Genome-wide identification and expression pattern analysis of the Aux/IAA (auxin/indole-3-acetic acid) gene family in alfalfa (Medicago sativa) and the potential functions under drought stress.

BACKGROUND: Auxin/induced-3-acetic acid (Aux/IAA) is an important plant hormone that affects plant growth and resistance to abiotic stresses. Drought stress is a vital factor in reducing plant biomass yield and production quality. Alfalfa (Medicago sativa L.) is the most widely planted leguminous forage and one of the most economically valuable crops in the world. Aux/IAA is one of the early responsive gene families of auxin, playing a crucial role in response to drought stress. However, the characteristics of the Aux/IAA gene family in alfalfa and its potential function in response to drought stress are still unknown. RESULT: A total of 41 Aux/IAA gene members were identified in alfalfa genome. The physicochemical, peptide structure, secondary and tertiary structure analysis of proteins encoded by these genes revealed functional diversity of the MsIAA gene. A phylogenetic analysis classified the MsIAA genes into I-X classes in two subgroups. And according to the gene domain structure, these genes were classified into typical MsIAA and atypical MsIAA. Gene structure analysis showed that the MsIAA genes contained 1-4 related motifs, and except for the third chromosome without MsIAAs, they were all located on 7 chromosomes. The gene duplication analysis revealed that segmental duplication and tandem duplication greatly affected the amplification of the MsIAA genes. Analysis of the Ka/Ks ratio of duplicated MsAux/IAA genes suggested purification selection pressure was high and functional differences were limited. In addition, identification and classification of promoter cis-elements elucidated that MsIAA genes contained numerous elements associated to phytohormone response and abiotic stress response. The prediction protein-protein interaction network showed that there was a complex interaction between the MsAux/IAA genes. Gene expression profiles were tissue-specific, and MsAux/IAA had a broad response to both common abiotic stress (ABA, salt, drought and cold) and heavy metal stress (Al and Pb). Furthermore, the expression patterns analysis of 41 Aux/IAA genes by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Aux/IAA genes can act as positive or negative factors to regulate the drought resistance in alfalfa. CONCLUSION: This study provides useful information for the alfalfa auxin signaling gene families and candidate evidence for further investigation on the role of Aux/IAA under drought stress. Future studies could further elucidate the functional mechanism of the MsIAA genes response to drought stress.

Genome-wide identification of B-box zinc finger (BBX) gene family in Medicago sativa and their roles in abiotic stress responses.

BACKGROUND: B-box (BBX) family is a class of zinc finger transcription factors (TFs) that play essential roles in regulating plant growth, development, as well as abiotic stress. However, no systematic analysis of BBX genes has yet been conducted in alfalfa (Medica go sativa L.), and their functions have not been elucidated up to now. RESULTS: In this study, 28 MsBBX genes were identified from the alfalfa genome, which were clustered into 4 subfamilies according to an evolutionary tree of BBX proteins. Exon-intron structure and conserved motif analysis reflected the evolutionary conservation of MsBBXs in alfalfa. Collinearity analysis showed that segmental duplication promoted the expansion of the MsBBX family. Analysis of cis-regulatory elements suggested that the MsBBX genes possessed many growth/development-, light-, phytohormone-, and abiotic stress-related elements. MsBBX genes were differentially expressed in leaves, flowers, pre-elongated stems, elongated stems, roots and nodules, and most MsBBXs were remarkably induced by drought, salt and various plant growth regulators (ABA, JA, and SA). Further functional verification demonstrated that overexpressing of the MsBBX11 gene clearly promoted salt tolerance in transgenic Arabidopsis by regulating growth and physiological processes of seedlings. CONCLUSIONS: This research provides insights into further functional research and regulatory mechanisms of MsBBX family genes under abiotic stress of alfalfa.

Genome-wide investigation and expression analysis of OSCA gene family in response to abiotic stress in alfalfa.

Alfalfa is an excellent leguminous forage crop that is widely cultivated worldwide, but its yield and quality are often affected by drought and soil salinization. Hyperosmolality-gated calcium-permeable channel (OSCA) proteins are hyperosmotic calcium ion (Ca2+) receptors that play an essential role in regulating plant growth, development, and abiotic stress responses. However, no systematic analysis of the OSCA gene family has been conducted in alfalfa. In this study, a total of 14 OSCA genes were identified from the alfalfa genome and classified into three groups based on their sequence composition and phylogenetic relationships. Gene structure, conserved motifs and functional domain prediction showed that all MsOSCA genes had the same functional domain DUF221. Cis-acting element analysis showed that MsOSCA genes had many cis-regulatory elements in response to abiotic or biotic stresses and hormones. Tissue expression pattern analysis demonstrated that the MsOSCA genes had tissue-specific expression; for example, MsOSCA12 was only expressed in roots and leaves but not in stem and petiole tissues. Furthermore, RT-qPCR results indicated that the expression of MsOSCA genes was induced by abiotic stress (drought and salt) and hormones (JA, SA, and ABA). In particular, the expression levels of MsOSCA3, MsOSCA5, MsOSCA12 and MsOSCA13 were significantly increased under drought and salt stress, and MsOSCA7, MsOSCA10, MsOSCA12 and MsOSCA13 genes exhibited significant upregulation under plant hormone treatments, indicating that these genes play a positive role in drought, salt and hormone responses. Subcellular localization results showed that the MsOSCA3 protein was localized on the plasma membrane. This study provides a basis for understanding the biological information and further functional analysis of the MsOSCA gene family and provides candidate genes for stress resistance breeding in alfalfa.

Effects of exogenous melatonin on growth and physiological characteristics of Agropyron mongolicum seedlings under drought stress.

To clarify the alleviation effect of exogenous melatonin (MT) on Agropyron mongolicum under drought stress, we examined the response of A. mongolicum 'Yanchi' seedlings to simulated drought stress with polyethylene glycol 6000 (PEG-6000), by investigating the effects of exogenous addition of different concentrations (0, 1, 10, 50, 100, 150 and 200 mg·L-1) of MT on seedlings growth and physiological characteristics under drought stress. The results showed that drought stress significantly inhibited the growth of A. mongolicum seedlings, and that exogenous addition of different concentrations of MT could alleviate the growth inhibition caused by drought stress, with the strongest mitigation effect observed at MT concentration of 100 mg·L-1. Compared with the drought stress treatment alone, exogenous addition of 100 mg·L-1 MT under drought stress increased plant height, aboveground dry weight, and leaf relative water content by 58.2%, 121.2% and 48.1%. The contents of chlorophyll a, chlorophyll b, carotenoids increased by 48.7%, 80.8% and 38.3%, superoxide dismutase, peroxidase and root activity increased by 12.6%, 33.9% and 39.1%, and the contents of ascorbic acid and glutathione increased by 19.5% and 18.3%, respectively. The contents of proline, soluble sugar and soluble protein were increased by 16.2%, 32.6% and 14.3%, while that of malondialdehyde, hydrogen peroxide and superoxide anion radical were decreased by 45.8%, 65.8% and 30.8%, respectively. In summary, exogenous addition of 100 mg·L-1 MT could improve drought tolerance of A. mongolicum seedlings by promoting growth, enhancing antioxidant capacity, increasing the content of osmoregulation substances, inhibiting the excessive production of reactive oxygen, and reducing membrane peroxide level.

Osmotic stress-induced lignin synthesis is regulated at multiple levels in alfalfa (Medicago sativa L.).

Alfalfa is an important forage crop. Yield and quality are frequently threatened by extreme environments such as drought and salt stress. As a component of the cell wall, lignin plays an important role in the abiotic stress response, the mechanisms of which have not been well clarified. In this study, we combined physiological, transcriptional, and metabolic analyses to reveal the changes in lignin content in alfalfa under mannitol-induced osmotic stress. Osmotic stress enhanced lignin accumulation by increasing G and S units, which was associated with increases in enzyme activities and decreases in 8 intermediate metabolites. Upon combined analysis of the transcriptome and metabolome, we identified five key structural genes and several coexpressed transcription factors, such as MYB and WRKY, which may play a core role in regulating lignin content and composition under osmotic stress. In addition, lignin synthesis was positively regulated by ABA but negatively regulated by ethylene under osmotic stress. These results provide new insight into the regulatory mechanism of lignin synthesis under abiotic stress.

Transcriptome analysis and physiological changes in the leaves of two Bromus inermis L. genotypes in response to salt stress.

Soil salinity is a major factor threatening the production of crops around the world. Smooth bromegrass (Bromus inermis L.) is a high-quality grass in northern and northwestern China. Currently, selecting and utilizing salt-tolerant genotypes is an important way to mitigate the detrimental effects of salinity on crop productivity. In our research, salt-tolerant and salt-sensitive varieties were selected from 57 accessions based on a comprehensive evaluation of 22 relevant indexes, and their salt-tolerance physiological and molecular mechanisms were further analyzed. Results showed significant differences in salt tolerance between 57 genotypes, with Q25 and Q46 considered to be the most salt-tolerant and salt-sensitive accessions, respectively, compared to other varieties. Under saline conditions, the salt-tolerant genotype Q25 not only maintained significantly higher photosynthetic performance, leaf relative water content (RWC), and proline content but also exhibited obviously lower relative conductivity and malondialdehyde (MDA) content than the salt-sensitive Q46 (p < 0.05). The transcriptome sequencing indicated 15,128 differentially expressed genes (DEGs) in Q46, of which 7,885 were upregulated and 7,243 downregulated, and 12,658 DEGs in Q25, of which 6,059 were upregulated and 6,599 downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the salt response differences between Q25 and Q46 were attributed to the variable expression of genes associated with plant hormone signal transduction and MAPK signaling pathways. Furthermore, a large number of candidate genes, related to salt tolerance, were detected, which involved transcription factors (zinc finger proteins) and accumulation of compatible osmolytes (glutathione S-transferases and pyrroline-5-carboxylate reductases), etc. This study offers an important view of the physiological and molecular regulatory mechanisms of salt tolerance in two smooth bromegrass genotypes and lays the foundation for further identification of key genes linked to salt tolerance.

Identification of Competing Endogenous RNAs (ceRNAs) Network Associated with Drought Tolerance in Medicago truncatula with Rhizobium Symbiosis.

Drought, bringing the risks of agricultural production losses, is becoming a globally environmental stress. Previous results suggested that legumes with nodules exhibited superior drought tolerance compared with the non-nodule group. To investigate the molecular mechanism of rhizobium symbiosis impacting drought tolerance, transcriptome and sRNAome sequencing were performed to identify the potential mRNA-miRNA-ncRNA dynamic network. Our results revealed that seedlings with active nodules exhibited enhanced drought tolerance by reserving energy, synthesizing N-glycans, and medicating systemic acquired resistance due to the early effects of symbiotic nitrogen fixation (SNF) triggered in contrast to the drought susceptible with inactive nodules. The improved drought tolerance might be involved in the decreased expression levels of miRNA such as mtr_miR169l-5p, mtr_miR398b, and mtr_miR398c and its target genes in seedlings with active nodules. Based on the negative expression pattern between miRNA and its target genes, we constructed an mRNA-miR169l-ncRNA ceRNA network. During severe drought stress, the lncRNA alternative splicings TCONS_00049507 and TCONS_00049510 competitively interacted with mtr_miR169l-5p, which upregulated the expression of NUCLEAR FACTOR-Y (NF-Y) transcription factor subfamily NF-YA genes MtNF-YA2 and MtNF-YA3 to regulate their downstream drought-response genes. Our results emphasized the importance of SNF plants affecting drought tolerance. In conclusion, our work provides insight into ceRNA involvement in rhizobium symbiosis contributing to drought tolerance and provides molecular evidence for future study.

Exogenous Proline Improves Salt Tolerance of Alfalfa through Modulation of Antioxidant Capacity, Ion Homeostasis, and Proline Metabolism.

Alfalfa (Medicago sativa L.) is an important forage crop, and its productivity is severely affected by salt stress. Although proline is a compatible osmolyte that plays an important role in regulating plant abiotic stress resistance, the basic mechanism of proline requires further clarification regarding the effect of proline in mitigating the harmful effects of salinity. Here, we investigate the protective effects and regulatory mechanisms of proline on salt tolerance of alfalfa. The results show that exogenous proline obviously promotes seed germination and seedling growth of salt-stressed alfalfa. Salt stress results in stunted plant growth, while proline application alleviates this phenomenon by increasing photosynthetic capacity and antioxidant enzyme activities and decreasing cell membrane damage and reactive oxygen species (ROS) accumulation. Plants with proline treatment maintain a better K+/Na+ ratio by reducing Na+ accumulation and increasing K+ content under salt stress. Additionally, proline induces the expression of genes related to antioxidant biosynthesis (Cu/Zn-SOD and APX) and ion homeostasis (SOS1, HKT1, and NHX1) under salt stress conditions. Proline metabolism is mainly regulated by ornithine-δ-aminotransferase (OAT) and proline dehydrogenase (ProDH) activities and their transcription levels, with the proline-treated plants displaying an increase in proline content under salt stress. In addition, OAT activity in the ornithine (Orn) pathway rather than Δ1-pyrroline-5-carboxylate synthetase (P5CS) activity in the glutamate (Glu) pathway is strongly increased under salt stress, made evident by the sharp increase in the expression level of the OAT gene compared to P5CS1 and P5CS2. Our study provides new insight into how exogenous proline improves salt tolerance in plants and that it might be used as a significant practical strategy for cultivating salt-tolerant alfalfa.

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Scientific irrigation is of great significance to plant seed production. With two excellent native plant species in desert steppe, Agropyron mongolicum and Lespedeza potaninii, as the objects, and full irrigation as the control, we explored the effects of deficit irrigation in different growth stages on the seed production and water use efficiency (WUE) of those two species. The results showed that, compared with the control, soil water content of both species decreased under deficit irrigation. The decrease of soil water content of A. mongolicum mainly occurred in the 0-60 cm soil layer, while there was no obvious stratification for the reduction of soil water content of L. potaninii. There were significant differences in the yield components of A. mongolica under deficit irrigation. The seed yield of deficit irrigation at the flowering stage was the highest. There were significant differences in the numbers of fertile tillers, florets and pods of L. potaninii among treatments, but no significant difference in seed yield. There were positive correlations between seed yield of A. mongolicum and the numbers of fertile tillers (r=0.776) and spikelets (r=0.717). The racemes of L. potaninii was significantly negatively correlated with the number of fertile tillers (r=-0.685), and positively correlated with the number of florets (r=0.412). Compared with full irrigation, water consumption of seed production of the two native plant species was reduced under deficit irrigation, but water use efficiency was improved, with the strongest improvement at the flowering stage of A. mongolicum (32.9%) and at the branching stage of L. potaninii (27.4%). Therefore, proper deficit irrigation could improve water use efficiency of both plant species. From the perspective of water use efficiency and seed yield, deficit irrigation could be used for artificial breeding of A. mongolicum and L. potaninii seeds in arid area, with the suitable growth stage for deficit being the flowering and the branching stages, respectively.

Comparative Physiological and Transcriptome Analysis Reveal the Molecular Mechanism of Melatonin in Regulating Salt Tolerance in Alfalfa (Medicago sativa L.).

As a high-quality legume forage, alfalfa is restricted by various abiotic stresses during its growth and development. Melatonin is a multifunctional signaling molecule that involves in plant defense against multiple stresses. However, little is known about its downstream signaling pathway and regulatory mechanisms in salt stress of alfalfa. In this study, we investigated the protective effects and key regulatory pathways of melatonin on alfalfa under salt tolerance. The results showed that melatonin promoted the growth of alfalfa seedlings under salt stress, as demonstrated by higher plant height, leaf area, and fresh weight. Melatonin treatment resulted in an increase in the photosynthetic capacity and starch content of alfalfa. Moreover, melatonin decreased cell membrane damage and reactive oxygen species (ROS) accumulation by enhancing antioxidant defense activity under salt stress conditions. Transcriptome sequencing (RNA-seq) analysis revealed that melatonin mainly induced the transcription of genes involved in Ca2+ signaling (cyclic nucleotide gated channel, CNGCs; cam modulin/calmodulin-like protein, CAM/CMLs and calcium-dependent protein kinase, CDPKs), starch and sucrose metabolism (α-amylase, AMYs; ß-amylase, BAMs; starch synthase, SSs and sucrose synthase, SUSs), plant hormone signal transduction (auxin/indole acetic acid protein, AUX/IAAs; ABA receptor, PYL4; protein phosphatase 2C, PP2Cs; scarecrow-like protein, SCLs and ethylene-responsive transcription factor 1B, ERF1B), and key transcription factors (C3Hs, MYBs, ERFs, and WRKYs). Specifically, we focused on starch and sucrose metabolism and plant hormone signal transduction pathways. The interactions between melatonin and other phytohormones occurred via regulation of the expression of genes involved in hormone signaling pathways. In addition, melatonin increased the contents of endogenous melatonin, auxin, gibberellic acid (GA3), salicylic acid, brassinosteroids, and ethylene, while decreasing the abscisic acid content under salt stress. In summary, this study established a regulatory network for melatonin-induced key signaling pathways and functional genes under salt stress and provided a theoretical basis for salt tolerance breeding in alfalfa.

Exogenous melatonin ameliorates drought stress in Agropyron mongolicum by regulating flavonoid biosynthesis and carbohydrate metabolism.

Drought is one of the most common abiotic stressors in plants. Melatonin (MT) is a high-efficiency and low-toxicity growth regulator that plays an important role in plant responses to drought stress. As a wild relative of wheat, Agropyron mongolicum has become an important species for the improvement of degraded grasslands and the replanting of sandy grasslands. However, the physiological and molecular mechanisms by which exogenous MT regulates drought stress in A. mongolicum remain unclear. To assess the effectiveness of MT intervention (100 mg·L-1), polyethylene glycol 6000 was used to simulate drought stress, and its ameliorating effects on drought stress in A. mongolicum seedlings were investigated through physiology, transcriptomics, and metabolomics. Physiological analysis indicated that MT treatment increased the relative water content and chlorophyll content and decreased the relative conductivity of A. mongolicum seedlings. Additionally, MT decreased malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation by enhancing antioxidant enzyme activities. The transcriptome and metabolite profiling analysis of A. mongolicum seedlings treated with and without MT under drought stress identified the presence of 13,466 differentially expressed genes (DEGs) and 271 differentially expressed metabolites (DEMs). The integrated analysis of transcriptomics and metabolomics showed that DEGs and DEMs participated in diverse biological processes, such as flavonoid biosynthesis and carbohydrate metabolism. Moreover, MT may be involved in regulating the correlation of DEGs and DEMs in flavonoid biosynthesis and carbohydrate metabolism during drought stress. In summary, this study revealed the physiological and molecular regulatory mechanisms of exogenous MT in alleviating drought stress in A. mongolicum seedlings, and it provides a reference for the development and utilization of MT and the genetic improvement of drought tolerance in plants from arid habitats.