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Long non-coding RNAs (lncRNAs) are pivotal regulators of the abiotic stress responses in plants, yet their specific involvement in salt/alkali stress during alfalfa germination remains incompletely understood. Here, we subjected Zhongmu No.1 alfalfa (Medicago sativa L.) seeds to salt stress (20 mM NaCl and 20 mM Na2SO4 solutions) or alkali stress (5 mM NaHCO3 and 5 mM Na2CO3 solutions) treatments for 3 days, followed by total RNA extraction and RNA-seq analysis to delineate stress-responsive alfalfa lncRNAs. We identified 17,473 novel alfalfa lncRNAs, among which 101 and 123 were differentially expressed lncRNAs (DElncRNAs) under salt and alkali stress, respectively, compared to the control. Furthermore, we predicted 16 and 237 differentially expressed target genes regulated by DElncRNAs through cis/trans-regulatory mechanisms under salt or alkali stress, respectively. A functional enrichment analysis of DElncRNA target genes indicated that lncRNAs were implicated in the fatty acid metabolism pathway under salt stress, while they played a significant role in the phenylpropanoid and flavonoid biosynthesis pathway under alkali stress. Notably, lncRNAs were found to participate in the plant hormone signal transduction pathway, a common regulatory mechanism in both salt and alkali stress responses. These findings contribute to a deeper understanding of the mechanisms underlying alfalfa's response to salt and alkali stresses. [ABSTRACT FROM AUTHOR]

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa (Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf–stem ratio (LSR) and repression of several lignin-synthesis genes (MsNST, MsPAL1, MsC4H, and Ms4CL). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage. [ABSTRACT FROM AUTHOR]

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I–EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress. [ABSTRACT FROM AUTHOR]

Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes (MsHSFs) in the tetraploid alfalfa genome ("Xinjiangdaye" reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSFs and tissue-wide expression for 28 MsHSFs. Based on transcriptomic data analysis, 21, 11 and 27 MsHSFs responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT–PCR, MsHSF27/33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSFs and for genetic improvement of stress resistance in alfalfa. [ABSTRACT FROM AUTHOR]

The PLATZ family is a novel class of plant-specific zinc finger transcription factors with important roles in plant growth and development and abiotic stress responses. PLATZ members have been identified in many plants, including Oryza sativa, Zea mays, Triticum aestivum, Fagopyrum tataricum, and Arabidopsis thaliana; however, due to the complexity of the alfalfa reference genome, the members of the PLATZ gene family in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed. In this study, 55 Medicago sativa PLATZ genes (MsPLATZs) were identified in the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatic analysis was performed, including the characterization of sequence lengths, protein molecular weights, genomic positions, and conserved motifs. Expression analysis reveals that 7 MsPLATZs are tissue-specifically expressed, and 10 MsPLATZs are expressed in all examined tissues. The transcriptomic expression of these genes is obvious, indicating that these MsPLATZs have different functions in the growth and development of alfalfa. Based on transcriptome data analysis and real-time quantitative PCR (RT-qPCR), we identified 22, 22, and 21 MsPLATZ genes that responded to salt, cold, and drought stress, respectively, with 20 MsPLATZs responding to all three stresses. This study lays a foundation for further exploring the functions of MsPLATZs, and provides ideas for the improvement of alfalfa varieties and germplasm innovation. [ABSTRACT FROM AUTHOR]

NAC (NAM, ATAF1/2, and CUC2) transcription factors compose one of the largest families of plant-specific transcription factors; they are widely involved in plant growth and development and have especially important roles in improving stress resistance in plants. However, NAC gene family members in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed genome-wide due to the complexity of the alfalfa reference genome. In this study, a total of 421 M. sativa NAC genes (MsNACs) were identified from the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatics analysis, including characterization of sequence length, protein molecular weight and genome position and conserved motif analysis, was conducted. Expression analysis showed that 47 MsNACs had tissue-specific expression, and 64 MsNACs were expressed in all tissues. The transcriptomic profiles of the genes were very different, indicating that these MsNACs have various functions in alfalfa growth and development. We identified 25, 42 and 47 MsNACs that respond to cold, drought and salt stress based on transcriptome data analysis and real-time quantitative PCR (RT–qPCR). Furthermore, 22 MsNACs were found to respond to both salt and drought stress, and 15 MsNACs were found to respond to cold, salt and drought stress. The results of this study could provide valuable information for further functional analysis of MsNACs and for the improvement of stress resistance in alfalfa. [ABSTRACT FROM AUTHOR]

Plant filamentation temperature-sensitive H (FtsH) proteins are ATP-dependent zinc proteases that play an important role in regulating abiotic stress adaptions. Here we explore their potential role in abiotic stress tolerance in alfalfa, an important legume crop. Genomic analysis revealed seventeen MsFtsH genes in five clusters, which generally featured conserved domains and gene structures. Furthermore, the expression of MsFtsHs was found to be tightly associated with abiotic stresses, including osmotic, salt and oxidative stress. In addition, numerous stress responsive cis -elements, including those related to ABA, auxin, and salicylic acid, were identified in their promoter regions. Moreover, MsFtsH8 overexpression was shown to confer tolerance to salt and oxidative stress which was associated with reduced levels of reactive oxygen species, and enhanced expression and activity of antioxidant enzymes. Our results highlight MsFtsHs as key factors in abiotic stress tolerance, and show their potential usefulness for breeding alfalfa and other crops with improved yield and stress tolerance. • Seventeen MsFtsHs were identified belonging to five groups, which feature conserved domain composition and gene structures. • Stress responsive elements were discovered and 5 MsFtsHs were up-regulated under drought, salt and oxidative stresses. • MsFtsH8 promoted salt and oxidative tolerance in Arabidopsis and alfalfa, providing its role in abiotic stress resistance. [ABSTRACT FROM AUTHOR]

Abstract Carex rigescens is an ornamental turfgrass in northern China which has a relatively low maintenance cost and robust tolerance to many adverse environmental conditions, so it could be considered a new material for researching into plant stress resistance. However, suitable reference genes are vacant for obtaining reliable results in quantitative real-time PCR (qRT-PCR) analysis of C. rigescens in adversity research. In this study, we tested the expression stability of nine potential reference genes in leaves and roots under five different abiotic stress conditions, including cold, salt, heat, osmotic and cadmium (Cd). We then selected the best reference genes according to the analysis results calculated by three algorithmic programs (geNorm, NormFinder and BestKeeper) and used the RankAggreg package to merge the outputted data. The results showed that combinations of at least two reference genes should be used for reliable normalization except in heat-treated root samples, which require three reference genes. eIF-4α , GADPH , SAND and PEPKR1 and their combination were found to be the most stably expressed reference genes, while SAM , TUA-α and UPL7 were the three least stable reference genes among most of experimental samples. In addition, five stress-induced genes (Cu-Zn SOD , P5CS , LEA , GST , and APX) were chosen to verify the stability of the selected reference genes in various tissues and under various stress conditions. The results of this study will provide an important fundamental basis both for gene expression verification for transcriptomic and proteomic analyses and for gene expression analysis for future gene function research in C. rigescens. Highlights • At least two reference genes should be used in qRT-PCR analysis in all experimental conditions. • eIF-4α , GADPH , SAND , PEPKR1 and their combination performed superiorly in this study. • SAM , TUA-α and UPL7 were the least stable reference genes in all experimental samples. • Cu-Zn SOD , P5CS , LEA , GST , and APX were chosen to verify reference genes stability. [ABSTRACT FROM AUTHOR]

Highlights • The physiological and phenotypic analyses indicated that Zhongmu-3 is more alkali tolerant than R108. • An iTRAQ-based quantitative proteomic analysis was applied to detect alkali-responsive proteins in Zhongmu-3 and R108. A total of 467 differentially changed alkali-responsive proteins were identified from them. Zhongmu-3 and R108 shared 97 common differentially changed proteins, but a large percentage of them showed different change patterns between Zhongmu-3 and R108. • Subsequent functional annotation indicated these proteins influenced diverse processes, such as catalytic activity, signaling, and antioxidant activity. • The data provide new insights into the regulatory mechanisms responsible for alkali stress responses in leguminous plants and have potential implications for breeding of alkaline-resistant alfalfa and other crops. Abstract Saline-alkaline stress is one of the primary abiotic stresses that limits crop yields worldwide. The early seedling stage of plants is the most vulnerable stage to stress conditions. In this study, the physiological and phenotypic changes induced by alkali treatments (Na 2 CO 3 and NaHCO 3 mixtures) were analyzed for alfalfa (Medicago sativa L. cv. Zhongmu-3) and barrel medic (Medicago truncatula line R108) seedlings. As expected, Zhongmu-3, which is alkali tolerant, and R108, which is alkali sensitive, responded differently to alkaline stress. To characterize the protein expression profiles of these two Medicago species in response to alkaline stress, an iTRAQ-based quantitative proteomic analysis was applied to detect alkali-responsive proteins. A total of 467 differentially changed alkali-responsive proteins were identified from Zhongmu-3 and R108. Compared with their levels in untreated control seedlings, the abundance of 349 proteins increased and 38 proteins decreased in alkali-treated Zhongmu-3 seedlings, whereas 142 proteins increased and 35 proteins decreased in R108 seedlings. Zhongmu-3 and R108 shared 97 common differentially changed proteins, but a large percentage of them showed different change patterns between Zhongmu-3 and R108. Subsequent functional annotation indicated these proteins influenced diverse processes, such as catalytic activity, signaling, and antioxidant activity. The transcript levels of genes encoding 10 differentially changed proteins were determined by quantitative PCR. The data provide new insights into the regulatory mechanisms responsible for alkali stress responses in leguminous plants and have potential implications for breeding of alkaline-resistant alfalfa and other crops. [ABSTRACT FROM AUTHOR]

Butanediol (BD) is a bacterial volatile compound which can activate induced-systemic resistance to diseases in plants, but its effects on abiotic stress tolerance are not well-known. The objectives of this study were to examine physiological effects of BD on heat tolerance in creeping bentgrass and to identify BD-responsive metabolites and genes contributing to effects of BD on heat tolerance. Creeping bentgrass plants (cv.’ PennA4’ and’ Penncross’) were treated with 2,3 -butanediol or water through foliar spray and were exposed to heat stress (35/30 °C, day/night) or optimal temperature (20/15 °C) in growth chambers. Creeping bentgrass plants treated with BD exhibited improved heat tolerance, demonstrated by higher visual quality and leaf photochemical efficiency when compared with the untreated control plants. Real-time PCR revealed that BD application resulted in up-regulation of genes related to cell elongation, metabolism, and stress responses in plants exposed to heat stress. Metabolite profiling identified a number of organic acids, sugars and sugar acids that accumulated due to BD treatment under heat stress. Results of the current study suggest that BD is effective in improving heat tolerance in creeping bentgrass, mainly through the enhancement of gene expression and metabolite accumulation involved in energy metabolism and stress signaling. [ABSTRACT FROM AUTHOR]