheepgrass, as a positive regulator of chilling and freezing tolerance in transgenic Arabidopsis

BackgroundSheepgrass (Leymus chinensis (Trin.) Tzvel) is a perennial forage grass that can survive extreme freezing winters (-47.5 degrees C) in China. In this study, we isolated an unknown function MYB transcription factor gene, LcMYB4, from sheepgrass. However, the function of LcMYB4 and its homologous genes has not been studied in other plants.ResultsThe expression of the LcMYB4 gene was upregulated in response to cold induction, and the LcMYB4 fusion protein was localized in the nucleus, with transcriptional activation activity. Biological function analysis showed that compared with WT plants, LcMYB4-overexpressing Arabidopsis presented significantly increased chilling and freezing tolerance as evidenced by increased germination rate, survival rate, and seed setting rate under conditions of low temperature stress. Furthermore, LcMYB4-overexpressing plants showed increased soluble sugar content, leaf chlorophyll content and superoxide dismutase activity but decreased malondialdehyde (MDA) under chilling stress. Moreover, the expression of the CBF1, KIN1, KIN2 and RCI2A genes were significantly upregulated in transgenic plants with chilling treatment. These results suggest that LcMYB4 overexpression increased the soluble sugar content and cold-inducible gene expression and alleviated oxidative damage and membrane damage, resulting in enhanced cold resistance in transgenic plants. Interestingly, our results showed that the LcMYB4 protein interacts with fructose-1,6-bisphosphate aldolase protein1 (LcFBA1) and that the expression of the LcFBA1 gene was also upregulated during cold induction in sheepgrass, similar to LcMYB4.ConclusionOur findings suggest that LcMYB4 encodes MYB transcription factor that plays a positive regulatory role in cold stress

bHLH92 from sheepgrass acts as a negative regulator of anthocyanin/proanthocyandin accumulation and influences seed dormancy

Sheepgrass (Leymus chinensis) is an important native forage grass, and it is widely grown in north China. Differential dormancy exists in sheepgrass germplasms with different seed colors. To decipher and find potential genes underlying this phenomenon, we compared the transcript profiles of yellow seeds with weak dormancy and brown seeds with strong dormancy. We identified a transcription factor gene LcbHLH92 which is negatively correlated with those of anthocyanin/proanthocyanidin-specific pathway genes, anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). LcbHLH92 had two transcripts, LcbHLH92a and LcbHLH92b, and their expression could be induced by abscisic acid, cold, and NaCI. Overexpression of LcbHLH92a or LcbHLH92b in Arabidopsis significantly inhibited the transcript levels of dihydroflavonol reductase (DFR) and ANS genes in leaves and seeds, which resulted in a decrease in anthocyanins and proanthocyanidins, respectively. Importantly, transgenic Arabidopsis seeds with a yellow color showed a higher germination rate than did the wild-type with a brown seed color. Moreover, LcbHLH92a and LcbHLH92b repressed the transcription of Transparent Testa8, ANS, DFR, and ANR, possibly by elevating the transcript levels of jasmonate-ZIM domain proteins through binding to their promoters. Together, our results demonstrate that LcbHLH92a and LcbHLH92b are negative regulators of the anthocyanin/proanthocyanidin pathway and influence seed dormancy

Ectopic Expression of a Salt-Inducible Gene, LcSAIN3, from Sheepgrass Improves Seed Germination and Seedling Growth under Salt Stress in Arabidopsis

Sheepgrass is a perennial native grass species in China, and it can tolerate high levels of salt stress with an aggressive and vigorous rhizome system. Many salt-stress-responsive genes have been identified in sheepgrass. In this study, we report the cloning and characterization of a novel salt-induced gene, LcSAIN3 (Leymus chinensis salt-induced 3), from sheepgrass. Expression analysis confirmed that LcSAIN3 was induced by PEG, ABA, and salt treatments, and the expression of LcSAIN3 was significantly increased in salt-tolerant germplasms under salt treatment. Subcellular localization analysis indicated that the GFP-LcSAIN3 protein was mainly localized in the chloroplasts. The heterologous expression of LcSAIN3 in Arabidopsis increased the seed germination rate of transgenic plants under salt, ABA, and mannitol treatments. The seedling survival rate, plant height, and fresh weight of the transgenic plants were higher than those of WT plants under salt stress. The overexpression of LcSAIN3 caused a relatively high accumulation of free proline, enhanced SOD activity, and led to the upregulation of several stress-responsive genes such as AtRD26, AtRD29B, AtSOS1, and AtP5CS1. These results suggest that LcSAIN3 could be a potential target for molecular breeding to improve plants' salt tolerance

Comparative transcriptome analysis provides insights into the distinct germination in sheepgrass (Leymus chinensis) during seed development

Sheepgrass (Leymus chinensis ((Trin.) Tzvel)) is an important perennial forage grass that is widely distributed in the Eurasia steppe. The seed germination percentage show significant variation among the different germplasm in sheepgrass. However, the underlying molecular mechanisms of distinct germination during seed development are still mostly unknown. Here, we performed comparative transcriptomic analyses of high seed germination percentage (H) and low seed germination percentage (L) at 14, 28, and 42 days after pollination. After comparing 3 consecutive development stages, 9255, 5366, and 4306 genes were found to be significantly differently expressed between H and L. Pathway analysis indicated that transcripts related to starch and sucrose metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, and photosynthesis were significantly changed between the two germplasm at three stages. ABA and GA metabolism- and signaling transduction-related genes were differentially expressed between two germplasm at development stages, suggesting that the reduced signaling of GA and ABA is likely to be related to seed germination and dormancy in sheepgrass. We also identified 81 transcription factor (TF) families, and some TFs genes such as NAC48, NAC78, WRICY80, ZnFP, C3H14 and ILR3 were significantly differential expressed in two germplasm. Our results provide insights into seed development, germination and dormancy in sheepgrass at the transcriptional level

MADS-box family genes in sheepgrass and their involvement in abiotic stress responses

Background: MADS-box genes are categorized into A, B, C, D and E classes and are involved in floral organ identity and flowering. Sheepgrass (Leymus chinensis (Trin.) Tzvel) is an important perennial forage grass and adapts well to many adverse environments. However, there are few studies on the molecular mechanisms of flower development in sheepgrass, especially studies on MADS-domain proteins. Results: In this study, we cloned 11 MADS-box genes from sheepgrass (Leymus chinensis (Trin.) Tzvel), and phylogenetic analysis of the 11 genes with their homologs revealed that they are divided into nine subclades. Tissue-specific expression profile analysis showed that most of these MADS-box genes were highly expressed in floral organs. LcMADS1 and LcMADS3 showed higher expression in the stamen than in the other tissues, and LcMADS7 showed high expression in the stamen, glume, lemma and palea, while expression of LcMADS2, LcMADS9 and LcMADS11 was higher in vegetative organs than floral organs. Furthermore, yeast two-hybrid analyses showed that LcMADS2 interacted with LcMADS7 and LcMADS9. LcMADS3 interacted with LcMADS4, LcMADS7 and LcMADS10, while LcMADS1 could interact with only LcMADS7. Interestingly, the expression of LcMADS1 and LcMADS2 were significantly induced by cold, and LcMADS9 was significantly up-regulated by NaCl. Conclusion: Hence, we proposed that LcMADS1, LcMADS2, LcMADS3, LcMADS7 and LcMADS9 play a pivotal role in sheepgrass sexual reproduction and may be involved in abiotic stress responses, and our findings provide useful information for further exploration of the functions of this gene family in rice, wheat and other graminaceous cereals

LcSAIN1, a Novel Salt-Induced Gene from SheepGrass, Confers Salt Stress Tolerance in Transgenic Arabidopsis and Rice

Previously, we identified > 1,500 genes that were induced by high salt stress in sheepgrass (Leymus chinensis, Gramineae: Triticeae) when comparing the changes in their transcription levels in response to high salt stress by next-generation sequencing. Among the identified genes, a gene of unknown function (designated as Leymus chinensis salt-induced 1, LcSAIN1) showed a high sequence identity to its homologs from wheat, Hordeum vulgare and Oryza sativa, but LcSAIN1 and its homologs produce hypothetical proteins with no conserved functional domains. Transcription of the LcSAIN1 gene was up-regulated by various stresses. The overexpression of LcSAIN1 in Arabidopsis and rice increased the greening rate of cotyledons, the fresh weight, root elongation, plant height and the plant survival rate when compared with control plants and conferred a tolerance against salt stress. Subcellular localization analysis indicated that LcSAIN1 is localized predominantly in the nucleus. Our results show that the LcSAIN1 gene might play an important positive modulation role in increasing the expression of transcription factors (MYB2 and DREB2A) and functional genes (P5CS and RAB18) in transgenic plants under salt stress and that it augments stress tolerance through the accumulation of compatible solutes (proline and soluble sugar) and the alleviation of changes in reactive oxygen species. The LcSAIN1 gene could be a potential resource for engineering salinity tolerance in important crop species

New Insights on Drought Stress Response by Global Investigation of Gene Expression Changes in Sheepgrass (Leymus chinensis)

Water is a critical environmental factor that restricts the geographic distribution of plants. Sheepgrass [Leyrnus chinensis, (Trin.) Tzvel] is an important forage grass in the Eurasia Steppe and a close germplasm for wheat and barley. This native grass adapts well to adverse environments such as cold, salinity, alkalinity and drought, and it can survive when the soil moisture may be less than 6% in dry seasons. However, little is known about how sheepgrass tolerates water stress at the molecular level. Here, drought stress experiment and RNA-sequencing (RNA-seq) was performed in three pools of RNA samples (control, drought stress, and rewatering). We found that sheepgrass seedlings could still survive when the soil water content (SWC) was reduced to 14.09%. Differentially expressed genes (DEGs) analysis showed that 7320 genes exhibited significant responses to drought stress. Of these DEGs, 2671 presented opposite expression trends before and after rewatering. Furthermore, 680 putative sheepgrass-specific water responsive genes were revealed that can be studied deeply. Gene ontology (GO) annotation revealed that stress-associated genes were activated extensively by drought treatment. Interestingly, cold stress-related genes were up-regulated greatly after drought stress. The DEGs of MAPK and calcium signal pathways, plant hormone ABA, jasmonate, ethylene, brassinosteroid signal pathways, cold response CBF pathway participated coordinatively in sheepgrass drought stress response. In addition, we identified 288 putative transcription factors (TFs) involved in drought response, among them, the WRKY, NAG, AP2/ERF, bHLH, bZIP, and MYB families were enriched, and might play crucial and significant roles in drought stress response of sheepgrass. Our research provided new and valuable information for understanding the mechanism of drought tolerance in sheepgrass. Moreover, the identification of genes involved in drought response can facilitate the genetic improvement of crops by molecular breeding

LcFIN2, a novel chloroplast protein gene from sheepgrass, enhances tolerance to low temperature in Arabidopsis and rice

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA-sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing-induced protein 2, encoded a chloroplast-targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (phi PSII), the non-photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal-related and photosynthesis-related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low-temperature tolerance in crops