| Abiotic stresses, such as cold, drought, and salinity stresses, significantly constrain plant growth and crop yield. Cold stress affects plant growth and development, spatial distribution and crop yield. Many important food crops, such as maize, rice, soybean, tomato, etc. are sensitive to cold stress and have poor adaption to cold stress. Maize possesses the main area in northeast of China, however, due to factor of geographical conditions, maize is often exposed to low temperatures in Spring, resulting in maize yield loss. Given that the demand for maize will continue to expand, so it is imperative to improve the overall yield of maize.A combination of conventional and molecular approaches is an effective strategy to generate stress-tolerant and widely-adapted maize varieties. To identify the potential genes involved in cold stress response and unravel the stress responsive mechanism, leaves and roots from the maize inbred line W9816(a cold-resistance genotype) were harvested at three-leaf stage and used to identify the differentially expressed genes(DEGs) between the cold stress(4 °C) and control conditions(25 °C). c DNA amplified fragments length polymorphism(c DNA-AFLP) technology was carried out to identify the DEGs. According to the c DNA-AFLP analysis, 6829 and 6955 DEGs were displayed using a total of 174 primer combinations in maize leaves and roots separately. A total of 30-50 AFLP bands were detected per primer combination that ranged from 70 to 600 bp. Although the majority of bands revealed no change after the cold treatment, we detected 620 and 531 DEGs that were responsive to cold stress in leaves and roots, respectively, which showed different expression patterns. Of those, only 61 up-regulated DEGs and 32 down-regulated DEGs showed the same expression trend in both maize leaves and roots between the two conditions. Through analysis using the NCBI BLASTX and Maize GDB databases, the DEGs were organized into five categories according to their functions: signal transduction(10, 15%), regulation of transcription(9, 13%), translation and posttranslational modifications(10, 15%), cellular metabolism and organization(24, 36%). Six DEGs were hypothetical proteins in maize(9%) and eight of the sequenced DEGs did not match in the databases(12%).Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer associated protein, Zm SEC14p(accession no. KT932998), isolated from a cold-tolerant maize inbred line using the rapid amplification of c DNA ends(RACE) PCR method. Full length c DNA that consisted of a single open reading frame(ORF) encoded a putative polypeptide of 295 amino acids. The chromosome location of the Sec14 phosphatidylinositol transfer associated protein homolog in the maize genome suggested that the candidate gene was located on chromosome 1, spanned 3420 bp and was comprised of five coding exons. The multiple sequence alignment displayed that Zm SEC14 p had a high amino acid identity with Sb SEC14 p from Sorghum bicolor. Phylogenetic analysis of diverse Sec14p-like demonstrated that Zm SEC14 p belonged to a member of UCSH and only contained an N-terminal SEC14 domain. Zm SEC14 comprised of ten ?-helics, five ?-strand, and three 310-helics, of which, seven ?-helics, five ?-strand, and two 310-helics were positioned directly with the predicted phospholipid binding pocket. Zm SEC14 p expression was induced by cold, salt, and ABA treatment. Tissue expression pattern of Zm SEC14 p displayed that the highest expression level was observed in the leaf. Subcellular localization of the Zm SEC14 p indicated that the Zm SEC14 p protein was mainly localized in the nucleus. We assayed phenotypic analysis for stress tolerance in overexpressed Zm SEC14 p Arabidopsis plants under different growth stages. The results displayed that during the seed germination stage, the transgenic plants were insensitive to cold stress and sensitive to Na Cl and ABA treatments. During seedling stage, the transgenic plants had a faster growth rate in the primary roots under cold stress compared to wild type. During the reproductive growth stage, survival rate of transgenic plants increased by about 38% compared to wild type under freezing shock. Tissue localization of ROS and activities of antioxidant enzymes assay revealed that transgenic plants can significantly improve the activities of POD and SOD compared to wild type under cold stress. The improved enzyme activities were associated with the expression of antioxidant-related gene which was regulated by Zm SEC14 p. The content of proline in transgenic plants was 1.35-and 1.61-fold higher than that of the wild type after 48 h cold treatment. Under cold stress, the expression of some stress-responsive genes in transgenic plants was significantly up-regulated compared to wild type. Zm SEC14 p overexpression regulated the expression of PLC and PLC activity, which may account for the upregulation of stress-responsive genes in transgenic plants.Herein, leaves from the maize inbred line W9816(a cold-resistance genotype) were harvested at three-leaf stage, and were used to identify the differential abundance protein species(DAPS) between chilling stress(4 °C) and control conditions(25 °C). i TRAQ-based quantitative proteomic was used in this study. As a result, 173 DAPS were identified after chilling stress. These DAPS were grouped into the following functional categories: carbohydrate and energy metabolism(38, 22.0%), posttranscriptional regulation(13, 7.51%), translation, ribosomal structure and biogenesis(15, 8.67%), posttranslational modifications, protein turnover, chaperones(14, 8.09%), amino acid metabolism(13, 7.51%), stress response(12, 6.94%), signal transduction(9, 5.20%), lipid metabolism(5, 2.90%), biosynthesis of secondary metabolites(6, 3.47%), inorganic ion transport and metabolism(2, 1.16%), replication, recombination and repair(2, 1.16%), other biological processes(22, 12.71%), and unknown biological processes(22, 12.71%). Bioinformatics analysis showed that 159 DAPS were annotated in 38 Gene Ontology(GO) functional groups, 108 DAPS were classified into 20 clusters of orthologous groups of protein categories, 99 DAPS were enrichment in 60 KEGG pathways. Antioxidants assays showed that the i TRAQ results were reliable. The conjoint analysis of DEGs and DAPS indicated the discrepancy resulted from posttranscriptional regulation, posttranslational modifications, etc. in chilling stress maize leaves. Based on functional analysis, we concluded that the adaptive response of maize seedlings to chilling stress might be related to alleviation of photodamage caused by the over-energized state of thylakoid membrane, more energy produced through glycolysis, increased abundance of stress-responsive protein species, and improvement in the overall ability to scavenge ROS. Posttranscriptional regulation and posttranslational modifications also played important roles for maize to adapt to chilling stress. To date, changes in protein species abundance of maize seedlings in response to cold stress are still unknown.Based on the above results, Zm SEC14 p functioned as a positive factor in stress regulatory networks. Moreover, our study represented comprehensive analysis of transcriptome and proteome profiles in response to chilling stress and enriched our understanding of the functional network responding to chilling stress in maize, which will provide important candidate genes for improving maize cold tolerance by genetic engineering. |
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