Chloroplast-targeted late embryogenesis abundant 1 increases alfalfa tolerance to drought and aluminum.

Late embryogenesis-abundant (LEA) proteins are important stress-response proteins that participate in protecting plants against abiotic stresses. Here, we investigated LEA group 3 protein MsLEA1, containing the typically disordered and α-helix structure, via overexpression and RNA interference (RNAi) approaches in alfalfa (Medicago sativa L.) under drought and aluminum (Al) stresses. MsLEA1 was highly expressed in leaves and localized in chloroplasts. Overexpressing MsLEA1 increased alfalfa tolerance to drought and Al stresses, but downregulating MsLEA1 decreased the tolerance. We observed a larger stomatal aperture and a lower water use efficiency in MsLEA1 RNAi lines compared with wild-type plants under drought stress. Photosynthetic rate, Rubisco activity, and superoxide dismutase (SOD) activity increased or decreased in MsLEA1-OE or MsLEA1-RNAi lines, respectively, under drought and Al stress. Copper/zinc SOD (Cu/Zn-SOD), iron SOD (Fe-SOD), and Rubisco large subunit proteins (Ms1770) were identified as binding partners of MsLEA1, which protected chloroplast structure and function under drought and Al stress. These results indicate that MsLEA1 recruits and protects its target proteins (SOD and Ms1770) and increases alfalfa tolerance against drought and Al stresses.

MsMYB741 is involved in alfalfa resistance to aluminum stress by regulating flavonoid biosynthesis.

Aluminum (Al) toxicity severely restricts plant growth in acidic soils (pH < 5.0). In this study, an R2R3-MYB transcription factor (TF) gene, MsMYB741, was cloned from alfalfa. Its function and gene regulatory pathways were studied via overexpression and RNA interference of MsMYB741 in alfalfa seedlings. Results showed that root elongation increased as a result of MsMYB741 overexpression (MsMYB741-OE) and decreased with MsMYB741 RNA interference (MsMYB741-RNAi) in alfalfa seedlings compared with the wild-type under Al stress. These were attributed to the reduced Al content in MsMYB741-OE lines, and increased Al content in MsMYB741-RNAi lines. MsMYB741 positively activated the expression of phenylalanine ammonia-lyase 1 (MsPAL1) and chalcone isomerase (MsCHI) by binding to MYB and ABRE elements in their promoters, respectively, which directly affected flavonoid accumulation in roots and secretion from root tips in plants under Al stress, eventually affecting Al accumulation in alfalfa. Additionally, MsABF2 TF directly activated the expression of MsMYB741 by binding to the ABRE element in its promoter. Taken together, our results indicate that MsMYB741 transcriptionally activates MsPAL1 and MsCHI expression to increase flavonoid accumulation in roots and secretion from root tips, leading to increased resistance of alfalfa to Al stress.

Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips.

A SK3 -type dehydrin MsDHN1 was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression and suppression of MsDHN1 in alfalfa seedlings or hairy roots. The results showed that MsDHN1 is a typical intrinsically disordered protein that exists in the form of monomers and homodimers in alfalfa. The plant growth rates increased as a result of MsDHN1 overexpression (MsDHN1-OE) and decreased upon MsDHN1 suppression (MsDHN1-RNAi) in seedlings or hairy roots of alfalfa compared with the wild-type or the vector line under Al stress. MsDHN1 interacting with aquaporin (AQP) MsPIP2;1 and MsTIP1;1 positively affected oxalate secretion from root tips and Al accumulation in root tips. MsABF2 was proven to be an upstream transcription factor of MsDHN1 and activated MsDHN1 expression by binding to the ABRE element of the MsDHN1 promoter. The transcriptional regulation of MsABF2 on MsDHN1 was dependent on the abscisic acid signaling pathway. These results indicate that MsDHN1 can increase alfalfa tolerance to Al stress via increasing oxalate secretion from root tips, which may involve in the interaction of MsDHN1 with two AQP.

Auxin Is Involved in Magnesium-Mediated Photoprotection in Photosystems of Alfalfa Seedlings Under Aluminum Stress.

The objective of this study was to investigate the effects of Mg and IAA on the photosystems of Al-stressed alfalfa (Medicago sativa L.). Alfalfa seedlings with or without apical buds were exposed to solutions fully mixed with 0 or 100 µM AlCl3 and 0 or 50 µM MgCl2 followed by foliar spray with water or IAA. Results from seedlings with apical buds showed that application of Mg and IAA either alone or combine greatly alleviated the Al-induced damage on photosystems. The values of photosynthetic rate (Pn), effective quantum yields [Y(I) and Y(II)] and electron transfer rates (ETRI and ETRII), proton motive force (pmf), cyclic electron flow (CEF), proton efflux rate (gH +), and activities of ATP synthase and PM H+-ATPase significantly increased, and proton gradient (ΔpH pmf ) between lumen and stroma decreased under Al stress. After removing apical buds of seedlings, the Y(I), Y(II), ETRI, ETRII, pmf, and gH + under exogenous spraying IAA significantly increased, and ΔpH pmf significantly decreased in Mg addition than Al treatment alone, but they were no significant difference under none spraying IAA. The interaction of Mg and IAA directly increased quantum yields and electron transfer rates, and decreased O2 - accumulation in Al-stressed seedlings with or without apical buds. These results suggest that IAA involves in Mg alleviation of Al-induced photosystem damage via increasing pmf and PM H+-ATPase activity, and decreasing ΔpH pmf .

Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway.

Dehydrin improves plant resistance to many abiotic stresses. In this study, the expression profiles of a dehydrin gene, CdDHN4, were estimated under various stresses and abscisic acid (ABA) treatments in two bermudagrasses (Cynodon dactylon L.): Tifway (drought-tolerant) and C299 (drought-sensitive). The expression of CdDHN4 was up-regulated by high temperatures, low temperatures, drought, salt and ABA. The sensitivity of CdDHN4 to ABA and the expression of CdDHN4 under drought conditions were higher in Tifway than in C299. A 1239-bp fragment, CdDHN4-P, the partial upstream sequence of the CdDHN4 gene, was cloned by genomic walking from Tifway. Bioinformatic analysis showed that the CdDHN4-P sequence possessed features typical of a plant promoter and contained many typical cis elements, including a transcription initiation site, a TATA-box, an ABRE, an MBS, a MYC, an LTRE, a TATC-box and a GT1-motif. Transient expression in tobacco leaves demonstrated that the promoter CdDHN4-P can be activated by ABA, drought and cold. These results indicate that CdDHN4 is regulated by an ABA-dependent signal pathway and that the high sensitivity of CdDHN4 to ABA might be an important mechanism enhancing the drought tolerance of bermudagrass.

Gene Expression Analysis of Alfalfa Seedlings Response to Acid-Aluminum.

Acid-Aluminum (Al) is toxic to plants and greatly affects crop production worldwide. To understand the responses of plants to acid soils and Aluminum toxicity, we examined global gene expression using microarray data in alfalfa seedlings with the treatment of acid-Aluminum. 3,926 genes that were identified significantly up- or downregulated in response to Al3+ ions with pH 4.5 treatment, 66.33% of which were found in roots. Their functional categories were mainly involved with phytohormone regulation, reactive oxygen species, and transporters. Both gene ontology (GO) enrichment and KEGG analysis indicated that phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis played a critical role on defense to Aluminum stress in alfalfa. In addition, we found that transcription factors such as the MYB and WRKY family proteins may be also involved in the regulation of reactive oxygen species reactions and flavonoid biosynthesis. Thus, the finding of global gene expression profile provided insights into the mechanisms of plant defense to acid-Al stress in alfalfa. Understanding the key regulatory genes and pathways would be advantageous for improving crop production not only in alfalfa but also in other crops under acid-Aluminum stress.