Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance.

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

Salinity Stress Alters the Secondary Metabolic Profile of M. sativa, M. arborea and Their Hybrid (Alborea).

Increased soil salinity, and therefore accumulation of ions, is one of the major abiotic stresses of cultivated plants that negatively affect their growth and yield. Among Medicago species, only Medicago truncatula, which is a model plant, has been extensively studied, while research regarding salinity responses of two important forage legumes of Medicago sativa (M. sativa) and Medicago arborea (M. arborea) has been limited. In the present work, differences between M. arborea, M. sativa and their hybrid Alborea were studied regarding growth parameters and metabolomic responses. The entries were subjected to three different treatments: (1) no NaCl application (control plants), (2) continuous application of 100 mM NaCl (acute stress) and (3) gradual application of NaCl at concentrations of 50-75-150 mM by increasing NaCl concentration every 10 days. According to the results, M. arborea maintained steady growth in all three treatments and appeared to be more resistant to salinity. Furthermore, results clearly demonstrated that M. arborea presented a different metabolic profile from that of M. sativa and their hybrid. In general, it was found that under acute and gradual stress, M. sativa overexpressed saponins in the shoots while M. arborea overexpressed saponins in the roots, which is the part of the plant where most of the saponins are produced and overexpressed. Alborea did not perform well, as more metabolites were downregulated than upregulated when subjected to salinity stress. Finally, saponins and hydroxycinnamic acids were key players of increased salinity tolerance.

Genome-wide identification, phylogenetic, and expression analysis under abiotic stress conditions of LIM gene family in Medicago sativa L.

The LIM (Lin-11, Isl-1 and Mec-3 domains) family is a key transcription factor widely distributed in animals and plants. The LIM proteins in plants are involved in the regulation of a variety of biological processes, including cytoskeletal organization, the development of secondary cell walls, and cell differentiation. It has been identified and analyzed in many species. However, the systematic identification and analysis of the LIM genes family have not yet been reported in alfalfa (Medicago sativa L.). Based on the genome-wide data of alfalfa, a total of 21 LIM genes were identified and named MsLIM01-MsLIM21. Comprehensive analysis of the chromosome location, physicochemical properties of the protein, evolutionary relationship, conserved motifs, and responses to abiotic stresses of the LIM gene family in alfalfa using bioinformatics methods. The results showed that these MsLIM genes were distributed unequally on 21 of the 32 chromosomes in alfalfa. Gene duplication analysis showed that segmental duplications were the major contributors to the expansion of the alfalfa LIM family. Based on phylogenetic analyses, the LIM gene family of alfalfa can be divided into four subfamilies: αLIM subfamily, ßLIM subfamily, γLIM subfamily, and δLIM subfamily, and approximately all the LIM genes within the same subfamily shared similar gene structure. The 21 MsLIM genes of alfalfa contain 10 Motifs, of which Motif1 and Motif3 are the conserved motifs shared by these genes. Furthermore, the analysis of cis-regulatory elements indicated that regulatory elements related to transcription, cell cycle, development, hormone, and stress response are abundant in the promoter sequence of MsLIM genes. Real-time quantitative PCR demonstrated that MsLIM gene expression is induced by low temperature and salt. The present study serves as a basic foundation for future functional studies on the alfalfa LIM family.

The impact of intra-specific diversity in the rhizobia-legume symbiosis.

Rhizobia - nitrogen-fixing, root-nodulating bacteria - play a critical role in both plant ecosystems and sustainable agriculture. Rhizobia form intracellular infections within legumes roots where they produce plant accessible nitrogen from atmospheric nitrogen and thus reduce the reliance on industrial inputs. The rhizobia-legume symbiosis is often treated as a pairwise relationship between single genotypes, both in research and in the production of rhizobial inoculants. However in nature individual plants are infected by a high diversity of rhizobia symbionts. How this diversity affects productivity within the symbiosis is unclear. Here, we use a powerful statistical approach to assess the impact of diversity within the Rhizobium leguminosarum - clover symbiosis using a biodiversity-ecosystem function framework. Statistically, we found no significant impact of rhizobium diversity. However this relationship was weakly positive - rather than negative - indicating that there is no significant cost to increasing inoculant diversity. Productivity was influenced by the identity of the strains within an inoculant; strains with the highest individual performance showed a significant positive contribution within mixed inoculants. Overall, inoculant effectiveness was best predicted by the individual performance of the best inoculant member, and only weakly predicted by the worst performing member. Collectively, our data suggest that the Rhizobium leguminosarum - clover symbiosis displays a weak diversity-function relationship, but that inoculant performance can be improved through the inclusion of high performing strains. Given the wide environmental dependence of rhizobial inoculant quality, multi-strain inoculants could be highly successful as they increase the likelihood of including a strain well adapted to local conditions across different environments.

AIR12 confers cold tolerance through regulation of the CBF cold response pathway and ascorbate homeostasis.

Auxin induced in root culture (AIR12) is a single gene in Arabidopsis and codes for a mono-heme cytochrome b, but it is unknown whether plant AIR12 is involved in abiotic stress responses. MfAIR12 was identified from Medicago falcata that is legume germplasm with great cold tolerance. Transcript levels of MfAIR12 and its homolog MtAIR12 from Medicago truncatula was induced under low temperature. Overexpression of MfAIR12 led to the accumulation of H2 O2 in apoplast and enhanced cold tolerance, which was blocked by H2 O2 scavengers, indicating that the increased cold tolerance was dependent upon the accumulated H2 O2 . In addition, declined cold tolerance was observed in Arabidopsis mutant air12, which could be restored by expressing MfAIR12. Compared to the wild type, higher levels of ascorbic acid and ascorbate redox state, as well as transcripts of the C repeat/dehydration responsive element-binding factor (CBF) transcription factors and their downstream cold-responsive genes, were observed in MfAIR12 transgenic lines, but lower levels of those in air12 mutant. It is suggested AIR12 confers cold tolerance as a result of the altered H2 O2 in the apoplast that is signaling in the regulation of CBF cold response pathway and ascorbate homeostasis.

Full-length transcriptome sequencing reveals the low-temperature-tolerance mechanism of Medicago falcata roots.

BACKGROUND: Low temperature is one of the main environmental factors that limits crop growth, development, and production. Medicago falcata is an important leguminous herb that is widely distributed worldwide. M. falcata is related to alfalfa but is more tolerant to low temperature than alfalfa. Understanding the low temperature tolerance mechanism of M. falcata is important for the genetic improvement of alfalfa. RESULTS: In this study, we explored the transcriptomic changes in the roots of low-temperature-treated M. falcata plants by combining SMRT sequencing and NGS technologies. A total of 115,153 nonredundant sequences were obtained, and 8849 AS events, 73,149 SSRs, and 4189 lncRNAs were predicted. A total of 111,587 genes from SMRT sequencing were annotated, and 11,369 DEGs involved in plant hormone signal transduction, protein processing in endoplasmic reticulum, carbon metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and endocytosis pathways were identified. We characterized 1538 TF genes into 45 TF gene families, and the most abundant TF family was the WRKY family, followed by the ERF, MYB, bHLH and NAC families. A total of 134 genes, including 101 whose expression was upregulated and 33 whose expression was downregulated, were differentially coexpressed at all five temperature points. PB40804, PB75011, PB110405 and PB108808 were found to play crucial roles in the tolerance of M. falcata to low temperature. WGCNA revealed that the MEbrown module was significantly correlated with low-temperature stress in M. falcata. Electrolyte leakage was correlated with most genetic modules and verified that electrolyte leakage can be used as a direct stress marker in physiological assays to indicate cell membrane damage from low-temperature stress. The consistency between the qRT-PCR results and RNA-seq analyses confirmed the validity of the RNA-seq data and the analysis of the regulatory mechanism of low-temperature stress on the basis of the transcriptome. CONCLUSIONS: The full-length transcripts generated in this study provide a full characterization of the transcriptome of M. falcata and may be useful for mining new low-temperature stress-related genes specific to M. falcata. These new findings could facilitate the understanding of the low-temperature-tolerance mechanism of M. falcata.

Cost-effective detection of genome-wide signatures for 2,4-D herbicide resistance adaptation in red clover.

Herbicide resistance is a recurrent evolutionary event that has been reported across many species and for all major herbicide modes of action. The synthetic auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely used since the 1940s, however the genetic variation underlying naturally evolving resistance remains largely unknown. In this study, we used populations of the forage legume crop red clover (Trifolium pratense L.) that were recurrently selected for 2,4-D resistance to detect genome-wide signatures of adaptation. Four susceptible and six derived resistant populations were sequenced using a less costly approach by combining targeted sequencing (Capture-Seq) with pooled individuals (Pool-Seq). Genomic signatures of selection were identified using: (i) pairwise allele frequency differences; (ii) genome scan for overly differentiated loci; and (iii) genome-wide association. Fifty significant SNPs were consistently detected, most located in a single chromosome, which can be useful for marker assisted selection. Additionally, we searched for candidate genes at these genomic regions to gain insights into potential molecular mechanisms underlying 2,4-D resistance. Among the predicted functions of candidate genes, we found some related to the auxin metabolism, response to oxidative stress, and detoxification, which are also promising for further functional validation studies.

Legacy of prior host and soil selection on rhizobial fitness in planta.

Measuring selection acting on microbial populations in natural or even seminatural environments is challenging because many microbial populations experience variable selection. The majority of rhizobial bacteria are found in the soil. However, they also live symbiotically inside nodules of legume hosts and each nodule can release thousands of daughter cells back into the soil. We tested how past selection (i.e., legacies) by two plant genotypes and by the soil alone affected selection and genetic diversity within a population of 101 strains of Ensifer meliloti. We also identified allelic variants most strongly associated with soil- and host-dependent fitness. In addition to imposing direct selection on rhizobia populations, soil and host environments had lasting effects across host generations. Host presence and genotype during the legacy period explained 22% and 12% of the variance in the strain composition of nodule communities in the second cohort, respectively. Although strains with high host fitness in the legacy cohort tended to be enriched in the second cohort, the diversity of the strain community was greater when the second cohort was preceded by host rather than soil legacies. Our results indicate the potential importance of soil selection driving the evolution of these plant-associated microbes.

An eukaryotic elongation factor 2 from Medicago falcata (MfEF2) confers cold tolerance.

BACKGROUND: An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study. RESULTS: MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (Fv/Fm) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into "photosynthesis" and "photosynthesis-antenna", such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into "spliceosome". CONCLUSIONS: The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature.

Comprehensive analysis of phenotype, microstructure and global transcriptional profiling to unravel the effect of excess copper on the symbiosis between nitrogen-fixing bacteria and Medicago lupulina.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. However, knowledge of the molecular mechanisms, especially the microstructure and global transcriptional profiling, of the symbiosis process under heavy metal contamination is limited. In this study, a heavy metal-tolerant legume, Medicago lupulina, was treated with different concentrations of copper (Cu). The results showed that the early infection process was inhibited and the nodule ultrastructure was changed under 200 mg kg-1 Cu stress. Most infection threads (ITs) were prevented from entering the nodule cells, and few rhizobia were released into the host cells, in which thickening of the plant cell wall and IT wall was observed, demonstrating that rhizobial invasion was inhibited under Cu stress. RNA-seq analysis indicated that a strong shift in gene expression occurred (3257 differentially expressed genes, DEGs). The most pronounced effect was the upregulation of a set of 71 of 73 DEGs for nodule-specific cysteine-rich peptides, which have been shown to control the terminal differentiation of rhizobia in the nodules and to have antimicrobial activity. Various genes for metal transport, chelation binding and antioxidant defence were regulated. In particular, the DEGs for Cu trafficking and detoxification were induced during nodule formation. The DEGs for ethylene (ET) biosynthesis and signalling were also differentially expressed during nodulation, suggesting that the inhibition of nodulation by Cu occurred partially through ET signalling. Furthermore, the genes related to the cell wall were mostly upregulated and most likely involved in cell wall thickening. These findings provide an integrated understanding of the effects of Cu on legume nodule symbiosis at the molecular and phenotypic levels.

Medicago LINC Complexes Function in Nuclear Morphology, Nuclear Movement, and Root Nodule Symbiosis.

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.

Rapid establishment of a flowering cline in Medicago polymorpha after invasion of North America.

To establish and spread in a new location, an invasive species must be able to carry out its life cycle in novel environmental conditions. A key trait underlying fitness is the shift from vegetative to reproductive growth through floral development. In this study, we used a common garden experiment and genotyping-by-sequencing to test whether the latitudinal flowering cline of the North American invasive plant Medicago polymorpha was translocated from its European native range through multiple introductions, or whether the cline rapidly established due to evolution following a genetic bottleneck. Analysis of flowering time in 736 common garden plants showed a latitudinal flowering time cline in both the native and invaded ranges where genotypes from lower latitudes flowered earlier. Genotyping-by-sequencing of 9,658 SNPs in 446 individuals revealed two major subpopulations of M. polymorpha in the native range, only one of which is present in the invaded range. Additionally, native range populations have higher genetic diversity than invaded range populations, suggesting that a genetic bottleneck occurred during invasion. All invaded range individuals are closely related to plants collected from native range populations in Portugal and southern Spain, and population assignment tests assigned invaded range individuals to this same narrow source region. Taken together, our results suggest that latitudinal clinal variation in flowering time has rapidly evolved across the invaded range despite a genetic bottleneck following introduction.

Physiological and structural modifications in snail medic (Medicago scutellata L.) plants exposed to salinity.

Seeds of snail medic (Medicago scutellata L.) were assessed for their response to salt at the germination and seedling stages. NaCl at concentrations 86 and 170 mM decreased the final germination percentage. Embryonic axis length, water content and dry weight of embryonic axis and cotyledons were also reduced by salt treatment. Furthermore, 28-d-old plants were grown hydroponically with different NaCl concentrations (0, 86 and 170 mM). After 7 days of treatment, growth, water content and development of the different organs of M. scutellata plant were affected especially at the highest NaCl concentration (170 mM). However, NaCl did not affect root length and the number of stem shoots but reduced stem length and total leaf area. Salt treatment increased markedly the concentration of Na+ in leaf and root tissues while reduced that of K+ only in root and stem tissues. Lipid peroxidation revealed the damage of the membranes of roots and leaves. Moreover, showed a more intense suberization and lignification at the cambial zone of roots of M. scutellata, were observed under the effect of NaCl.

A comparison of lateral root patterning among dicot and monocot plants.

Lateral root branching along the primary root involves complex gene regulatory networks in model plant Arabidopsis. However, it is largely unclarified whether different plant species share a common mechanism to pattern the lateral root along the primary axis. In this study, we assessed the development pattern of lateral root among several dicot and monocot plants, including Arabidopsis, tomato, Medicago, Nicotiana, rice, and ryegrass by using an agar-gel culture system. Our results reveal a regular-spaced distribution pattern of lateral roots along the primary root axis of both dicot and monocot plants. Meanwhile, the root patterning is tightly controlled by root bending and the plant hormone auxin. However, nitrogen and phosphate starvations trigger distinguished root growth patterns among different plant species. Our studies strongly suggest a partially shared signaling pathway underlying root patterning of various plant species, and also provide a foundation for further identification of genes associated with root development.

Yak and Tibetan sheep trampling inhibit reproductive and photosynthetic traits of Medicago ruthenica var. inschanica.

Livestock grazing affects grassland stability, resilience, and productivity owing to trampling, foraging, and excretion. Over time, trampling influences a wide range of grassland components and can have lasting effects. Trampling helps maintain grassland health but may also cause its degradation. In a field experiment over two growing seasons, we simulated yak and sheep trampling at different intensities and investigated their effects on the reproductive and photosynthetic characteristics of Medicago ruthenica var. inschanica in a Tianzhu alpine meadow in Gansu Province, China. Our results show that simulated trampling inhibited the asexual and sexual reproduction and growth of M. ruthenica. The root surface area, root volume, root biomass, pod length, pod number per unit area, number of seeds per pod, thousand-seed weight, and seed yield were significantly reduced under simulated trampling in the upper 30 cm of soil (P < 0.05) but were not reduced in the deeper soil layers (> 30 cm). Light trampling by both yak and Tibetan sheep promoted photosynthesis, while heavy trampling by both species inhibited photosynthesis. Yak trampling inhibited photosynthesis more than Tibetan sheep trampling, and overall, the adverse effects of yak trampling on asexual and sexual reproduction and growth of M. ruthenica were greater than those of Tibetan sheep trampling. Thus, the effect of yak trampling is greater than the effect of trampling by Tibetan sheep, where the different trampling intensities of yak and Tibetan sheep can result in direct but varied influences on grasslands, potentially leading to grassland differentiation.

Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments.

Assays to accurately estimate relative fitness of bacteria growing in multistrain communities can advance our understanding of how selection shapes diversity within a lineage. Here, we present a variant of the "evolve and resequence" approach both to estimate relative fitness and to identify genetic variants responsible for fitness variation of symbiotic bacteria in free-living and host environments. We demonstrate the utility of this approach by characterizing selection by two plant hosts and in two free-living environments (sterilized soil and liquid media) acting on synthetic communities of the facultatively symbiotic bacterium Ensifer meliloti We find (i) selection that hosts exert on rhizobial communities depends on competition among strains, (ii) selection is stronger inside hosts than in either free-living environment, and (iii) a positive host-dependent relationship between relative strain fitness in multistrain communities and host benefits provided by strains in single-strain experiments. The greatest changes in allele frequencies in response to plant hosts are in genes associated with motility, regulation of nitrogen fixation, and host/rhizobia signaling. The approach we present provides a powerful complement to experimental evolution and forward genetic screens for characterizing selection in bacterial populations, identifying gene function, and surveying the functional importance of naturally occurring genomic variation.

A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover, antioxidant protection, and proline accumulation.

Ethylene responsive factor (ERF) subfamily transcription factors play an important role in plant abiotic and biotic stress tolerance. A cold responsive ERF, MfERF1, was isolated from Medicago falcata, an important forage legume that has great cold tolerance. Overexpression of MfERF1 resulted in an increased tolerance to freezing and chilling in transgenic tobacco plants, whereas down-regulation of the ortholog of MfERF1 in Medicago truncatula resulted in reduced freezing tolerance in RNAi plants. Higher transcript levels of some stress responsive genes (CHN50, OSM, ERD10C, and SAMS) and those involved in spermidine (Spd) and spermine (Spm) synthesis (SAMDC1, SAMDC2, SPDS1, SPDS2, and SPMS) and catabolism (PAO) were observed in transgenic plants than in wild type. However, neither Spd nor Spm level was accumulated in transgenic plants as a result of promoted polyamine oxidase activity. Transgenic plants had higher activities of antioxidants associated with the induced encoding genes including Cu, Zn-SOD, CAT1, CAT2, CAT3, and cpAPX and accumulated more proline associated with induced P5CS and reduced PROX2 transcription as compared with wild type. The results suggest that MfERF1 confers cold tolerance through promoted polyamine turnover, antioxidant protection, and proline accumulation.

Metabolic Pathways Regulated by Chitosan Contributing to Drought Resistance in White Clover.

Increased endogenous chitosan (CTS) could be associated with improved drought resistance in white clover (Trifolium repens). Plants were pretreated with or without 1 mg/mL CTS and then were subjected to optimal or water-limited condition in controlled growth chambers for 6 days. Phenotypic and physiological results indicated that exogenous CTS significantly improved drought resistance of white clover. Metabolome results showed that exogenous CTS induced a significant increase in endogenous CTS content during dehydration accompanied by the maintenance of greater accumulation of sugars, sugar alcohols, amino acids, organic acids, and other metabolites (ascorbate, glutathione, flavonoids, putrescine, and spermidine). These compounds are associated with osmotic adjustment, antioxidant defense, stress signaling, and energy metabolism under stress condition. Similarly, transcriptome revealed that many genes in relation to amino acid and carbohydrate metabolism, energy production and conversion, and ascorbate-glutathione and flavonoid metabolism were significantly up-regulated by CTS in response to dehydration stress. CTS-induced drought resistance was associated with the accumulation of stress protective metabolites, the enhancement of ascorbate-glutathione and tricarboxylic acid cycle, and increases in the γ-aminobutyric acid shunt, polyamine synthesis, and flavonoids metabolism contributing to improved osmotic adjustment, antioxidant capacity, stress signaling, and energy production for stress defense, thereby maintaining metabolic homeostasis under dehydration stress.

A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play a vital role in the response to drought stress. Here, we report a lipid-anchored NACsa TF in Medicago falcata MfNACsa is an essential regulator of plant tolerance to drought stress, resulting in the differential expression of genes involved in oxidation reduction and lipid transport and localization. MfNACsa is associated with membranes under unstressed conditions and, more specifically, is targeted to the plasma membrane through S-palmitoylation. However, a Cys26-to-Ser mutation or inhibition of S-palmitoylation results in MfNACsa retention in the endoplasmic reticulum/Golgi. Under drought stress, MfNACsa translocates to the nucleus through de-S-palmitoylation mediated by the thioesterase MtAPT1, as coexpression of APT1 results in the nuclear translocation of MfNACsa, whereas mutation of the catalytic site of APT1 results in colocalization with MfNACsa and membrane retention of MfNACsa. Specifically, the nuclear MfNACsa binds the glyoxalase I (MtGlyl) promoter under drought stress, resulting in drought tolerance by maintaining the glutathione pool in a reduced state, and the process is dependent on the APT1-NACsa regulatory module. Our findings reveal a novel mechanism for the nuclear translocation of an S-palmitoylated NAC in response to stress.

Physiological and iTRAQ-Based Proteomic Analyses Reveal the Function of Spermidine on Improving Drought Tolerance in White Clover.

Endogenous spermidine interacting with phytohormones may be involved in the regulation of differentially expressed proteins (DEPs) associated with drought tolerance in white clover. Plants treated with or without spermidine (50 µM) were subjected to 20% PEG 6000 nutrient solution to induce drought stress (50% leaf-relative water content). The results showed that increased endogenous spermidine induced by exogenous spermidine altered endogenous phytohormones in association with improved drought tolerance, as demonstrated by the delay in water-deficit development, improved photosynthesis and water use efficiency, and lower oxidative damage. As compared to untreated plants, Spd-treated plants maintained a higher abundance of DEPs under drought stress involved in (1) protein biosynthesis (ribosomal and chaperone proteins); (2) amino acids synthesis; (3) the carbon and energy metabolism; (4) antioxidant and stress defense (ascorbate peroxidase, glutathione peroxidase, and dehydrins); and (5) GA and ABA signaling pathways (gibberellin receptor GID1, ABA-responsive protein 17, and ABA stress ripening protein). Thus, the findings of proteome could explain the Spd-induced physiological effects associated with drought tolerance. The analysis of functional protein-protein networks further proved that the alteration of endogenous spermidine and phytohormones induced the interaction among ribosome, photosynthesis, carbon metabolism, and amino acid biosynthesis. These differences could contribute to improved drought tolerance.