Identification and characterization of the RcTCP gene family and its expression in response to abiotic stresses in castor bean.

BACKGROUND: The TCP (teosinte branched1/cincinnata/proliferating cell factor) family plays a prominent role in plant development and stress responses. However, TCP family genes have thus far not been identified in castor bean, and therefore an understanding of the expression and functional aspects of castor bean TCP genes is lacking. To identify the potential biological functions of castor bean (RcTCP) TCP members, the composition of RcTCP family members, their basic physicochemical properties, subcellular localizations, interacting proteins, miRNA target sites, and gene expression patterns under stress were assessed. RESULTS: The presence of 20 RcTCP genes on the nine chromosomes of castor bean was identified, all of which possess TCP domains. Phylogenetic analysis indicated a close relationship between RcTCP genes and Arabidopsis AtTCP genes, suggesting potential functional similarity. Subcellular localization experiments confirmed that RcTC01/02/03/10/16/18 are all localized in the nucleus. Protein interaction analysis revealed that the interaction quantity of RcTCP03/06/11 proteins is the highest, indicating a cascade response in the functional genes. Furthermore, it was found that the promoter region of RcTCP genes contains a large number of stress-responsive elements and hormone-induced elements, indicating a potential link between RcTCP genes and stress response functions. qRT-PCR showed that all RcTCP genes exhibit a distinct tissue-specific expression pattern and their expression is induced by abiotic stress (including low temperature, abscisic acid, drought, and high salt). Among them, RcTCP01/03/04/08/09/10/14/15/18/19 genes may be excellent stress-responsive genes. CONCLUSION: We discovered that RcTCP genes play a crucial role in various activities, including growth and development, the stress response, and transcription. This study provides a basis for studying the function of RcTCP gene in castor.

Genome-wide analysis of LOG family genes in castor and RcLOG5 enhances drought, salt, and cold stress tolerance in Arabidopsis thaliana.

The gene encoding the specific phosphohydrolase LONELY GUY (LOG) plays an important role in the activation of cytokinin and the stress response in plant cells. However, the role of LOG genes in castor bean (Ricinus communis) has not been reported. In this study, we identified a total of nine members of the LOG gene family in the castor bean genome and investigated the upregulated expression of the RcLOG5 gene using transcriptome data analysis. We found that the RcLOG5 gene exhibited tissue-specific expression and was activated by polyethylene glycol, NaCl, low temperature, and abscisic acid stress. The subcellular localization results showed that the RcLOG5 gene is mainly located in the cytoplasm. Based on phenotypic and physiological indicators, namely root length, peroxidase activity, and malondialdehyde content, overexpression of the RcLOG5 gene not only improved the drought resistance, salt tolerance, and cold tolerance of transgenic Arabidopsis, but also shortened the dormancy period of the transgenic plants. Transcriptomic sequencing revealed that the overexpression of the RcLOG5 gene led to the enrichment of differentially expressed genes in the glutathione metabolism pathway in transgenic Arabidopsis. Moreover, the overexpression plants had higher levels of glutathione and a higher GSH/GSSG ratio under stress compared to the wild type. Therefore, we inferred that the RcLOG5 gene may be responsible for regulating cell membrane homeostasis by reducing the accumulation of reactive oxygen species through the glutathione pathway. Overall, the overexpression of the RcLOG5 gene positively regulated the stress resistance of transgenic Arabidopsis. This study provides valuable gene resources for breeding stress-tolerant castor bean varieties.

[Identification and expression pattern analysis of RcACA gene family in castor under abiotic stresses].

Auto-inhibited Ca2+-ATPase (ACA) is one of the Ca2+-ATPase subfamilies that plays an important role in maintaining Ca2+ concentration balance in plant cells. To explore the function and gene expression pattern of the RcACA gene family in castor, bioinformatics analysis was used to identify the members of the RcACA gene family in castor. The basic physical and chemical properties, subcellular location, protein secondary and tertiary structure, conserved domain, conserved motif, gene structure, chromosome location and collinear relationship, as well as the evolutionary characteristics and promoter cis-acting elements were predicted and analyzed. The expression pattern of the RcACA gene under abiotic stress was analyzed by expression (fragments per kilobase of exon model per million mapped fragments, FPKM) in castor transcriptome data. The results showed that 8 RcACA gene family members were identified in castor, acidic proteins located in the plasma membrane. In the secondary structure of all proteins, the α-helix and random coil is more; the RcACA genes were clustered into three categories, and the design of the genes in the same category was similar to the conserved motif. Both of them had four typical domains, RcACA3-RcACA8 had a Ca2+-ATPase N-terminal autoinhibitory domain. The RcACA gene is mostly located on the long arm of the chromosome and has 2 pairs of collinear relationships. There are more light response elements but fewer hormone-induced elements located upstream of the RcACA coding region. Interspecific clustering showed that the evolution of ACA genes among species was conservative. Tissue expression pattern analysis showed that RcACA genes showed apparent tissue expression specificity, and most of the genes showed the highest expression level in male flowers. Expression analysis under abiotic stress showed that RcACA2-RcACA8 were up-regulated under high salt and drought stress, and RcACA1 was up-regulated at 0-24 h under low-temperature stress, indicating that RcACA genes positively responded to abiotic stresses. The above results provide a theoretical basis for exploring the role of the RcACA gene in castor growth, development and stress response.

Effect of LuxS/AI-2-mediated quorum sensing system on bacteriocin production of Lactobacillus plantarum NMD-17.

Lactobacillus plantarum NMD-17 separated from koumiss could produce a bacteriocin named plantaricin MX against Gram-positive bacteria and Gram-negative bacteria. The bacteriocin synthesis of L. plantarum NMD-17 was remarkably induced in co-cultivation with Lactobacillus reuteri NMD-86 as the increase of cell numbers and AI-2 activity, and the expressions of luxS encoding signal AI-2 synthetase, plnB encoding histidine protein kinase, plnD encoding response regulator, and plnE and plnF encoding structural genes of bacteriocin were significantly upregulated in co-cultivation, showing that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation may be regulated by LuxS/AI-2-mediated quorum sensing system. In order to further demonstrate the role of LuxS/AI-2-mediated quorum sensing system in the bacteriocin synthesis of L. plantarum NMD-17, plasmids pUC18 and pMD18-T simple were used as the skeleton to construct the suicide plasmids pUC18-UF-tet-DF and pMD18-T simple-plnB-tet-plnD for luxS and plnB-plnD gene deletion, respectively. luxS and plnB-plnD gene knockout mutants were successfully obtained by homologous recombination. luxS gene knockout mutant lost its AI-2 synthesis ability, suggesting that LuxS protein encoded by luxS gene is key enzyme for AI-2 synthesis. plnB-plnD gene knockout mutant lost the ability to synthesize bacteriocin against Salmonella typhimurium ATCC14028, indicating that plnB-plnD gene was a necessary gene for bacteriocin synthesis of L. plantarum NMD-17. Bacteriocin synthesis, cell numbers, and AI-2 activity of luxS or plnB-plnD gene knockout mutants in co-cultivation with L. reuteri NMD-86 were obviously lower than those of wild-type strain in co-cultivation at 6-9 h (P < 0.01). The results showed that LuxS/AI-2-mediated quorum sensing system played an important role in the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation.

LuxS-mediated quorum sensing system in Lactobacillus plantarum NMD-17 from koumiss: induction of plantaricin MX in co-cultivation with certain lactic acid bacteria.

A bacteriocin termed plantaricin MX with a broad antimicrobial spectrum was produced by Lactobacillus plantarum NMD-17, which was isolated from Inner Mongolia traditional koumiss of china. Among 300 strains of lactic acid bacteria (LAB) belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, and Enterococcus, five strains including Lactobacillus reuteri NMD-86, Lactobacillus helveticus NMD-137, Lactococcus lactis NMD-152, Enterococcus faecalis NMD-178, and Enterococcus faecium NMD-219 were revealed to significantly induce the bacteriocin synthesis and greatly increase the cell numbers of Lactobacillus plantarum NMD-17 and activity of AI-2 signaling molecule. Bacteriocin synthesis was not increased by cell-free supernatants and autoclaved cultures of inducing strains, demonstrating that intact cells of inducing strains were essential to the induction of bacteriocin synthesis. The existence of bacteriocin structural plnEF genes and the plnD and luxS genes involved in quorum sensing was confirmed by PCR, and the presence of plnB gene encoding histidine protein kinase was determined by single oligonucleotide nested PCR (Son-PCR). Quantitative real-time PCR demonstrated that plnB, plnD, luxS, plnE, and plnF genes of L. plantarum NMD-17 were upregulated significantly (P < 0.01) in co-cultivation with L. reuteri NMD-86. The results showed that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation might have a close relationship with LuxS-mediated quorum sensing system.

Heterologous expression of an Agropyron cristatum SnRK2 protein kinase gene (AcSnRK2.11) increases freezing tolerance in transgenic yeast and tobacco.

The sucrose non-fermenting-1 related protein kinase 2 (SnRK2) family plays an important role in the response to abiotic stress. To characterize the function of the SnRK2 gene from Agropyron cristatum in stress protection, we cloned the complete coding sequence of the AcSnRK2.11 gene from A. cristatum and generated AcSnRK2.11-overexpressing tobacco lines. The open reading frame of AcSnRK2.11 was 1083 bp in length and encoded a polypeptide of 360 amino acid residues. The sequence analysis results showed that AcSnRK2.11 contained conserved domains typified in SnRK2 protein kinases. Subcellular localization analysis showed that AcSnRK2.11 was located in the nucleus. AcSnRK2.11 was constitutively expressed in all of the examined tissues, and its transcription was induced by cold, dehydration, and salt stress, but not by abscisic acid treatment. Overexpression of the AcSnRK2.11 gene conferred freezing tolerance in yeast. AcSnRK2.11-overexpressing tobacco lines showed higher tolerance to freezing stress than did wild-type (WT) based on higher survival rates, lower malondialdehyde content and increased relative water content retention, chlorophyll yields, superoxide dismutase activities, reactive oxygen species content, peroxidase levels, and soluble carbohydrates under low-temperature conditions. The transcripts of NtDREB1, NtDREB2, NtERD10A, NtERD10B, NtERD10C, NtERD10D, NtMnSOD, NtCDPK15, and NtMPK9 in AcSnRK2.11-overexpressing tobacco lines were more abundant than in WT plants under low-temperature stress. These results suggest that AcSnRK2.11 may function as a regulatory factor associated with a cold-response pathway and could be used in plant breeding for cold resistance.

Characterization of a broad spectrum bacteriocin produced by Lactobacillus plantarum MXG-68 from Inner Mongolia traditional fermented koumiss.

An agar well diffusion assay (AWDA) was used to isolate a high bacteriocin-producing strain with a broad spectrum of antibacterial activity, strain MXG-68, from Inner Mongolia traditional fermented koumiss. Lactobacillus plantarum MXG-68 was identified by morphological, biochemical, and physiological characteristics and 16S rDNA analysis. The production of antibacterial substance followed a growth-interrelated model, starting at the late lag phase of 4 h and arriving at a maximum value in the middle of the stationary phase at 24 h. Antibacterial activity was abolished or decreased in the presence of pepsin, chymotrypsin, trypsin, proteinase, and papain K. The results showed that antibacterial substances produced by L. plantarum MXG-68 were proteinaceous and could thus be classified as the bacteriocin, named plantaricin MXG-68. The molar mass of plantaricin MXG-68 was estimated to be 6.5 kDa, and the amino acid sequence of its N-terminal was determined to be VYGPAGIFNT. The mode of plantaricin MXG-68 action was determined to be bactericidal. Bacteriocin in cell-free supernatant (CFS) at pH 7 was stable at different temperatures (60 °C, 80 °C, 100 °C, 121 °C for 30 min; 4 °C and - 20 °C for 30 days), as well as at pH 2.0-10.0. Antibacterial activity maintained stable after treatment with organic solvents, surfactants, and detergents but increased in response to EDTA. Response surface methodology (RSM) revealed the optimum conditions of bacteriocin production in L. plantarum MXG-68, and the bacteriocin production in medium optimized by RSM was 26.10% higher than that in the basal MRS medium.

Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6.

The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO4 and CaCl2 increased B. subtilis MX-6 nattokinase production.

A new Em-like protein from Lactuca sativa, LsEm1, enhances drought and salt stress tolerance in Escherichia coli and rice.

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.

Stress-responsive gene RsICE1 from Raphanus sativus increases cold tolerance in rice.

The ICE1 transcription factor plays a critical role in plant cold tolerance via triggering CBF/DREB1 cold-regulated signal networks. In this work, a novel MYC-type ICE1-like gene, RsICE1, was isolated from radish (Raphanus sativus L.), and its function in cold tolerance was characterized in rice. The RsICE1 gene was expressed constitutively with higher transcriptional levels in the roots and stems of radish seedlings. The NaCl, cold, and ABA treatments could significantly upregulate RsICE1 expression levels, but dehydration stress had a weak effect on its expression. Ectopic expression of the RsICE1 gene in rice conferred enhanced tolerance to low-temperature stress grounded on a higher survival rate, higher accumulation of soluble sugars and free proline content, a decline in electrolyte leakage and MDA levels, and higher chlorophyll levels relative to control plants. OsDREBL and OsTPP1, downstream cold-regulated genes, were remarkably upregulated at transcription levels in rice overexpressing RsICE1 under low-temperature stress, which indicated that RsICE1 was involved in CBF/DREB1 cold-regulated signal networks. Overall, the above data showed that RsICE1 played an active role in improving rice cold tolerance, most likely resulting from the upregulation of OsDREBL and OsTPP1 expression levels by interacting with the RsICE1 gene under low-temperature stress.