MsWRKY44 regulates Mg-K homeostasis of shoots and promotes alfalfa sensitivities to acid and Al stresses.

Mg-K homeostasis is essential for plant response to abiotic stress, but its regulation remains largely unknown. MsWRKY44 cloned from alfalfa was highly expressed in leaves and petioles. Overexpression of it inhibited alfalfa growth, and promoted leaf senescence and alfalfa sensitivities to acid and Al stresses. The leaf tips, margins and interveins of old leaves occurred yellow spots in MsWRKY44-OE plants under pH4.5 and pH4.5 +Al conditions. Meanwhile, Mg-K homeostasis was substantially changed with reduction of K accumulation and increases of Mg as well as Al accumulation in shoots of MsWRKY44-OE plants. Further, MsWRKY44 was found to directly bind to the promoters of MsMGT7 and MsCIPK23, and positively activated their expression. Transiently overexpressed MsMGT7 and MsCIPK23 in tobacco leaves increased the Mg and Al accumulations but decreased K accumulation. These results revealed a novel regulatory module MsWRKY44-MsMGT7/MsCIPK23, which affects the transport and accumulation of Mg and K in shoots, and promotes alfalfa sensitivities to acid and Al stresses.

PECTIN ACETYLESTERASE12 regulates shoot branching via acetic acid and auxin accumulation in alfalfa shoots.

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa.

The MsDHN1-MsPIP2;1-MsmMYB module orchestrates the trade-off between growth and survival of alfalfa in response to drought stress.

Dehydrins and aquaporins play crucial roles in plant growth and stress responses by acting as protector and controlling water transport across membranes, respectively. MsDHN1 (dehydrin) and MsPIP2;1 (aquaporin) were demonstrated to interact with a membrane-anchored MYB protein, MsmMYB (as mMYB) in plasma membrane under normal condition. MsDHN1, MsPIP2;1 and MsDHN1-MsPIP2;1 positively regulated alfalfa tolerance to water deficiency. Water deficiency caused phosphorylation of MsPIP2;1 at Ser 272, which led to release C terminus of mMYB (mMYBΔ83) from plasma membrane and translocate to nucleus, where C terminus of MsDHN1 interacted with mMYBΔ83, and promoted mMYBΔ83 transcriptional activity in response to water deficiency. Overexpression of mMYB and mMYBΔ83 down-regulated the expression of MsCESA3, but up-regulated MsCESA7 expression by directly binding to their promoters, and resulted in high drought tolerance in transgenic hairy roots. These results indicate that the MsDHN1-MsPIP2;1-MsMYB module serves as a key regulator in alfalfa against drought stress.

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.

Genome-wide identification and analysis of LEA_2 gene family in alfalfa (Medicago sativa L.) under aluminum stress.

Late embryonic development abundant proteins (LEAs) are a large family of proteins commonly existing in plants. LEA_2 is the largest subfamily in the LEA, it plays an important role in plant resistance to abiotic stress. In order to explore the characteristics of LEA_2 gene family members in alfalfa (Medicago sativa L.), 155 members of LEA_2 (MsLEA_2) family were identified from alfalfa genome. Bioinformatics analysis was conducted from the aspects of phylogenetic relationship, chromosome distribution, chromosome colinearity, physical and chemical properties, motif composition, exon-intron structure, cis-element and so on. Expression profiles of MsLEA_2 gene were obtained based on Real-time fluorescent quantitative PCR (qRT-PCR) analysis and previous RNA-seq data under aluminum (Al) stress. Bioinformatics results were shown that the MsLEA_2 genes are distributed on all 32 chromosomes. Among them, 85 genes were present in the gene clusters, accounting for 54.83%, and chromosome Chr7.3 carries the largest number of MsLEA_2 (19 LEA_2 genes on Chr7.3). Chr7.3 has a unique structure of MsLEA_2 distribution, which reveals a possible special role of Chr7.3 in ensuring the function of MsLEA_2. Transcriptional structure analysis revealed that the number of exons in each gene varies from 1 to 3, and introns varies from 0 to 2. Cis-element analysis identified that the promoter region of MsLEA_2 is rich in ABRE, MBS, LTR, and MeJARE, indicating MsLEA_2 has stress resistance potential under abiotic stress. RNA-seq data and qRT-PCR analyses showed that most of the MsLEA_2 members were up-regulated when alfalfa exposed to Al stress. This study revealed that phylogenetic relationship and possible function of LEA_ 2 gene in alfalfa, which were helpful for the functional analysis of LEA_ 2 proteins in the future and provided a new theoretical basis for improving Al tolerance of alfalfa.

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.

Analysis of the Function of the Alfalfa Mslea-D34 Gene in Abiotic Stress Responses and Flowering Time.

A novel late embryogenesis abundant (LEA) gene, MsLEA-D34, was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression (OE) and RNA interference (RNAi) of the gene in Arabidopsis and in hairy roots of alfalfa, as well as via analyzing key genes related to MsLEA-D34 during developmental phases in alfalfa. The results showed that MsLEA-D34 was a typical intrinsically disordered protein with a high capability for protein protection. Overexpression of MsLEA-D34 increased plant tolerance to osmotic and salt stresses, and caused Arabidopsis early flowering under drought and well-watered conditions. Overexpressing MsLEA-D34 induced up-regulation of FLOWERING LOCUS T (FT) and GIGANTEA (GI) at the flowering phase of Arabidopsis and hairy roots of alfalfa, but only FT was down-regulated in MsLEA-D34-RNAi lines. A positive effect of MsLEA-D34 on FT accumulation was demonstrated in alfalfa hairy roots. An ABA-responsive element (ABRE)-binding transcription factor (MsABF2), a novel transcription factor cloned from alfalfa, directly bound to the RY element in the MsLEA-D34 promoter and activated MsLEA-D34 expression. The above results indicate that MsLEA-D34 can regulate abiotic stress response in plants and influence flowering time of Arabidopsis.

Interaction of zinc and IAA alleviate aluminum-induced damage on photosystems via promoting proton motive force and reducing proton gradient in alfalfa.

BACKGROUND: In acidic soils, aluminum (Al) competing with Zn results in Zn deficiency in plants. Zn is essential for auxin biosynthesis. Zn-mediated alleviation of Al toxicity has been rarely studied, the mechanism of Zn alleviation on Al-induced photoinhibition in photosystems remains unclear. The objective of this study was to investigate the effects of Zn and IAA on photosystems of Al-stressed alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or100 µM AlCl3 combined with 0 or 50 µM ZnCl2, and then foliar spray with water or 6 mg L- 1 IAA. RESULTS: Our results showed that Al stress significantly decreased plant growth rate, net photosynthetic rate (Pn), quantum yields and electron transfer rates of PSI and PSII. Exogenous application of Zn and IAA significantly alleviated the Al-induced negative effects on photosynthetic machinery, and an interaction of Zn and IAA played an important role in the alleviative effects. After removing apical buds of Al-stressed alfalfa seedlings, the values of pmf, gH+ and Y(II) under exogenous spraying IAA were significantly higher, and ΔpHpmf was significantly lower in Zn addition than Al treatment alone, but the changes did not occur under none spraying IAA. The interaction of Zn and IAA directly increased Y(I), Y(II), ETRI and ETRII, and decreased O2- content of Al-stressed seedlings. In addition, the transcriptome analysis showed that fourteen functionally noted genes classified into functional category of energy production and conversion were differentially expressed in leaves of alfalfa seedlings with and without apical buds. CONCLUSION: Our results suggest that the interaction of zinc and IAA alleviate aluminum-induced damage on photosystems via increasing pmf and decreasing ΔpHpmf between lumen and stroma.

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 .

Knockdown of STAYGREEN in Perennial Ryegrass (Lolium perenne L.) Leads to Transcriptomic Alterations Related to Suppressed Leaf Senescence and Improved Forage Quality.

Chl breakdown is a hallmark of leaf senescence. Protein degradation is tightly associated with accelerated Chl catabolism during leaf senescence. Therefore, blocking or reducing Chl breakdown and thereby improving Chl and leaf protein contents is desirable for agronomic improvement in perennial forage grasses. Perennial ryegrass (Lolium perenne L.) is one principle cool-season forage grass in temperate areas throughout the world. In this study, the perennial ryegrass STAY-GREEN gene (LpSGR) was cloned and characterized. LpSGR was highly expressed in developmentally or dark-induced senescent leaves. LpSGR was subcellularly localized in chloroplast and interacted with the other Chl catabolic enzymes. RNA interference (RNAi) of LpSGR in perennial ryegrass blocked the degradation of Chl, resulting in increased Chl content and photochemical efficiency in senescent leaves. The RNAi transgenic plants had significantly improved forage quality, with up to 46.1% increased protein content in the harvested biomass. Transcriptome comparison revealed that suppression of LpSGR led to multiple alterations in metabolic pathways in locations inside the chloroplast. Most transcription factors of senescence-associated hormonal signaling pathways (e.g. ABA, ethylene and jasmonic acid) had decreased expression levels in the RNAi plants. These results provided a foundation for the further study on the regulatory mechanism of LpSGR in perennial ryegrass for the purpose of forage improvement with delayed leaf senescence and higher forage quality.

Switchgrass PvDREB1C plays opposite roles in plant cold and salt tolerance in transgenic tobacco.

BACKGROUND: The C-repeat-binding factors/DRE-binding factors (CBF/DREBs) comprise a key transcription factor family involved in plant stress tolerance. Yet, there is limited information about switchgrass DREB genes and their functional roles. RESULTS: In this study, four cold-inducible PvDREB1s were identified from switchgrass (Panicum virgatum), among which PvDREB1C was the one responded to cold stress later than the other three PvDREB1s. Yet, ectopic overexpression of PvDREB1C led to significantly compromised, instead of improved cold tolerance in transgenic tobacco. On the other hand, PvDREB1C was transcriptionally down-regulated in response to salt stress, but overexpression of PvDREB1C improved plant salt tolerance in transgenic tobacco. The improved salt tolerance was associated with increased K+/Na+ ratio and Ca2+ content, higher cellular osmotic potential, and activation of stress-related functional genes in the leaves of transgenic plants under salt stress. CONCLUSIONS: The current results implied that PvDREB1C played opposite roles in plant cold and salt tolerance. Although DREB1s were known as positive stress regulators, particular attentions shall be paid to their potential negative regulatory role(s).

Functional characterization and hormonal regulation of the PHEOPHYTINASE gene LpPPH controlling leaf senescence in perennial ryegrass.

Chlorophyll (Chl) degradation occurs naturally during leaf maturation and senescence, and can be induced by stresses, both processes involving the regulation of plant hormones. The objective of this study was to determine the functional roles and hormonal regulation of a gene encoding pheophytin pheophorbide hydrolyase (PPH) that catabolizes Chl degradation during leaf senescence in perennial grass species. A PPH gene, LpPPH, was cloned from perennial ryegrass (Lolium perenne L.). LpPPH was localized in the chloroplast. Overexpressing LpPPH accelerated Chl degradation in wild tobacco, and rescued the stay-green phenotype of the Arabidopsis pph null mutant. The expression level of LpPPH was positively related to the extent of leaf senescence. Exogenous application of abscisic acid (ABA) and ethephon (an ethylene-releasing agent) accelerated the decline in Chl content in leaves of perennial ryegrass, whereas cytokinin (CK) and aminoethoxyvinylglycine (AVG; an ethylene biosynthesis inhibitor) treatments suppressed leaf senescence, corresponding to the up- or down-regulation of LpPPH expression. The promoters of five orthologous PPH genes were predicted to share conserved cis-elements potentially recognized by transcription factors in the ABA and CK pathways. Taken together, the results suggested that LpPPH-mediated Chl breakdown could be regulated positively by ABA and ethylene, and negatively by CK, and LpPPH could be a direct downstream target gene of transcription factors in the ABA and CK signaling pathways.