Drought Tolerance In Alfalfa Research Articles

MsFtsH8 Enhances the Tolerance of PEG-Simulated Drought Stress by Boosting Antioxidant Capacity in Medicago sativa L.

Drought is a major abiotic stress that limits the growth and yield of alfalfa, a vital forage legume. The plant metalloproteinase Filamentation temperature-sensitive H (FtsH) is an ATP- and Zn2+-dependent enzyme that plays a significant character in the plant's response to environmental stress. However, its functional role in drought resistance remains largely unexplored. This study investigates the drought tolerance role of alfalfa MsFtsH8 by analyzing the growth, physiology, and gene expression of overexpressing plants under drought conditions. The results demonstrated that both MsFtsH8-overexpressing Arabidopsis and alfalfa plants exhibited superior growth condition and enhanced membrane stability. The overexpressing alfalfa plants also showed reduced MDA levels, higher proline content, lower H2O2 accumulation, an increased activity of antioxidant-related enzymes (SOD, POD, and CAT) activity, and an elevated expression of antioxidant-related genes. These results indicated that the overexpression of MsFtsH8 enhanced growth, improved osmotic regulation, reduced ROS levels, and increased antioxidative capacity, ultimately leading to greater drought tolerance in alfalfa. Our findings suggest that MsFtsH8 mitigates oxidative damage caused by drought by modulating the plant's antioxidant system, thus improving drought tolerance in alfalfa. This study provides a molecular basis and candidate genes for enhancing drought resistance in alfalfa through genetic engineering.

Constitutive expression of MsPROPEP1 enhances salinity and drought tolerance in alfalfa (Medicago sativa)

Constitutive expression of MsPROPEP1 enhances salinity and drought tolerance in alfalfa (Medicago sativa)

Drought-induced recruitment of specific root-associated bacteria enhances adaptation of alfalfa to drought stress.

Drought is a major abiotic stress that threatens crop production. Soil microbiomes are thought to play a role in enhancing plant adaptation to various stresses. However, it remains unclear whether soil microbiomes play a key role when plants are challenged by drought and whether different varieties are enriched with specific bacteria at the rhizosphere. In this study, we measured changes in growth phenotypes, physiological and biochemical characteristics of drought-tolerant alfalfa (AH) and drought-sensitive (QS) under sterilized and unsterilized soil conditions with adequate watering and with drought stress, and analyzed the rhizosphere bacterial community composition and changes using 16S rRNA high-throughput sequencing. We observed that the unsterilized treatment significantly improved the growth, and physiological and biochemical characteristics of alfalfa seedlings under drought stress compared to the sterilized treatment. Under drought stress, the fresh and dry weight of seedlings increased by 35.24, 29.04, and 11.64%, 2.74% for unsterilized AH and QS, respectively, compared to sterilized treatments. The improvement was greater for AH than for QS. AH and QS recruited different rhizosphere bacteria when challenged by drought. Interestingly, under well-watered conditions, the AH rhizosphere was already rich in drought-tolerant bacterial communities, mainly Proteobacteria and Bacteroidetes, whereas these bacteria started to increase only when QS was subjected to drought. When drought stress was applied, AH was enriched with more drought-tolerant bacteria, mainly Acidobacteria, while the enrichment was weaker in QS rhizosphere. Therefore, the increase in drought tolerance of the drought-tolerant variety AH was greater than that of the drought-sensitive variety QS. Overall, this study confirmed the key role of drought-induced rhizosphere bacteria in improving the adaptation of alfalfa to drought stress, and clarified that this process is significantly related to the variety (genotype). The results of this study provide a basis for improving drought tolerance in alfalfa by regulating the rhizosphere microbiome.

Identification of QTL and candidate genes associated with biomass yield and Feed Quality in response to water deficit in alfalfa (Medicago sativa L.) using linkage mapping and RNA-Seq

Biomass yield and Feed Quality are the most important traits in alfalfa (Medicago sativa L.), which directly affect its economic value. Drought stress is one of the main limiting factors affecting alfalfa production worldwide. However, the genetic and especially the molecular mechanisms for drought tolerance in alfalfa are poorly understood. In this study, linkage mapping was performed in an F1 population by combining 12 phenotypic data (biomass yield, plant height, and 10 Feed Quality-related traits). A total of 48 significant QTLs were identified on the high-density genetic linkage maps that were constructed in our previous study. Among them, nine main QTLs, which explained more than 10% phenotypic variance, were detected for biomass yield (one), plant height (one), CP (two), ASH (one), P (two), K(one), and Mg (one). A total of 31 candidate genes were identified in the nine main QTL intervals based on the RNA-seq analysis under the drought condition. Blast-P was further performed to screen candidate genes controlling drought tolerance, and 22 functional protein candidates were finally identified. The results of the present study will be useful for improving drought tolerance of alfalfa varieties by marker-assisted selection (MAS), and provide promising candidates for further gene cloning and mechanism study.

NUCLEAR TRANSPORT FACTOR 2-LIKE improves drought tolerance by modulating leaf water loss in alfalfa (Medicago sativa L.).

Drought is a major environmental factor that limits the production of alfalfa (Medicago sativa). In the present study, M. sativa NUCLEAR TRANSPORT FACTOR 2-LIKE (MsNTF2L) was identified as a nucleus-, cytoplasm-, and plasma membrane-localized protein. Its transcriptional expression was highly induced by ABA and drought stress. Overexpression of MsNTF2L in Arabidopsis resulted in hypersensitivity to ABA during both the seed germination and seedling growth stages. However, transgenic Arabidopsis plants exhibited enhanced tolerance to drought stress by reducing the levels of reactive oxygen species (ROS) and increasing the expression of stress/ABA-inducible genes. Consistently, analysis of MsNTF2L overexpression (OE) and RNA interference (RNAi) alfalfa plants revealed that MsNTF2L confers drought tolerance through promoting ROS scavenging, a decrease in stomatal density, ABA-induced stomatal closure, and epicuticular wax crystal accumulation. MsNTF2L highly affected epicuticular wax deposition, as a large group of wax biosynthesis and transport genes were influenced in the alfalfa OE and RNAi lines. Furthermore, transcript profiling of drought-treated alfalfa WT, OE, and RNAi plants showed a differential drought response for genes related to stress/ABA signaling, antioxidant defense, and photosynthesis. Taken together, these results reveal that MsNTF2L confers drought tolerance in alfalfa via modulation of leaf water loss (by regulating both stomata and wax deposition), antioxidant defense, and photosynthesis.

Pretreatment with Chitosan Arbitrates Physiological Processes and Antioxidant Defense System to Increase Drought Tolerance in Alfalfa (Medicago sativa L.)

Pretreatment with Chitosan Arbitrates Physiological Processes and Antioxidant Defense System to Increase Drought Tolerance in Alfalfa (Medicago sativa L.)

Exogenous melatonin protects alfalfa (Medicago sativa L.) seedlings from drought-induced damage by modulating reactive oxygen species metabolism, mineral balance and photosynthetic efficiency

• Data on melatonin-mediated drought tolerance in alfalfa was provided. • Drought is a constraint on the growth and establishment of seedlings. • The results accurately characterize the risk of abiotic stress in the environment. • These findings help to find sustainable strategy-enabled successful cultivation of alfalfa. Drought stress is a severe global environmental concern for the growth and development of alfalfa ( Medicago sativa L.), which is crucial for agricultural sustainability owing to its high feed value and economic prospects. Melatonin (MT) has emerged as a multifaceted biomolecule that plays a fundamental role in regulating plants' multiple environmental stresses. However, the significant effect of exogenous MT on drought stress tolerance in the alfalfa forage crops remains inexpressible. We investigated the effects of foliar-sprayed melatonin at different doses (50, 100, 200 μM) on alfalfa seedlings' growth and physiological responses under drought stress. Our results showed that drought stress reduced morphological growth attributes (by 12 to 54%), gas exchange parameters (by 37 to 88%), photosynthetic pigments (Chl a, and Chl b contents by 29%, and 40%), mineral contents, and increased lipid peroxidation (by 69%) and accumulation of reactive oxygen species (ROS) in alfalfa plants. In contrast, exogenous foliar application of MT significantly revived growth characteristics, boosted antioxidant enzyme activities, improved photosynthetic performance, decreased ROS-induced oxidative damage by increasing plant mineral elements, and lowered lipid peroxidation in plants under drought stress. In addition, MT also stimulated the accumulation of total soluble sugar and proline content by 48% and by 75% respectively, which were positively associated with improved leaf water status in drought-stressed plants. Meanwhile, our findings revealed that the highest efficiency was achieved at 100 μM melatonin. In conclusion, these findings bring a new perspective on melatonin-mediated drought stress tolerance and provide a foundational understanding of alfalfa's future cultivation in drought-prone regions globally.

Transcriptome-IPMS analysis reveals a tissue-dependent miR156/SPL13 regulatory mechanism in alfalfa drought tolerance

BackgroundWe previously reported on the interplay between miR156/SPL13 and WD40–1/DFR to improve response to drought stress in alfalfa (Medicago sativa L.). Here we aimed to investigate whether the role of miR156/SPL13 module in drought response is tissue-specific, and to identify SPL13-interacting proteins. We analyzed the global transcript profiles of leaf, stem, and root tissues of one-month old RNAi-silenced SPL13 (SPL13RNAi) alfalfa plants exposed to drought stress and conducted protein-protein interaction analysis to identify SPL13 interacting partners.ResultTranscript analysis combined with weighted gene co-expression network analysis showed tissue and genotype-specific gene expression patterns. Moreover, pathway analysis of stem-derived differentially expressed genes (DEG) revealed upregulation of genes associated with stress mitigating primary and specialized metabolites, whereas genes associated with photosynthesis light reactions were silenced in SPL13RNAi plants. Leaf-derived DEG were attributed to enhanced light reactions, largely photosystem I, II, and electron transport chains, while roots of SPL13RNAi plants upregulated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. Using immunoprecipitation combined with mass spectrometry (IPMS) we showed that SPL13 interacts with proteins involved in photosynthesis, specialized metabolite biosynthesis, and stress tolerance.ConclusionsWe conclude that the miR156/SPL13 module mitigates drought stress in alfalfa by regulating molecular and physiological processes in a tissue-dependent manner.

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions.

Over-expression of a \u03b3-tocopherol methyltransferase gene in vitamin E pathway confers PEG-simulated drought tolerance in alfalfa

Backgroundα-Tocopherol is one of the most important vitamin E components present in plant. α-Tocopherol is a potent antioxidant, which can deactivate photoproduced reactive oxygen species (ROS) and prevent lipids from oxidation when plants suffer drought stress. γ-Tocopherol methyltransferase (γ-TMT) catalyzes the formation of α-tocopherol in the tocopherol biosynthetic pathway. Our previous studies showed that over-expression of γ-TMT gene can increase the accumulation of α-tocopherol in alfalfa (Medicago sativa). However, whether these transgenic plants confer increased drought tolerance and the underlying mechanism are still unknown.ResultsIn the present study, we further evaluate transgenic alfalfa lines, and found that over-expression of MsTMT led to an increase in α-tocopherol and total tocopherol level in the transgenic lines compared with the control plant. It was revealed that drought tolerance of the transgenic alfalfa was remarkably increased, with alleviated oxidative damage and accumulation of more osmolytic substances. The stomatal development in transgenic plants was significantly inhibited on both sides of leaves, which may be resulted from the repression of MsSPCHLESS (MsSPCH) gene. The reduced stomatal density of transgenic plants contributes to a lower stomatal conductance and higher water use efficiency (WUE). Moreover, both RNA-seq and qRT-PCR analyses indicate that regulatory mechanism of MsTMT in drought involved in both ABA-dependent and ABA-independent pathways.ConclusionOur results suggest that MsTMT gene plays a positive role in regulating alfalfa response to PEG-simulated drought stress, which might involve complex mechanisms, including ROS scavenging system, stomatal development and multiple phytohormone signaling pathways. This study will broaden our view on the function of γ-TMT gene and provide new strategy for genetic engineering in alfalfa breeding.

The interplay between miR156/SPL13 and DFR/WD40\u20131 regulate drought tolerance in alfalfa

BackgroundDeveloping Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop’s sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors.ResultsTo understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40–1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40–1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40–1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression.ConclusionsTaken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40–1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Endogenous NO-mediated transcripts involved in photosynthesis and carbohydrate metabolism in alfalfa (Medicago sativa L.) seedlings under drought stress

Alfalfa (Medicago sativa L.) is an important perennial legume and used as a forage crop worldwide, and has extensive resistance to various abiotic stresses. Nitric oxide (NO) plays a critical role in response to external and internal cues to regulate plant growth and development. However, endogenous NO-mediated molecular mechanisms of drought tolerance in alfalfa is poorly understood. To get a deeper insight into the regulate pathway of NO, RNA-Seq was used to profile transcriptome changes of alfalfa seedlings, which were treated with NO scavenger under normal and drought conditions. A total of 1,025 and 3,461 differently-expressed genes (FDR < 0.0001; fold change ≥ 2) were observed while NO absence under normal and drought conditions, respectively. Based on GO enrich and KEGG pathway analysis, we found NO absence induced photosynthesis, carbon fixation in photosynthetic organisms and primary metabolism were significantly up-enriched. Most oxidoreductase, dehydrogenase, reductase and transferase genes were down-regulated in the above processes. Moreover, NO absence restrained chlorophyll biosynthesis and decreased different sugar content. Therefore, this work provides insights into the mechanism that NO-mediated enhanced photosynthesis and carbohydrate metabolism in alfalfa under drought stress.

The constitutive expression of alfalfa MsMYB2L enhances salinity and drought tolerance of Arabidopsis thaliana

MYB-type transcription factors are known to participate in the response of plants to a number of stress agents. MsMYB2L is an alfalfa member of this large gene family. Its transcription in alfalfa seedlings was found to be rapidly and strongly induced by salinity, moisture deficiency and exogenously supplied abscisic acid. An analysis based on a yeast one hybrid assay indicated that its product is able to activate transcription, consistent with its function as a transcription factor. When the gene was constitutively expressed in Arabidopsis thaliana, both germination and seedling growth were more sensitive to ABA treatment than wild type, and growth was less strongly compromised by salinity and moisture deficiency stress, presumably as a result of the induction of certain stress-related genes active in ABA-dependent pathways. The transgenic seedlings’ enhanced the synthesis of many osmotic regulatory substances such as proline and soluble sugar, and decreased the lipid peroxidation. In all, MsMYB2L represents a potential candidate gene for manipulating the salinity and drought tolerance of alfalfa.

Drought tolerance in alfalfa (Medicago sativa L.) varieties is associated with enhanced antioxidative protection and declined lipid peroxidation

Drought stress is considered the most adverse factor restricting plant survival, growth, and productivity. The identification of the key adaptive mechanisms to drought stress is essential to enhance the drought resistance of plants. In this study, differential responses of three alfalfa varieties to drought, including Medicago sativa L. cv. Longzhong (drought-tolerant), Longdong (moderate drought-tolerant), and Gannong No. 3 (drought-sensitive), were comparatively studied at morphological, physio-biochemical, and transcriptional levels after a 12-day period of drought stress simulated by −1.2 MPa polyethylene glycol (PEG-6000). The results showed that prolonged drought stress dramatically decreased growth and photosynthetic capacity of three alfalfa varieties while it increased the accumulation of malondialdehyde (MDA), reactive oxygen species (ROS), osmolytes and antioxidants including reduced ascorbate and glutathione, ascorbate peroxidase (APX) activities, and gene expression of antioxidative enzymes (MsCu/Zn-SOD, MsFeSOD, MtPOD, MsGPX, MsAPX, MsMDAR, MtDHAR, and MsGR). Nine days of treatment and some key traits, including the maximum quantum yield of photosystem II (Fv/Fm), the levels of MDA, O2−, and H2O2, the redox states of ascorbate and glutathione, APX activity, and the transcript levels of MsFeSOD, MsGR, and MsMDAR, might contribute to differentiating the drought stress tolerance in alfalfa. Overall, drought-tolerant Longzhong showed the highest water retention, photosynthetic performance, and osmoregulation capacity, the lowest lipid peroxidation, and the highest antioxidant enzyme activities and gene expression, which were mainly involved in the ascorbate-glutathione cycle to maintain the balance between the generation and scavenging of intracellular ROS. These findings highlight that enhanced antioxidative protection and declined lipid peroxidation play an important role in alfalfa tolerance against drought.

Transcriptome analysis of microRNA156 overexpression alfalfa roots under drought stress

Drought is one of the major abiotic stresses that negatively impact alfalfa growth and productivity. The role of microRNA156 (miR156) in drought has been demonstrated in plants. To date, there are no published studies investigating the role of miR156 in regulating global gene expression in alfalfa under drought. In our study, alfalfa genotypes overexpressing miR156 (miR156OE) exhibited reduced water loss, and enhanced root growth under drought. Our RNA-seq data showed that in response to drought, a total of 415 genes were upregulated and 169 genes were downregulated specifically in miR156OE genotypes. Genotypic comparison revealed that 285 genes were upregulated and 253 genes were downregulated in miR156OE genotypes relative to corresponding WT under drought. Gene Ontology enrichment analysis revealed that the number of differentially expressed genes belonging to biological process, molecular function and cell component functional groups was decreased in miR156OE genotypes under drought. Furthermore, RNA-Seq data showed downregulation of a gene encoding WD40 repeat in a miR156-specific manner. 5′ RACE experiments verified cleavage of WD40-2 transcript under drought. Moreover, alfalfa plants overexpressing WD40-2 showed drought sensitive, whereas those with silenced WD40-2 exhibited drought tolerant phenotypes. These findings suggest that miR156 improves drought tolerance in alfalfa by targeting WD40-2.

Physiological and Proteomic Responses of Contrasting Alfalfa (Medicago sativa L.) Varieties to PEG-Induced Osmotic Stress.

Drought severely limits global plant distribution and agricultural production. Elucidating the physiological and molecular mechanisms governing alfalfa stress responses will contribute to the improvement of drought tolerance in leguminous crops. In this study, the physiological and proteomic responses of two alfalfa (Medicago sativa L.) varieties contrasting in drought tolerance, Longzhong (drought-tolerant) and Gannong No. 3 (drought-sensitive), were comparatively assayed when seedlings were exposed to -1.2 MPa polyethylene glycol (PEG-6000) treatments for 15 days. The results showed that the levels of proline, malondialdehyde (MDA), hydrogen peroxide (H2O2), hydroxyl free radical (OH•) and superoxide anion free radical (O2•-) in both varieties were significantly increased, while the root activity, the superoxide dismutase (SOD) and glutathione reductase (GR) activities, and the ratios of reduced/oxidized ascorbate (AsA/DHA) and reduced/oxidized glutathione (GSH/GSSG) were significantly decreased. The soluble protein and soluble sugar contents, the total antioxidant capability (T-AOC) and the activities of peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) first increased and then decreased with the increase in treatment days. Under osmotic stress, Longzhong exhibited lower levels of MDA, H2O2, OH• and O2•- but higher levels of SOD, CAT, APX, T-AOC and ratios of AsA/DHA and GSH/GSSG compared with Gannong No.3. Using isobaric tags for relative and absolute quantification (iTRAQ), 142 differentially accumulated proteins (DAPs) were identified from two alfalfa varieties, including 52 proteins (34 up-regulated and 18 down-regulated) in Longzhong, 71 proteins (28 up-regulated and 43 down-regulated) in Gannong No. 3, and 19 proteins (13 up-regulated and 6 down-regulated) shared by both varieties. Most of these DAPs were involved in stress and defense, protein metabolism, transmembrane transport, signal transduction, as well as cell wall and cytoskeleton metabolism. In conclusion, the stronger drought-tolerance of Longzhong was attributed to its higher osmotic adjustment capacity, greater ability to orchestrate its enzymatic and non-enzymatic antioxidant systems and thus avoid great oxidative damage in comparison to Gannong No. 3. Moreover, the involvement of other pathways, including carbohydrate metabolism, ROS detoxification, secondary metabolism, protein processing, ion and water transport, signal transduction, and cell wall adjustment, are important mechanisms for conferring drought tolerance in alfalfa.

Over-Expression of Arabidopsis EDT1 Gene Confers Drought Tolerance in Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa L.) is an important legume forage crop with great economic value. However, as the growth of alfalfa is seriously affected by an inadequate supply of water, drought is probably the major abiotic environmental factor that most severely affects alfalfa production worldwide. In an effort to enhance alfalfa drought tolerance, we transformed the Arabidopsis Enhanced Drought Tolerance 1 (AtEDT1) gene into alfalfa via Agrobacterium-mediated transformation. Compared with wild type plants, drought stress treatment resulted in higher survival rates and biomass, but reduced water loss rates in the transgenic plants. Furthermore, transgenic alfalfa plants had increased stomatal size, but reduced stomatal density, and these stomatal changes contributed greatly to reduced water loss from leaves. Importantly, transgenic alfalfa plants exhibited larger root systems with larger root lengths, root weight, and root diameters than wild type plants. The transgenic alfalfa plants had reduced membrane permeability and malondialdehyde content, but higher soluble sugar and proline content, higher superoxide dismutase activity, higher chlorophyll content, enhanced expression of drought-responsive genes, as compared with wild type plants. Notably, transgenic alfalfa plants grew better in a 2-year field trial and showed enhanced growth performance with increased biomass yield. All of our morphological, physiological, and molecular analyses demonstrated that the ectopic expression of AtEDT1 improved growth and enhanced drought tolerance in alfalfa. Our study provides alfalfa germplasm for use in forage improvement programs, and may help to increase alfalfa production in arid lands.

MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13

Alfalfa (Medicago sativa) is an important forage crop that is often grown in areas that frequently experience drought and water shortage. MicroRNA156 (miR156) is an emerging tool for improving various traits in plants. We tested the role of miR156d in drought response of alfalfa, and observed a significant improvement in drought tolerance of miR156 overexpression (miR156OE) alfalfa genotypes compared to the wild type control (WT). In addition to higher survival and reduced water loss, miR156OE genotypes also maintained higher stomatal conductance compared to WT during drought stress. Furthermore, we observed an enhanced accumulation of compatible solute (proline) and increased levels of abscisic acid (ABA) and antioxidants in miR156OE genotypes. Similarly, alfalfa plants with reduced expression of miR156-targeted SPL13 showed reduced water loss and enhanced stomatal conductance, chlorophyll content and photosynthetic assimilation. Several genes known to be involved in drought tolerance were differentially expressed in leaf and root of miR156 overexpression plants. Taken together, our findings reveal that miR156 improves drought tolerance in alfalfa at least partially by silencing SPL13.

Identify Physiological Markers for Drought Tolerance in Alfalfa

Physiological markers may provide a shortcut for identifying drought tolerance in alfalfa (Medicago sativa L.). Thus, the objective of this study was to understand physiological properties of alfalfa to assess drought tolerance of its cultivars. Ten alfalfa cultivars (Qomi, Isfahani, Hamedani, Bami, Ordobadi, Gharayonje, Nikshahri, Yazdi, Baghdadi, and Cody) and four irrigation regimes (55, 65, 75, and 85% depletion of available soil water) were arranged as split plot in a completely randomized block design with three replications to determine the chlorophyll, carotenoid, soluble sugars, proline, relative water contents, and herbage yield response of alfalfa cultivars to drought stress. Physiological and herbage yield traits were drought level‐cultivar‐specific and discriminated the cultivars based on their drought tolerance. Based on the correlation between drought tolerance and measured traits at the highest drought level, carotenoid content (r = 0.94), was the most important marker followed by relative water content (r = 0.92) and soluble sugars content (r = 0.89), respectively, and Baghdadi was the most drought tolerant cultivar. The results showed that the physiological markers may be used to identify alfalfa germplasms for drought tolerance.

System responses to long‐term drought and re‐watering of two contrasting alfalfa varieties

Systems analysis of two alfalfa varieties, Wisfal (Medicago sativa ssp. falcata var. Wisfal) and Chilean (M.sativa ssp. sativa var. Chilean), with contrasting tolerance/sensitivity to drought revealed common and divergent responses to drought stress. At a qualitative level, molecular, biochemical, and physiological responses to drought stress were similar in the two varieties, indicating that they employ the same strategies to cope with drought. However, quantitative differences in responses at all levels were revealed that may contribute to greater drought tolerance in Wisfal. These included lower stomatal density and conductance in Wisfal; delayed leaf senescence compared with Chilean; greater root growth following a drought episode, and greater accumulation of osmolytes, including raffinose and galactinol, and flavonoid antioxidants in roots and/or shoots of Wisfal. Genes encoding transcription factors and other regulatory proteins, and genes involved in the biosynthesis of osmolytes and (iso)flavonoids were differentially regulated between the two varieties and represent potential targets for improving drought tolerance in alfalfa in the future.