Genome-wide identification of SHMT family genes in alfalfa (Medicago sativa) and its functional analyses under various abiotic stresses.

BACKGROUND: Alfalfa (Medicago sativa L.) is the most widely planted legume forage and one of the most economically valuable crops in the world. Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays crucial roles in plant growth, development, and stress responses. To date, there has been no comprehensive bioinformatics investigation conducted on the SHMT genes in M. sativa. RESULTS: Here, we systematically analyzed the phylogenetic relationship, expansion pattern, gene structure, cis-acting elements, and expression profile of the MsSHMT family genes. The result showed that a total of 15 SHMT members were identified from the M. sativa genome database. Phylogenetic analysis demonstrated that the MsSHMTs can be divided into 4 subgroups and conserved with other plant homologues. Gene structure analysis found that the exons of MsSHMTs ranges from 3 to 15. Analysis of cis-acting elements found that each of the MsSHMT genes contained different kinds of hormones and stress-related cis-acting elements in their promoter regions. Expression and function analysis revealed that MsSHMTs expressed in all plant tissues. qRT-PCR analysis showed that MsSHMTs induced by ABA, Salt, and drought stresses. CONCLUSIONS: These results provided definite evidence that MsSHMTs might involve in growth, development and adversity responses in M. sativa, which laid a foundation for future functional studies of MsSHMTs.

Genome-wide identification and expression analysis of the Auxin-Response factor (ARF) gene family in Medicago sativa under abiotic stress.

BACKGROUND: Alfalfa (Medicago sativa) is the most widely planted legume forage and one of the most economically valuable crops in the world. The periodic changes in its growth and development and abiotic stress determine its yield and economic benefits. Auxin controls many aspects of alfalfa growth by regulating gene expression, including organ differentiation and stress response. Auxin response factors (ARF) are transcription factors that play an essential role in auxin signal transduction and regulate the expression of auxin-responsive genes. However, the function of ARF transcription factors is unclear in autotetraploid-cultivated alfalfa. RESULT: A total of 81 ARF were identified in the alfalfa genome in this study. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed, identifying that ARF genes are mainly involved in transcriptional regulation and plant hormone signal transduction pathways. Phylogenetic analysis showed that MsARF was divided into four clades: I, II, III, and IV, each containing 52, 13, 7, and 9 genes, respectively. The promoter region of the MsARF gene contained stress-related elements, such as ABRE, TC-rich repeats, MBS, LTR. Proteins encoded by 50 ARF genes were localized in the nucleus without guide peptides, signal peptides, or transmembrane structures, indicating that most MsARF genes are not secreted or transported but only function in the nucleus. Protein structure analysis revealed that the secondary and tertiary structures of the 81 MsARF genes varied. Chromosomal localization analysis showed 81 MsARF genes were unevenly distributed on 25 chromosomes, with the highest distribution on chromosome 5. Furthermore, 14 segmental duplications and two sets of tandem repeats were identified. Expression analysis indicated that the MsARF was differentially expressed in different tissues and under various abiotic stressors. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that the expression profiles of 23 MsARF genes were specific to abiotic stresses such as drought, salt, high temperature, and low temperature, as well as tissue-specific and closely related to the duration of stress. CONCLUSION: This study identified MsARF in the cultivated alfalfa genome based on the autotetraploid level, which GO, KEGG analysis, phylogenetic analysis, sequence characteristics, and expression pattern analysis further confirmed. Together, these findings provide clues for further investigation of MsARF functional verification and molecular breeding of alfalfa. This study provides a novel approach to systematically identify and characterize ARF transcription factors in autotetraploid cultivated alfalfa, revealing 23 MsARF genes significantly involved in response to various stresses.

Transcriptome expression profiles reveal response mechanisms to drought and drought-stress mitigation mechanisms by exogenous glycine betaine in maize.

Drought stress is one of the major abiotic stresses that limit growth, development and yield of maize crops. To better understand the responses of maize inbred lines with different levels of drought resistance and the molecular mechanism of exogenous glycine betaine (GB) in alleviating drought stress, the responses of two maize inbred lines to drought stress and to the stress-mitigating effects of exogenous GB were investigated. Seedling morphology, physiological and biochemical indexes, root cell morphology and root transcriptome expression profiles were compared between a drought-tolerant inbred line Chang 7-2 and drought-sensitive inbred line TS141. Plants of both lines were subjected to treatments of drought stress alone and drought stress with application of exogenous GB. The results showed that with the increase of drought treatment time, the growth and development of TS141 were inhibited, while those of Chang 7-2 were not significantly different from those of the control (no drought stress and GB). Compared with the corresponding data of the drought-stress group, every index measured from the two inbred lines indicated mitigating effects from exogenous GB, and TS141 produced stronger mitigating responses due to the GB. Transcriptome analysis showed that 562 differentially expressed genes (DEGs) were up-regulated and 824 DEGs were down-regulated in both inbred lines under drought stress. Due to the exogenous GB, 1061 DEGs were up-regulated and 424 DEGs were down-regulated in both lines. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify 10 DEGs screened from the different treatments. These results showed that the expression of 9 DEGs were basically consistent with their respective transcriptome expression profiles. The results of this study provide models of potential mechanisms of drought tolerance in maize as well as potential mechanisms of how exogenous GB may regulate drought tolerance.

Mutant lpa1 Analysis of ZmLPA1 Gene Regulates Maize Leaf-Angle Development through the Auxin Pathway.

Maize plant type is one of the main factors determining maize yield, and leaf angle is an important aspect of plant type. The rice Loose Plant Architecture1 (LPA1) gene and Arabidopsis AtIDD15/SHOOT GRAVITROPISM5 (SGR5) gene are related to their leaf angle. However, the homologous ZmLPA1 in maize has not been studied. In this study, the changing of leaf angle, as well as gene expression in leaves in maize mutant lpa1 and wild-type 'B73' under different IAA concentrations were investigated. The regulation effect of IAA on the leaf angle of lpa1 was significantly stronger than that of the wild type. Transcriptome analysis showed that different exogenous IAA treatments had a common enrichment pathway-the indole alkaloid biosynthesis pathway-and among the differentially expressed genes, four genes-AUX1, AUX/IAA, ARF and SAUR-were significantly upregulated. This study revealed the regulation mechanism of ZmLPA1 gene on maize leaf angle and provided a promising gene resource for maize breeding.

Comparative Proteomics Analysis of the Seedling Root Response of Drought-sensitive and Drought-tolerant Maize Varieties to Drought Stress.

The growth and development of maize roots are closely related to drought tolerance. In order to clarify the molecular mechanisms of drought tolerance between different maize (Zea mays L.) varieties at the protein level, the isobaric tags for relative and absolute quantitation (iTRAQ) quantitative proteomics were used for the comparative analysis of protein expression in the seedling roots of the drought-tolerant Chang 7-2 and drought-sensitive TS141 maize varieties under 20% polyethylene glycol 6000 (PEG 6000)-simulated drought stress. We identified a total of 7723 differentially expressed proteins (DEPs), 1243 were significantly differentially expressed in Chang 7-2 following drought stress, 572 of which were up-regulated and 671 were down-regulated; 419 DEPs were identified in TS141, 172 of which were up-regulated and 247 were down-regulated. In Chang 7-2, the DEPs were associated with ribosome pathway, glycolysis/gluconeogenesis pathway, and amino sugar and nucleotide sugar metabolism. In TS141, the DEPs were associated with metabolic pathway, phenylpropanoid biosynthesis pathway, and starch and sucrose metabolism. Compared with TS141, the higher drought tolerance of Chang 7-2 root system was attributed to a stronger water retention capacity; the synergistic effect of antioxidant enzymes; the strengthen cell wall; the osmotic stabilization of plasma membrane proteins; the effectiveness of recycling amino acid; and an improvement in the degree of lignification. The common mechanisms of the drought stress response between the two varieties included: The promotion of enzymes in the glycolysis/gluconeogenesis pathway; cross-protection against the toxicity of aldehydes and ammonia; maintenance of the cell membrane stability. Based on the proteome sequencing information, the coding region sequences of eight DEP-related genes were analyzed at the mRNA level by quantitative real-time PCR (qRT-PCR). The findings of this study can inform the future breeding of drought-tolerant maize varieties.

Comparative Proteomics of Salt-Tolerant and Salt-Sensitive Maize Inbred Lines to Reveal the Molecular Mechanism of Salt Tolerance.

Salt stress is one of the key abiotic stresses that causes great loss of yield and serious decrease in quality in maize (Zea mays L.). Therefore, it is very important to reveal the molecular mechanism of salt tolerance in maize. To acknowledge the molecular mechanisms underlying maize salt tolerance, two maize inbred lines, including salt-tolerant 8723 and salt-sensitive P138, were used in this study. Comparative proteomics of seedling roots from two maize inbred lines under 180 mM salt stress for 10 days were performed by the isobaric tags for relative and absolute quantitation (iTRAQ) approach. A total of 1056 differentially expressed proteins (DEPs) were identified. In total, 626 DEPs were identified in line 8723 under salt stress, among them, 378 up-regulated and 248 down-regulated. There were 473 DEPs identified in P138, of which 212 were up-regulated and 261 were down-regulated. Venn diagram analysis showed that 17 DEPs were up-regulated and 12 DEPs were down-regulated in the two inbred lines. In addition, 8 DEPs were up-regulated in line 8723 but down-regulated in P138, 6 DEPs were down-regulated in line 8723 but up-regulated in P138. In salt-stressed 8723, the DEPs were primarily associated with phenylpropanoid biosynthesis, starch and sucrose metabolism, and the mitogen-activated protein kinase (MAPK) signaling pathway. Intriguingly, the DEPs were only associated with the nitrogen metabolism pathway in P138. Compared to P138, the root response to salt stress in 8723 could maintain stronger water retention capacity, osmotic regulation ability, synergistic effects of antioxidant enzymes, energy supply capacity, signal transduction, ammonia detoxification ability, lipid metabolism, and nucleic acid synthesis. Based on the proteome sequencing information, changes of 8 DEPs abundance were related to the corresponding mRNA levels by quantitative real-time PCR (qRT-PCR). Our results from this study may elucidate some details of salt tolerance mechanisms and salt tolerance breeding of maize.

Analysis on morphological characteristics and identification of candidate genes during the flowering development of alfalfa.

Flower development is a crucial and complex process in the reproductive stage of plants, which involves the interaction of multiple endogenous signals and environmental factors. However, regulatory mechanism of flower development was unknown in alfalfa (Medicago sativa). In this study, the three stages of flower development of 'M. sativa cv. Gannong No. 5' (G5) and its early flowering and multi flowering mutant (MG5) were comparatively analyzed by transcriptomics. The results showed that compared with late bud stage (S1), 14287 and 8351 differentially expressed genes (DEGs) were identified at early flower stage (S2) in G5 and MG5, and 19941 and 19469 DEGs were identified at late flower stage (S3). Compared with S2, 9574 and 10870 DEGs were identified at S3 in G5 and MG5, respectively. Venn analysis revealed that 547 DEGs were identified among the three comparison groups. KEGG pathway enrichment analysis showed that these genes were involved in the development of alfalfa flowers through redox pathways and plant hormone signaling pathways. Key candidate genes including SnRK2, BSK, GID1, DELLA and CRE1, for regulating the development from buds to mature flowers in alfalfa were screened. In addition, differential expression of transcription factors such as MYB, AP2, bHLH, C2C2, MADS-box, NAC, bZIP, B3 and AUX/IAA also played an important role in this process. The results laid a theoretical foundation for studying the molecular mechanisms of the development process from buds to mature flowers in alfalfa.

Network Analysis of Different Exogenous Hormones on the Regulation of Deep Sowing Tolerance in Maize Seedlings.

The planting method of deep sowing can make the seeds make full use of water in deep soil, which is considered to be an effective way to respond to drought stress. However, deep sowing will affect the growth and development of maize (Zea mays L.) at seedling stage. To better understand the response of maize to deep sowing stress and the mechanism of exogenous hormones [Gibberellin (GA3), Brassinolide (BR), Strigolactone (SL)] alleviates the damaging effects of deep-sowing stress, the physiological and transcriptome expression profiles of seedlings of deep sowing sensitive inbred line Zi330 and the deep-tolerant inbred line Qi319 were compared under deep sowing stress and the conditions of exogenous hormones alleviates stress. The results showed that mesocotyl elongated significantly after both deep sowing stress and application of exogenous hormones, and its elongation was mainly through elongation and expansion of cell volume. Hormone assays revealed no significant changes in zeatin (ZT) content of the mesocotyl after deep sowing and exogenous hormone application. The endogenous GA3 and auxin (IAA) contents in the mesocotyl of the two inbred lines increased significantly after the addition of exogenous GA3, BR, and SL under deep sowing stress compared to deep sowing stress, while BR and SL decreased significantly. Transcriptome analysis showed that the deep seeding stress was alleviated by GA3, BR, and SLs, the differentially expressed genes (DEGs) mainly included cellulose synthase, expansin and glucanase, oxidase, lignin biosynthesis genes and so on. We also found that protein phosphatase 2C and GA receptor GID1 enhanced the ability of resist deep seeding stress in maize by participating in the abscisic acid (ABA) and the GA signaling pathway, respectively. In addition, we identified two gene modules that were significantly related to mesocotyl elongation, and identified some hub genes that were significantly related to mesocotyl elongation by WGCNA analysis. These genes were mainly involved in transcription regulation, hydrolase activity, protein binding and plasma membrane. Our results from this study may provide theoretical basis for determining the maize deep seeding tolerance and the mechanism by which exogenous hormones regulates deep seeding tolerance.

Comparing transcriptome expression profiles to reveal the mechanisms of salt tolerance and exogenous glycine betaine mitigation in maize seedlings.

Salt stress is a common abiotic stress that limits the growth, development and yield of maize (Zea mays L.). To better understand the response of maize to salt stress and the mechanism by which exogenous glycine betaine (GB) alleviates the damaging effects of salt stress, the morphology, physiological and biochemical indexes, and root transcriptome expression profiles of seedlings of salt-sensitive inbred line P138 and salt-tolerant inbred line 8723 were compared under salt stress and GB-alleviated salt stress conditions. The results showed that under salt stress the growth of P138 was significantly inhibited and the vivo ion balance was disrupted, whereas 8723 could prevent salt injury by maintaining a high ratio of K+ to Na+. The addition of a suitable concentration of GB could effectively alleviate the damage caused by salt stress, and the mitigating effect on salt-sensitive inbred line P138 was more obvious than that on 8723. Transcriptome analysis revealed that 219 differentially expressed genes (DEGs) were up-regulated and 153 DEGs were down-regulated in both P138 and 8723 under NaCl treatment, and that 487 DEGs were up-regulated and 942 DEGs were down-regulated in both P138 and 8723 under salt plus exogenous GB treatment. In 8723 the response to salt stress is mainly achieved through stabilizing ion homeostasis, strong signal transduction activation, increasing reactive oxygen scavenging. GB alleviates salt stress in maize mainly by inducing gene expression changes to enhance the ion balance, secondary metabolic level, reactive oxygen scavenging mechanism, signal transduction activation. In addition, the transcription factors involved in the regulation of salt stress response and exogenous GB mitigation mainly belong to the MYB, MYB-related, AP2-EREBP, bHLH, and NAC families. We verified 10 selected up-regulated DEGs by quantitative real-time polymerase chain reaction (qRT-PCR), and the expression results were basically consistent with the transcriptome expression profiles. Our results from this study may provide the theoretical basis for determining maize salt tolerance mechanisms and the mechanism by which GB regulates salt tolerance.