Insights into the Impact of Trans-Zeatin Overproduction-Engineered Sinorhizobium meliloti on Alfalfa (Medicago sativa L.) Tolerance to Drought Stress.

Plant growth-promoting rhizobacteria have been shown to enhance plant tolerance to drought stress through various mechanisms. However, there is limited research on improving drought resistance in alfalfa by genetically modifying PGPR to produce increased levels of cytokinins. Herein, we employed synthetic biology approaches to engineer two novel strains of Sinorhizobium meliloti capable of overproducing trans-Zeatin and investigated their potential in enhancing drought tolerance in alfalfa. Our results demonstrate that alfalfa plants inoculated with these engineered S. meliloti strains exhibited reduced wilting and yellowing while maintaining higher relative water content under drought conditions. The engineered S. meliloti-induced tZ activated the activity of antioxidant enzymes and the accumulation of osmolytes. Additionally, the increased endogenous tZ content in plants alleviated the impact of drought stress on the alfalfa photosynthetic rate. However, under nondrought conditions, inoculation with the engineered S. meliloti strains had no significant effect on alfalfa biomass and nodule formation.

Overexpression of the alfalfa (Medicago sativa) gene, MsKMS1, negatively regulates seed germination in transgenic tobacco (Nicotiana tabacum).

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-associated proteins are a class of transmembrane proteins involved in intracellular trafficking pathways. However, the functions of many SNARE domain-containing proteins remain unclear. We have previously identified a SNARE-associated gene in alfalfa (Medicago sativa ) KILLING ME SLOWLY1 (MsKMS1 ), which is involved in various abiotic stresses. In this study, we investigated the function of MsKMS1 in the seed germination of transgenic tobacco (Nicotiana tabacum ). Phylogenetic analysis showed that MsKMS1 was homologous to the SNARE-associated or MAPR component-related proteins of other plants. Germination assays revealed that MsKMS1 negatively regulated seed germination under normal, D-mannitol and abscisic acid-induced stress conditions, yet MsKMS1 -overexpression could confer enhanced heat tolerance in transgenic tobacco. The suppressive effect on germination in MsKMS1 -overexpression lines was associated with higher abscisic acid and salicylic acid contents in seeds. This was accompanied by the upregulation of abscisic acid biosynthetic genes (ZEP and NCED ) and the downregulation of gibberellin biosynthetic genes (GA20ox2 and GA20ox3 ). Taken together, these results suggested that MsKMS1 negatively regulated seed germination by increasing abscisic acid and salicylic acid contents through the expression of genes related to abscisic acid and gibberellin biosynthesis. In addition, MsKMS1 could improve heat tolerance during the germination of transgenic tobacco seeds.

Genome assembly and multi-omic analyses reveal the mechanisms underlying flower color formation in Torenia fournieri.

Torenia fournieri Lind. is an ornamental plant that is popular for its numerous flowers and variety of colors. However, its genomic evolutionary history and the genetic and metabolic bases of flower color formation remain poorly understood. Here, we report the first T. fournieri reference genome, which was resolved to the chromosome scale and was 164.4 Mb in size. Phylogenetic analyses clarified relationships with other plant species, and a comparative genomic analysis indicated that the shared ancestor of T. fournieri and Antirrhinum majus underwent a whole genome duplication event. Joint transcriptomic and metabolomic analyses identified many metabolites related to pelargonidin, peonidin, and naringenin production in rose (TfR)-colored flowers. Samples with blue (TfB) and deep blue (TfD) colors contained numerous derivatives of petunidin, cyanidin, quercetin, and malvidin; differences in the abundances of these metabolites and expression levels of the associated genes were hypothesized to be responsible for variety-specific differences in flower color. Furthermore, the genes encoding flavonoid 3-hydroxylase, anthocyanin synthase, and anthocyanin reductase were differentially expressed between flowers of different colors. Overall, we successfully identified key genes and metabolites involved in T. fournieri flower color formation. The data provided by the chromosome-scale genome assembly establish a basis for understanding the differentiation of this species and will facilitate future genetic studies and genomic-assisted breeding.

Integrated physiological, metabolomic, and transcriptomic analyses elucidate the regulation mechanisms of lignin synthesis under osmotic stress in alfalfa leaf (Medicago sativa L.).

Alfalfa, an essential forage crop known for its high yield, nutritional value, and strong adaptability, has been widely cultivated worldwide. The yield and quality of alfalfa are frequently jeopardized due to environmental degradation. Lignin, a constituent of the cell wall, enhances plant resistance to abiotic stress, which often causes osmotic stress in plant cells. However, how lignin responds to osmotic stress in leaves remains unclear. This study explored the effects of osmotic stress on lignin accumulation and the contents of intermediate metabolites involved in lignin synthesis in alfalfa leaves. Osmotic stress caused an increase in lignin accumulation and the alteration of core enzyme activities and gene expression in the phenylpropanoid pathway. We identified five hub genes (CSE, CCR, CADa, CADb, and POD) and thirty edge genes (including WRKYs, MYBs, and UBPs) by integrating transcriptome and metabolome analyses. In addition, ABA and ethylene signaling induced by osmotic stress regulated lignin biosynthesis in a contradictory way. These findings contribute to a new theoretical foundation for the breeding of high-quality and resistant alfalfa varieties.

Genome-wide identification and expression analysis of the glycosyl hydrolase family 1 genes in Medicago sativa revealed their potential roles in response to multiple abiotic stresses.

Glycoside hydrolase family 1 (GH1) ß-glucosidases (BGLUs), are encoded by a large number of genes, which participate in the development and stress response of plants, particularly under biotic and abiotic stresses through the activation of phytohormones. However, there are few studies systematically analyzing stress or hormone-responsive BGLU genes in alfalfa. In this study, a total of 179 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of alfalfa, and then were classified into five distinct clusters. Sequence alignments revealed several conserved and unique motifs among these MsBGLU proteins. Many cis-acting elements related to abiotic stresses and phytohormones were identified in the promoter of some MsBGLUs. Moreover, RNA-seq and RT-qPCR analyses showed that these MsBGLU genes exhibited distinct expression patterns in response to different abiotic stress and hormonal treatments. In summary, this study suggests that MsBGLU genes play crucial roles in response to various abiotic stresses and hormonal responses, and provides candidate genes for stress tolerance breeding in alfalfa.

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

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

A retrotransposon insertion in the Mao1 promoter results in erect pubescence and higher yield in soybean.

Adaptive changes in crops contribute to the diversity of agronomic traits, which directly or indirectly affect yield. The change of pubescence form from appressed to erect is a notable feature during soybean domestication. However, the biological significance and regulatory mechanism underlying this transformation remain largely unknown. Here, we identified a major-effect locus, PUBESCENCE FORM 1 (PF1), the upstream region of Mao1, that regulates pubescence form in soybean. The insertion of a Ty3/Gypsy retrotransposon in PF1 can recruit the transcription factor GAGA-binding protein to a GA-rich region, which up-regulates Mao1 expression, underpinning soybean pubescence evolution. Interestingly, the proportion of improved cultivars with erect pubescence increases gradually with increasing latitude, and erect-pubescence cultivars have a higher yield possibly through a higher photosynthetic rate and photosynthetic stability. These findings open an avenue for molecular breeding through either natural introgression or genome editing toward yield improvement and productivity.

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

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

Proteomic Analysis of Alfalfa (Medicago sativa L.) Roots in Response to Rhizobium Nodulation and Salt Stress.

(1) Background: Alfalfa is an important legume forage throughout the world. Although alfalfa is considered moderately tolerant to salinity, its production and nitrogen-fixing activity are greatly limited by salt stress. (2) Methods: We examined the physiological changes and proteomic profiles of alfalfa with active nodules (NA) and without nodules (NN) under NaCl treatment. (3) Results: Our data suggested that NA roots showed upregulation of the pathways of abiotic and biotic stress responses (e.g., heat shock proteins and pathogenesis-related proteins), antioxidant enzyme synthesis, protein synthesis and degradation, cell wall degradation and modification, acid phosphatases, and porin transport when compared with NN plants under salt stress conditions. NA roots also upregulated the processes or proteins of lipid metabolism, heat shock proteins, protein degradation and folding, and cell cytoskeleton, downregulated the DNA and protein synthesis process, and vacuolar H+-ATPase proteins under salt stress. Besides, NA roots displayed a net H+ influx and low level of K+ efflux under salt stress, which may enhance the salt tolerance of NA plants. (4) Conclusions: The rhizobium symbiosis conferred the host plant salt tolerance by regulating a series of physiological processes to enhance stress response, improve antioxidant ability and energy use efficiency, and maintain ion homeostasis.

Functional Analysis of the Phosphate Transporter Gene MtPT6 From Medicago truncatula.

Phosphorus is one of the essential macronutrients required by plant growth and development, but phosphate resources are finite and diminishing rapidly because of the huge need in global agriculture. In this study, 11 genes were found in the Phosphate Transporter 1 (PHT1) family of Medicago truncatula. Seven genes of the PHT1 family were available by qRT-PCR. Most of them were expressed in roots, and almost all genes were induced by low-phosphate stress in the nodule. The expression of MtPT6 was relatively high in nodules and induced by low-phosphate stress. The fusion expression of MtPT6 promoter-GUS gene in M. truncatula suggested that the expression of MtPT6 was induced in roots and nodules by phosphate starvation. In roots, MtPT6 was mainly expressed in vascular tissue and tips, and it was also expressed in cortex under low-phosphate stress; in nodules, it was mainly expressed in vascular bundles, cortical cells, and fixation zone cells. MtPT6 had a close relationship with other PHT1 family members according to amino acid alignment and phylogenetic analysis. Subcellular localization analysis in tobacco revealed that MtPT6 protein was localized to the plasma membrane. The heterologous expression of MtPT6 in Arabidopsis knockout mutants of pht1.1 and pht1.4 made seedlings more susceptible to arsenate treatment, and the phosphate concentrations in pht1.1 were higher in high phosphate condition by expressing MtPT6. We conclude that MtPT6 is a typical phosphate transporter gene and can promote phosphate acquisition efficiency of plants.

Overexpression of zeaxanthin epoxidase gene from Medicago sativa enhances the tolerance to low light in transgenic tobacco.

Zeaxanthin epoxidase (ZEP) plays an important role in xanthophyll cycle which is a process closely related to photosynthesis. However, an impact of ZEP on low-light stress has not been studied. In this study, the functions of an alfalfa (Medicago sativa) zeaxanthin epoxidase gene, MsZEP, in response to low-light stress were investigated by heterologous expression in tobacco (Nicotiana tabacum). Under normal light conditions, the measured parameters were not significantly different between transgenic and wild-type (WT) plants except for non-photochemical quenching value and chlorophyll a content. However, the differences were detected under low-light stress. We found that MsZEP-overexpression tobacco grew faster than WT (p≤0.05). The leaf fresh weight and leaf area of transgenic plants were significantly higher, and the number of stomata was greater in MsZEP-overexpression tobacco. As for photosynthetic characteristics, quantum yield of PSII (ΦPSII) and maximal photochemical efficiency of PSII (Fv/Fm) were not significantly different, whereas non-photochemical quenching (NPQ), net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of MsZEP-overexpression tobacco were significantly higher than in WT plants. However, no significant difference was detected between the two types of tobacco in chlorophyll and carotenoids content. In conclusion, MsZEP can improve the ability of tobacco to withstand low-light stress, which might be due to its stronger photosynthetic activity and the improvement of stomatal density under low light.

MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers drought and salt tolerance in transgenic tobacco.

KEY MESSAGE: The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

Isolation and characterization of a buffalograss (Buchloe dactyloides) dehydration responsive element binding transcription factor, BdDREB2.

Dehydration responsive element binding (DREB) transcription factors play an important role in the regulation of stress-related genes. These factors contribute to resistance to different abiotic stresses. In the present study, a novel DREB transcription factor, BdDREB2, isolated from Buchloe dactyloides, was cloned and characterized. The BdDREB2 protein was estimated to have a molecular weight of 28.36kDa, a pI of 5.53 and a typical AP2/ERF domain. The expression of BdDREB2 was involved in responses to drought and salt stresses. Overexpression of BdDREB2 in tobacco showed higher relative water and proline content, and was associated with lower MDA content under drought stress, suggesting that the transgenic tobacco may tolerate drought stress better. Results demonstrate that BdDREB2 may play an important role in the regulation of abiotic stress responses, and mediate many physiological pathways that enhance stress tolerance in plants.