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1.
Physiol Plant ; 176(4): e14446, 2024.
Article in English | MEDLINE | ID: mdl-39092508

ABSTRACT

Drought has a devastating impact, presenting a formidable challenge to agricultural productivity and global food security. Among the numerous ABC transporter proteins found in plants, the ABCG transporters play a crucial role in plant responses to abiotic stress. In Medicago sativa, the function of ABCG transporters remains elusive. Here, we report that MsABCG1, a WBC-type transporter highly conserved in legumes, is critical for the response to drought in alfalfa. MsABCG1 is localized on the plasma membrane, with the highest expression observed in roots under normal conditions, and its expression is induced by drought, NaCl and ABA signalling. In transgenic tobacco, overexpression of MsABCG1 enhanced drought tolerance, evidenced by increased osmotic regulatory substances and reduced lipid peroxidation. Additionally, drought stress resulted in reduced ABA accumulation in tobacco overexpressing MsABCG1, demonstrating that overexpression of MsABCG1 enhanced drought tolerance was not via an ABA-dependent pathway. Furthermore, transgenic tobacco exhibited increased stomatal density and reduced stomatal aperture under drought stress, indicating that MsABCG1 has the potential to participate in stomatal regulation during drought stress. In summary, these findings suggest that MsABCG1 significantly enhances drought tolerance in plants and provides a foundation for developing efficient drought-resistance strategies in crops.


Subject(s)
Droughts , Medicago sativa , Nicotiana , Plant Proteins , Plants, Genetically Modified , Medicago sativa/genetics , Medicago sativa/physiology , Medicago sativa/metabolism , Nicotiana/genetics , Nicotiana/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Abscisic Acid/metabolism , Stress, Physiological/genetics , Plant Stomata/physiology , Plant Stomata/genetics , Drought Resistance
2.
BMC Plant Biol ; 24(1): 741, 2024 Aug 03.
Article in English | MEDLINE | ID: mdl-39095692

ABSTRACT

BACKGROUND: Daye No.3 is a novel cultivar of alfalfa (Medicago sativa L.) that is well suited for cultivation in high-altitude regions such as the Qinghai‒Tibet Plateau owing to its high yield and notable cold resistance. However, the limited availability of transcriptomic information has hindered our investigation into the potential mechanisms of cold tolerance in this cultivar. Consequently, we conducted de novo transcriptome assembly to overcome this limitation. Subsequently, we compared the patterns of gene expression in Daye No. 3 during cold acclimatization and exposure to cold stress at various time points. RESULTS: A total of 15 alfalfa samples were included in the transcriptome assembly, resulting in 141.97 Gb of clean bases. A total of 441 DEGs were induced by cold acclimation, while 4525, 5016, and 8056 DEGs were identified at 12 h, 24 h, and 36 h after prolonged cold stress at 4 °C, respectively. The consistency between the RT‒qPCR and transcriptome data confirmed the accuracy and reliability of the transcriptomic data. KEGG enrichment analysis revealed that many genes related to photosynthesis were enriched under cold stress. STEM analysis demonstrated that genes involved in nitrogen metabolism and the TCA cycle were consistently upregulated under cold stress, while genes associated with photosynthesis, particularly antenna protein genes, were downregulated. PPI network analysis revealed that ubiquitination-related ribosomal proteins act as hub genes in response to cold stress. Additionally, the plant hormone signaling pathway was activated under cold stress, suggesting its vital role in the cold stress response of alfalfa. CONCLUSIONS: Ubiquitination-related ribosomal proteins induced by cold acclimation play a crucial role in early cold signal transduction. As hub genes, these ubiquitination-related ribosomal proteins regulate a multitude of downstream genes in response to cold stress. The upregulation of genes related to nitrogen metabolism and the TCA cycle and the activation of the plant hormone signaling pathway contribute to the enhanced cold tolerance of alfalfa.


Subject(s)
Cold-Shock Response , Gene Expression Profiling , Medicago sativa , Transcriptome , Medicago sativa/genetics , Medicago sativa/physiology , Cold-Shock Response/genetics , Gene Expression Regulation, Plant , Acclimatization/genetics , Cold Temperature , Plant Proteins/genetics , Plant Proteins/metabolism
3.
Int J Mol Sci ; 25(13)2024 Jun 30.
Article in English | MEDLINE | ID: mdl-39000350

ABSTRACT

Low temperature is the most common abiotic factor that usually occurs during the seed germination of alfalfa (Medicago sativa L.). However, the potential regulatory mechanisms involved in alfalfa seed germination under low temperature stress are still ambiguous. Therefore, to determine the relevant key genes and pathways, the phenotypic and transcriptomic analyses of low-temperature sensitive (Instict) and low-temperature tolerant (Sardi10) alfalfa were conducted at 6 and 15 h of seed germination under normal (20 °C) and low (10 °C) temperature conditions. Germination phenotypic results showed that Sardi10 had the strongest germination ability under low temperatures, which was manifested by the higher germination-related indicators. Further transcriptome analysis indicated that differentially expressed genes were mainly enriched in galactose metabolism and carbon metabolism pathways, which were the most commonly enriched in two alfalfa genotypes. Additionally, fatty acid metabolism and glutathione metabolism pathways were preferably enriched in Sardi10 alfalfa. The Weighted Gene Co-Expression Network Analysis (WGCNA) suggested that genes were closely related to galactose metabolism, fatty acid metabolism, and glutathione metabolism in Sardi10 alfalfa at the module with the highest correlation (6 h of germination under low temperature). Finally, qRT-PCR analysis further validated the related genes involved in the above pathways, which might play crucial roles in regulating seed germination of alfalfa under low temperature conditions. These findings provide new insights into the molecular mechanisms of seed germination underlying the low temperature stress in alfalfa.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Plant , Germination , Medicago sativa , Phenotype , Seeds , Transcriptome , Medicago sativa/genetics , Medicago sativa/physiology , Medicago sativa/metabolism , Germination/genetics , Seeds/genetics , Seeds/growth & development , Gene Expression Profiling/methods , Cold Temperature , Cold-Shock Response/genetics , Gene Regulatory Networks
4.
BMC Plant Biol ; 24(1): 633, 2024 Jul 06.
Article in English | MEDLINE | ID: mdl-38971752

ABSTRACT

BACKGROUND: Alfalfa (Medicago sativa L.) experiences many negative effects under salinity stress, which may be mediated by recurrent selection. Salt-tolerant alfalfa may display unique adaptations in association with rhizobium under salt stress. RESULTS: To elucidate inoculation effects on salt-tolerant alfalfa under salt stress, this study leveraged a salt-tolerant alfalfa population selected through two cycles of recurrent selection under high salt stress. After experiencing 120-day salt stress, mRNA was extracted from 8 random genotypes either grown in 0 or 8 dS/m salt stress with or without inoculation by Ensifer meliloti. Results showed 320 and 176 differentially expressed genes (DEGs) modulated in response to salinity stress or inoculation x salinity stress, respectively. Notable results in plants under 8 dS/m stress included upregulation of a key gene involved in the Target of Rapamycin (TOR) signaling pathway with a concomitant decrease in expression of the SNrK pathway. Inoculation of salt-stressed plants stimulated increased transcription of a sulfate-uptake gene as well as upregulation of the Lysine-27-trimethyltransferase (EZH2), Histone 3 (H3), and argonaute (AGO, a component of miRISC silencing complexes) genes related to epigenetic and post-transcriptional gene control. CONCLUSIONS: Salt-tolerant alfalfa may benefit from improved activity of TOR and decreased activity of SNrK1 in salt stress, while inoculation by rhizobiumstimulates production of sulfate uptake- and other unique genes.


Subject(s)
Gene Expression Regulation, Plant , Medicago sativa , Salt Tolerance , Medicago sativa/genetics , Medicago sativa/physiology , Medicago sativa/microbiology , Salt Tolerance/genetics , Salt Stress/genetics , Salinity , Sinorhizobium meliloti/physiology , Salt-Tolerant Plants/genetics , Salt-Tolerant Plants/physiology
5.
Physiol Plant ; 176(4): e14448, 2024.
Article in English | MEDLINE | ID: mdl-39082126

ABSTRACT

The ascorbate-glutathione pathway plays an essential role in the physiology of vascular plants, particularly in their response to environmental stresses. This pathway is responsible for regulating the cellular redox state, which is critical for maintaining cell function and survival under adverse conditions. To study the involvement of the alfalfa monodehydroascorbate reductase (MsMDHAR) in water stress processes, Arabidopsis thaliana plants constitutively expressing the sequence encoding MsMDHAR were developed. Transgenic events with low and high MsMDHAR expression and ascorbate levels were selected for further analysis of drought and waterlogging tolerance. Under water stress, Arabidopsis transgenic plants generated higher biomass, produced more seeds, and had larger roots than wild type ones. This higher tolerance was associated with increased production of waxes and chlorophyll a at the basal level, greater stomatal opening and stability in regulating the relative water content and reduced H2O2 accumulation under stress conditions in transgenic plants. Overall, these results show that MsMDHAR is involved in plant tolerance to abiotic stresses. The data presented here also emphasises the potential of the MsMDHAR enzyme as a plant breeding tool to improve water stress tolerance.


Subject(s)
Arabidopsis , Gene Expression Regulation, Plant , Medicago sativa , Plants, Genetically Modified , Arabidopsis/genetics , Arabidopsis/physiology , Medicago sativa/genetics , Medicago sativa/physiology , Droughts , NADH, NADPH Oxidoreductases/metabolism , NADH, NADPH Oxidoreductases/genetics , Water/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Stress, Physiological/genetics , Hydrogen Peroxide/metabolism , Dehydration , Ascorbic Acid/metabolism , Plant Stomata/physiology , Plant Stomata/genetics
6.
Sci Rep ; 14(1): 17588, 2024 Jul 30.
Article in English | MEDLINE | ID: mdl-39080407

ABSTRACT

Alfalfa is widely recognized as an important forage crop. To understand the morphological characteristics and genetic basis of seed morphology in alfalfa, we screened 318 Medicago spp., including 244 Medicago sativa subsp. sativa (alfalfa) and 23 other Medicago spp., for seed area size, length, width, length-to-width ratio, perimeter, circularity, the distance between the intersection of length & width (IS) and center of gravity (CG), and seed darkness & red-green-blue (RGB) intensities. The results revealed phenotypic diversity and correlations among the tested accessions. Based on the phenotypic data of M. sativa subsp. sativa, a genome-wide association study (GWAS) was conducted using single nucleotide polymorphisms (SNPs) called against the Medicago truncatula genome. Genes in proximity to associated markers were detected, including CPR1, MON1, a PPR protein, and Wun1(threshold of 1E-04). Machine learning models were utilized to validate GWAS, and identify additional marker-trait associations for potentially complex traits. Marker S7_33375673, upstream of Wun1, was the most important predictor variable for red color intensity and highly important for brightness. Fifty-two markers were identified in coding regions. Along with strong correlations observed between seed morphology traits, these genes will facilitate the process of understanding the genetic basis of seed morphology in Medicago spp.


Subject(s)
Genome-Wide Association Study , Machine Learning , Medicago , Polymorphism, Single Nucleotide , Seeds , Seeds/genetics , Medicago/genetics , Phenotype , Quantitative Trait Loci , Medicago sativa/genetics , Medicago truncatula/genetics , Genome, Plant
7.
Int J Mol Sci ; 25(11)2024 May 26.
Article in English | MEDLINE | ID: mdl-38891967

ABSTRACT

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.


Subject(s)
Gene Expression Regulation, Plant , Multigene Family , Phylogeny , Plant Proteins , Salt Stress , Transcription Factors , Gene Expression Regulation, Plant/drug effects , Plant Proteins/genetics , Plant Proteins/metabolism , Salt Stress/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Genome, Plant , Medicago sativa/genetics , Medicago sativa/metabolism , Medicago sativa/drug effects , Medicago/genetics , Plant Growth Regulators/pharmacology , Plant Growth Regulators/metabolism , Gene Expression Profiling , Genome-Wide Association Study , Stress, Physiological/genetics
8.
Int J Mol Sci ; 25(11)2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38892413

ABSTRACT

The stem base of alfalfa is a critical part for its overwintering, regeneration, and yield. To better understand the specificity and importance of the stem base, we analyzed the structure, metabolic substances, and transcriptome of the stem base using anatomical techniques, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and RNA sequencing (RNA-seq), and compared it with stems and roots. The anatomical structure shows that the ratio of xylem to phloem changes at the base of the stem. A total of 801 compounds involved in 91 metabolic pathways were identified from the broadly targeted metabolome. Transcriptome analysis revealed 4974 differentially expressed genes (DEGs) at the stem base compared to the stem, and 5503 DEGs compared to the root. Comprehensive analyses of differentially accumulated compounds (DACs) and DEGs, in the stem base vs. stem, identified 10 valuable pathways, including plant hormone signal transduction, zeatin biosynthesis, α-Linolenic acid metabolism, histidine metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, pentose phosphate pathway, galactose metabolism, and fructose and mannose metabolism. The pathways of plant hormone signal transduction and carbon metabolism were also identified by comparing the stem base with the roots. Taken together, the stem base of alfalfa is the transition region between the stem and root in morphology; in terms of material metabolism, its growth, development, and function are regulated through hormones and sugars.


Subject(s)
Gene Expression Regulation, Plant , Medicago sativa , Plant Stems , Medicago sativa/metabolism , Medicago sativa/genetics , Plant Stems/metabolism , Metabolic Networks and Pathways , Plant Roots/metabolism , Plant Roots/genetics , Transcriptome , Gene Expression Profiling , Metabolome , Tandem Mass Spectrometry , Chromatography, High Pressure Liquid , Plant Growth Regulators/metabolism
9.
J Hazard Mater ; 474: 134851, 2024 Aug 05.
Article in English | MEDLINE | ID: mdl-38852253

ABSTRACT

Nanoparticle (NP) pollution has negative impacts and is a major global environmental problem. However, the molecular response of alfalfa (Medicago sativa L.) to titanium dioxide nanoparticles (TiO2 NPs) is limited. Herein, the dual effects of TiO2 NPs (0-1000 mg L-1) on carbon (C) and nitrogen (N) metabolisms in alfalfa were investigated. The results showed that 500 mg L-1 TiO2 NPs (Ti-500) had the highest phytotoxicity in the C/N metabolizing enzymes; and it significantly increased total soluble sugar, starch, sucrose, and sucrose-phosphate synthase. Furthermore, obvious photosynthesis responses were found in alfalfa exposed to Ti-500. By contrast, 100 mg L-1 TiO2 NPs (Ti-100) enhanced N metabolizing enzymes. RNA-seq analyses showed 4265 and 2121 differentially expressed genes (DEGs) in Ti-100 and Ti-500, respectively. A total of 904 and 844 differentially expressed proteins (DEPs) were identified in Ti-100 and Ti-500, respectively. Through the physiological, transcriptional, and proteomic analyses, the DEGs and DEPs related to C/N metabolism, photosynthesis, chlorophyll synthesis, starch and sucrose metabolism, and C fixation in photosynthetic organisms were observed. Overall, TiO2 NPs at low doses improve photosynthesis and C/N regulation, but high doses can cause toxicity. It is valuable for the safe application of NPs in agriculture.


Subject(s)
Carbon , Medicago sativa , Nitrogen , Photosynthesis , Titanium , Transcriptome , Medicago sativa/drug effects , Medicago sativa/genetics , Medicago sativa/metabolism , Titanium/toxicity , Nitrogen/metabolism , Carbon/metabolism , Transcriptome/drug effects , Photosynthesis/drug effects , Proteomics , Plant Proteins/genetics , Plant Proteins/metabolism , Metal Nanoparticles/toxicity , Gene Expression Regulation, Plant/drug effects , Nanoparticles/toxicity
10.
BMC Genomics ; 25(1): 636, 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38926665

ABSTRACT

BACKGROUND: Jasmonate ZIM-domain (JAZ) proteins, which act as negative regulators in the jasmonic acid (JA) signalling pathway, have significant implications for plant development and response to abiotic stress. RESULTS: Through a comprehensive genome-wide analysis, a total of 20 members of the JAZ gene family specific to alfalfa were identified in its genome. Phylogenetic analysis divided these 20 MsJAZ genes into five subgroups. Gene structure analysis, protein motif analysis, and 3D protein structure analysis revealed that alfalfa JAZ genes in the same evolutionary branch share similar exon‒intron, motif, and 3D structure compositions. Eight segmental duplication events were identified among these 20 MsJAZ genes through collinearity analysis. Among the 32 chromosomes of the autotetraploid cultivated alfalfa, there were 20 MsJAZ genes distributed on 17 chromosomes. Extensive stress-related cis-acting elements were detected in the upstream sequences of MsJAZ genes, suggesting that their response to stress has an underlying function. Furthermore, the expression levels of MsJAZ genes were examined across various tissues and under the influence of salt stress conditions, revealing tissue-specific expression and regulation by salt stress. Through RT‒qPCR experiments, it was discovered that the relative expression levels of these six MsJAZ genes increased under salt stress. CONCLUSIONS: In summary, our study represents the first comprehensive identification and analysis of the JAZ gene family in alfalfa. These results provide important information for exploring the mechanism of JAZ genes in alfalfa salt tolerance and identifying candidate genes for improving the salt tolerance of autotetraploid cultivated alfalfa via genetic engineering in the future.


Subject(s)
Gene Expression Regulation, Plant , Medicago sativa , Multigene Family , Phylogeny , Plant Proteins , Tetraploidy , Medicago sativa/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Salt Stress/genetics , Cyclopentanes/metabolism , Genome, Plant , Oxylipins/pharmacology , Gene Expression Profiling
11.
Int J Mol Sci ; 25(12)2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38928203

ABSTRACT

The morphological architecture of inflorescence influences seed production. The regulatory mechanisms underlying alfalfa (Medicago sativa) inflorescence elongation remain unclear. Therefore, in this study, we conducted a comparative analysis of the transcriptome, proteome, and metabolome of two extreme materials at three developmental stages to explore the mechanisms underlying inflorescence elongation in alfalfa. We observed the developmental processes of long and short inflorescences and found that the elongation capacity of alfalfa with long inflorescence was stronger than that of alfalfa with short inflorescences. Furthermore, integrative analysis of the transcriptome and proteome indicated that the phenylpropanoid biosynthesis pathway was closely correlated with the structural formation of the inflorescence. Additionally, we identified key genes and proteins associated with lignin biosynthesis based on the differential expressed genes and proteins (DEGs and DEPs) involved in phenylpropanoid biosynthesis. Moreover, targeted hormone metabolome analysis revealed that IAA, GA, and CK play an important role in the peduncle elongation of alfalfa inflorescences. Based on omics analysis, we detected key genes and proteins related to plant hormone biosynthesis and signal transduction. From the WGCNA and WPCNA results, we furthermore screened 28 candidate genes and six key proteins that were correlated with lignin biosynthesis, plant hormone biosynthesis, and signaling pathways. In addition, 19 crucial transcription factors were discovered using correlation analysis that might play a role in regulating candidate genes. This study provides insight into the molecular mechanism of inflorescence elongation in alfalfa and establishes a theoretical foundation for improving alfalfa seed production.


Subject(s)
Gene Expression Regulation, Plant , Inflorescence , Lignin , Medicago sativa , Plant Proteins , Transcriptome , Medicago sativa/genetics , Medicago sativa/growth & development , Medicago sativa/metabolism , Inflorescence/growth & development , Inflorescence/genetics , Inflorescence/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Lignin/biosynthesis , Lignin/metabolism , Plant Growth Regulators/metabolism , Plant Growth Regulators/genetics , Proteome/metabolism , Gene Expression Profiling , Proteomics/methods , Metabolome , Multiomics
12.
BMC Plant Biol ; 24(1): 544, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38872112

ABSTRACT

BACKGROUND: Plant height (PH) is an important agronomic trait influenced by a complex genetic network. However, the genetic basis for the variation in PH in Medicago sativa remains largely unknown. In this study, a comprehensive genome-wide association analysis was performed to identify genomic regions associated with PH using a diverse panel of 220 accessions of M. sativa worldwide. RESULTS: Our study identified eight novel single nucleotide polymorphisms (SNPs) significantly associated with PH evaluated in five environments, explaining 8.59-12.27% of the phenotypic variance. Among these SNPs, the favorable genotype of chr6__31716285 had a low frequency of 16.4%. Msa0882400, located proximal to this SNP, was annotated as phosphate transporter 3;1, and its role in regulating alfalfa PH was supported by transcriptome and candidate gene association analysis. In addition, 21 candidate genes were annotated within the associated regions that are involved in various biological processes related to plant growth and development. CONCLUSIONS: Our findings provide new molecular markers for marker-assisted selection in M. sativa breeding programs. Furthermore, this study enhances our understanding of the underlying genetic and molecular mechanisms governing PH variations in M. sativa.


Subject(s)
Genome-Wide Association Study , Medicago sativa , Polymorphism, Single Nucleotide , Medicago sativa/genetics , Phenotype , Genes, Plant , Quantitative Trait Loci/genetics , Genotype
13.
Protein Expr Purif ; 222: 106521, 2024 Oct.
Article in English | MEDLINE | ID: mdl-38852714

ABSTRACT

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/µg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/µg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.


Subject(s)
Bacterial Proteins , Elastin , Nicotiana , Protein Sorting Signals , Recombinant Fusion Proteins , Shigella dysenteriae , Nicotiana/genetics , Nicotiana/metabolism , Protein Sorting Signals/genetics , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/biosynthesis , Bacterial Proteins/metabolism , Elastin/genetics , Elastin/chemistry , Elastin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Shigella dysenteriae/genetics , Medicago sativa/genetics , Medicago sativa/metabolism , Medicago sativa/chemistry , Medicago sativa/microbiology , Gene Expression , Plant Proteins/genetics , Plant Proteins/biosynthesis , Plant Proteins/isolation & purification , Plant Proteins/chemistry , Plant Proteins/metabolism , Glycoproteins/genetics , Glycoproteins/chemistry , Glycoproteins/isolation & purification , Glycoproteins/biosynthesis , Glycoproteins/metabolism , Elastin-Like Polypeptides
14.
Plant Physiol Biochem ; 213: 108868, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38917738

ABSTRACT

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes' expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring Fv/Fm and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species.


Subject(s)
Chloroplasts , Gene Expression Regulation, Plant , Germination , Medicago sativa , Photosynthesis , Chloroplasts/metabolism , Chloroplasts/ultrastructure , Medicago sativa/genetics , Medicago sativa/metabolism , Medicago sativa/growth & development , Seeds/metabolism , Seeds/growth & development , Seeds/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Chlorophyll/metabolism
15.
J Agric Food Chem ; 72(25): 14448-14465, 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38864675

ABSTRACT

Alfalfa (Medicago sativa subsp. sativa), the "queen of forage," is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29, a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.


Subject(s)
Droughts , Flavonoids , Gene Expression Regulation, Plant , Medicago sativa , Plant Proteins , Medicago sativa/genetics , Medicago sativa/metabolism , Medicago sativa/growth & development , Plant Proteins/genetics , Plant Proteins/metabolism , Flavonoids/metabolism , Drought Resistance , Multiomics
16.
Plant Physiol Biochem ; 213: 108764, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38879983

ABSTRACT

The phosphoenolpyruvate carboxylase kinase of Medicago sativa L. (MsPPCK1) modulates the phosphorylation status and activity of the C4 pathway phosphoenolpyruvate carboxylase enzyme, which is pivotal for photosynthetic carbon assimilation in plants. This study investigated the role of MsPPCK1 in alfalfa by creating transgenic plants overexpressing MsPPCK1 under the control of the CaMV35S promoter. The enhanced alkali tolerance of transgenic plants indicated an important role of MsPPCK1 gene in regulating plant alkali tolerance. Transgenic plants exhibited heightened antioxidant activity (SOD, POD, and CAT), reduced MDA, H2O2, OFR and REC% content, increased activity of key photosynthetic enzymes (PEPC, PPDK, NADP-ME, and NADP-MDH), and enhanced photosynthetic parameters (Pn, E, Gs, and Ci). Moreover, MsPPCK1 overexpression increased the content of organic acids (oxaloacetic, malic, citric, and succinic acids) in the plants. The upregulation of MsPPCK1 under rhizobial inoculation showcased its other role in nodule development. In transgenic plants, MsDMI2, MsEnod12, and MsNODL4 expression increased, facilitating root nodule development and augmenting plant nodulation. Accelerated root nodule growth positively influences plant growth and yield and enhances alfalfa resistance to alkali stress. This study highlights the pivotal role of MsPPCK1 in fortifying plant alkali stress tolerance and improving yield, underscoring its potential as a key genetic target for developing alkali-tolerant and high-yielding alfalfa varieties.


Subject(s)
Medicago sativa , Photosynthesis , Plant Proteins , Plants, Genetically Modified , Medicago sativa/genetics , Medicago sativa/enzymology , Medicago sativa/growth & development , Photosynthesis/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Root Nodules, Plant/genetics , Root Nodules, Plant/growth & development , Root Nodules, Plant/metabolism , Gene Expression Regulation, Plant , Alkalies , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Serine-Threonine Kinases
17.
J Hazard Mater ; 473: 134610, 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-38776812

ABSTRACT

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.


Subject(s)
Aluminum , Homeostasis , Magnesium , Medicago sativa , Plant Proteins , Plant Shoots , Potassium , Stress, Physiological , Medicago sativa/genetics , Medicago sativa/metabolism , Medicago sativa/drug effects , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Shoots/metabolism , Plant Shoots/drug effects , Potassium/metabolism , Aluminum/toxicity , Magnesium/metabolism , Plants, Genetically Modified , Gene Expression Regulation, Plant/drug effects , Plant Leaves/metabolism , Plant Leaves/drug effects , Nicotiana/genetics , Nicotiana/metabolism , Nicotiana/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Acids/metabolism
18.
J Plant Physiol ; 297: 154262, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38703548

ABSTRACT

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.


Subject(s)
Aluminum , Gene Expression Profiling , Phylogeny , Plant Proteins , Aluminum/toxicity , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant/drug effects , Multigene Family , Medicago truncatula/genetics , Medicago truncatula/drug effects , Medicago truncatula/metabolism , Medicago sativa/genetics , Medicago sativa/drug effects , Medicago sativa/physiology , Plant Roots/genetics , Plant Roots/drug effects , Plant Roots/metabolism , Genome, Plant , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Medicago/genetics , Medicago/physiology
19.
Funct Plant Biol ; 512024 05.
Article in English | MEDLINE | ID: mdl-38739736

ABSTRACT

The forage quality of alfalfa (Medicago sativa ) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.


Subject(s)
Medicago sativa , Plant Stems , Starch , Sucrose , Medicago sativa/genetics , Medicago sativa/metabolism , Medicago sativa/growth & development , Starch/metabolism , Plant Stems/metabolism , Plant Stems/growth & development , Plant Stems/genetics , Sucrose/metabolism , Gene Expression Regulation, Plant , Transcriptome , Carbohydrate Metabolism/genetics , Gene Expression Profiling
20.
BMC Genomics ; 25(1): 497, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38773372

ABSTRACT

BACKGROUND: Alfalfa (Medicago sativa L.) is the most cultivated forage legume around the world. Under a variety of growing conditions, forage yield in alfalfa is stymied by biotic and abiotic stresses including heat, salt, drought, and disease. Given the sessile nature of plants, they use strategies including, but not limited to, differential gene expression to respond to environmental cues. Transcription factors control the expression of genes that contribute to or enable tolerance and survival during periods of stress. Basic-leucine zipper (bZIP) transcription factors have been demonstrated to play a critical role in regulating plant growth and development as well as mediate the responses to abiotic stress in several species, including Arabidopsis thaliana, Oryza sativa, Lotus japonicus and Medicago truncatula. However, there is little information about bZIP transcription factors in cultivated alfalfa. RESULT: In the present study, 237 bZIP genes were identified in alfalfa from publicly available sequencing data. Multiple sequence alignments showed the presence of intact bZIP motifs in the identified sequences. Based on previous phylogenetic analyses in A. thaliana, alfalfa bZIPs were similarly divided and fell into 10 groups. The physico-chemical properties, motif analysis and phylogenetic study of the alfalfa bZIPs revealed high specificity within groups. The differential expression of alfalfa bZIPs in a suite of tissues indicates that bZIP genes are specifically expressed at different developmental stages in alfalfa. Similarly, expression analysis in response to ABA, cold, drought and salt stresses, indicates that a subset of bZIP genes are also differentially expressed and likely play a role in abiotic stress signaling and/or tolerance. RT-qPCR analysis on selected genes further verified these differential expression patterns. CONCLUSIONS: Taken together, this work provides a framework for the future study of bZIPs in alfalfa and presents candidate bZIPs involved in stress-response signaling.


Subject(s)
Basic-Leucine Zipper Transcription Factors , Gene Expression Regulation, Plant , Medicago sativa , Phylogeny , Stress, Physiological , Medicago sativa/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Stress, Physiological/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Computer Simulation , Gene Expression Profiling , Computational Biology/methods
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