- response to drought stress. - Background: Drought is one of the most adverse environmental factors limiting crop productions and it is important to identify key genetic determinants for food safety. - In our previous study, abundance of the wheat CPK34 (TaCPK34) protein was remarkably upregulated in wheat plants suffering from drought stress, inferring that it could be involved in this stress. - Therefore, here we further detected its function and mechanism in response to drought stress.. - Results: Transcripts of the TaCPK34 gene were significantly induced after PEG-stimulated water deficiency (20%. - The iTRAQ-based quantitative proteomics was employed to measure the protein expression profiles in leaves of the transiently TaCPK34-silenced wheat plants after drought stress. - Conclusions: Our study suggested that the TaCPK34 gene played positive roles in wheat response to drought stress through directly or indirectly regulating the expression of ABA-dependent manner genes, which were encoding identified proteins from iTRAQ-based quantitative proteomics. - Because of the ever-increasing global population and the climate change, the food safety becomes a major issue be- cause it needs an annual increase (approximately 2.4%) in food yields, whereas yields of the major crops wheat, rice, and maize are presently increased merely only at 0.9, 1 and 1.6%, respectively [1]. - calcium-dependent protein kinases (CPKs or CDPKs), cal- modulins (CaMs) and CaM-like proteins and calcineurin B-like proteins (CBLs) have been reported to involve in the perception and transmission of intracellular Ca 2+ con- centrations [3]. - Bread wheat ( Triti- cum aestivum L., hexaploid species, AABBDD) is one of the most important global cereal crops and offers a rich source of carbohydrates, proteins, minerals, and other. - Identification of TaCPK34. - Dur- ing the late stages of wheat grain filling period (from 6 to 31 May, 2015, corresponding to 15 ~ 35 days after an- thesis), there was only 25.5 mm rainfall, which was enough for wheat growth and caused drought stress in this growth season (Table S1). - seedlings to monitor the transcriptional responses of the TaCPK34 gene. - Under water deficiency condition, transcripts of the TaCPK34 gene increased quickly, reached the highest level at 3 h, and then decreased quickly to low levels at 6 h (Fig. - After a 100 μM ABA treatment, the transcripts of the TaCPK34 gene quickly increased, remarkably higher than that in. - It provides a concise description of the experimental results, their interpretation as well as the experimental conclusions are drawn. - 3 Transcripts of the TaCPK34 gene in leaves. - Barley stripe mosaic virus-induced silencing (BSMV- VIGS) method was used to verify the function of TaCPK34 under drought stress. - 505 to + 723, the translation start site “ ATG ” is + 1) of TaCPK34 gene, which shared high identity (100%) with the other two copies (Figure S3), was used to construct its silencing vector (BSMV-VIGS- TaCPK34), and BSMV-VIGS-GFP vector was used as the control. - The silencing vectors of BSMV-VIGS-TaCPK34 and BSMV-VIGS-GFP were separately inoculated into the leaves of wheat seedlings. - At 6 days after inoculation, BSMV-VIGS-induced chlorosis emerged on the leaves of both BSMV-VIGS-TaCPK34- or BSMV-VIGS-GFP-inoc- ulated wheat seedlings (Figure S4A). - Transcripts of the TaCPK34 gene were remarkably inhibited in leaves of BSMV-VIGS-TaCPK34-inoculated wheat seedlings with chlorotic symptoms (Figure S4B).. - At this timepoint, the BSMV-VIGS-TaCPK34- inoculated wheat plants exhibited visual drought- induced damage symptoms such as leaf rolling and wilt- ing, whereas the BSMV-VIGS-GFP-inoculated wheat seedlings remained vigorous (Fig. - Coincident with these qualitative phenotypic changes, plant biomasses and relative water contents in BSMV-VIGS-TaCPK34- inoculated wheat plants were significantly lower than those in BSMV-VIGS-GFP-inoculated wheat seedlings (Fig. - The iTRAQ-based quantitative proteomics was employed to identify differences in the leaf protein pro- files between the BSMV-VIGS-TaCPK34- and the. - 4 Function of the TaCPK34 gene in response to drought stress. - a, Phenotypes of BSMV-VIGS-TaCPK34- or BSMV-VIGS-GFP-inoculated wheat seedlings are observed at 14 days after drought stress. - Biomass of dry weight (b), relative water content (c), soluble sugar content (d), and MDA content (e) of leaves in BSMV-VIGS-TaCPK34-inoculated wheat seedlings and BSMV-VIGS-GFP-inoculated wheat seedlings are determined at 14 days after drought stress. - BSMV-VIGS-GFP-inoculated wheat seedlings at 14 days after drought stress. - Ratios were used to assess the fold changes in the abundance of proteins between the BSMV-VIGS-TaCPK34- and BSMV-VIGS-GFP-inocu- lated wheat plants. - 0.05) were identified between BSMV-VIGS-TaCPK34- and BSMV-VIGS- GFP-inoculated wheat seedlings suffering from the above drought stress (Table 1).. - The promoters of the genes encoding the above function-known proteins were retrieved from the Inter- national Wheat Genome Sequencing Consortium (IWGSC) database (RefSeq 1.0 version) from Ensembl- plants (http://plants.ensembl.org/index.html). - 5 Volcano plot (a) and functional categorization (b) of the identified proteins based on iTRAQ proteomics. - The iTRAQ proteomics is performed on leaves of BSMV-VIGS-TaCPK34- or BSMV-VIGS-GFP-inoculated wheat plants suffering drought stress for 14 days. - Table 1 Identification of the differentially expressed proteins in leaves of BSMV-VIGS-TaCPK34-inoculated wheat plants. - P30110.1 Glutathione S-transferase E-03 2.04E-02. - EMS51097.1 Hypothetical protein TRIUR E-03 2.75E-02. - Table 1 Identification of the differentially expressed proteins in leaves of BSMV-VIGS-TaCPK34-inoculated wheat plants (Continued). - XP Uncharacterized protein LOC E-03 4.43E-02. - XP Uncharacterized protein LOC E-03 2.67E-02. - 6 BiFC assay of the interaction between TaCPK34 and TaGST1 or TaPrx113 proteins. - Of the reported 20 wheat CPKs, only 4 (CPK1, 6, 9 and 18) have been found to be drought-responsive, but their drought tolerance has not been tested [30]. - In our previous study, the abundance of TaCPK34 protein was upregulated during the late stages of wheat grain filling period (from 6 to 31 May, 2015, corresponding to 15 ~ 35 days after anthesis) under natural field condition (There was only 25.5 mm rainfall, from 6 to 31 May, 2015, thus drought stress oc- curred), in which wheat plants suffered from various stresses including drought [17]. - Transcripts of the TaCPK34 gene were significantly induced by the PEG-. - stimulated drought stress (Fig. - BSMV-VIGS- TaCPK34- and BSMV-VIGS-GFP-inoculated wheat plants displayed a drought sensitive phenotype with inhibited growth and physiological parameters (Fig. - TaCPK34 could regulate expression of the target genes in response to drought stress. - 0.05) in BSMV-VIGS-TaCPK34-inoculated wheat plants (Table 1). - A large proportion of the un- known function proteins were not annotated (Fig. - It has been reported that several plant CPKs function in drought stress responses through direct interaction with some proteins. - In Arabidopsis , CPK8 has been found to serve as a positive regulator in plant responses to drought stress through the direct interaction with CAT3 protein to regulate its activity [4].. - OsCPK14 has been identified to interact with OsDi19–4, one member of the drought-induced 19 (Di19) transcrip- tion factor family, whose overexpressing Arabidopsis plants display enhanced drought tolerance. - Among our identi- fied proteins of known function, many proteins or their isoforms have been reported to function in drought stress and they included dehydrin [39], LEAs [40], per- oxidases [18], GST1 [19], germin-like proteins [41], low- temperature-induced 65 kDa protein [42]. - These implied that TaCPK34 could directly or indirectly regulate the expression of the genes encoding these proteins. - 6), while not with peroxid- ase, suggesting that it could directly regulate the expres- sion of the formers, while indirectly control the latter.. - ABA is the most critical phytohormone in plant drought stress responses, and several plant CPKs are in- volved in the mechanisms of drought stress response through an ABA-dependent manner [43]. - results, we propose that TaCPK34 functions in a signal transduction pathway that is involved in wheat response to drought stress (Fig. - Our data showed that transcripts of the TaCPK34 gene were significantly induced by PEG-stimulated drought stress or ABA application, and TaCPK34- silenced wheat plants exhibited more sensitive to drought stress, sug- gesting that TaCPK34 has a positive regulatory role in response to drought stress. - Further proteomics analysis and BiFC assay suggested that TaCPK34 protein medi- ated the wheat drought tolerance by directly regulate the expression of the target genes.. - 7 Schematic figure showing signal pathway of TaCPK34 in wheat response to drought stress. - Based on the TuCPK34 sequence, a pair of primers was designed to amplify the coding sequence (CDS) of the TaCPK34 gene using the cDNA from Zhoumai 18. - Transcripts of the TaCPK34 gene were measured by using quantitative real-time PCR (qPCR) method follow- ing reaction conditions: 95 °C for 5 min (1 cycle), 95 °C for 10 s, 58 ~ 60 °C for 15 s, and finally 72 °C for 20 s (40 cycles). - Relative transcripts abundance of the TaCPK34 gene was calculated using the 2 −ΔΔ Ct method with the wheat β-actin gene as the internal control. - Consequently, in this study, all three copies of the TaCPK34 gene could be silenced simultaneously with the BSMV-VIGS-TaCPK34 vector, and this helped alleviate functional complementation among the three TaCPK34 copies. - wheat cultivar Zhoumai 18 were sown in soil (nutritive soil: vermiculite with 3:2 ratio), and wheat seedlings with two fully expanded leaves and almost identical heights (12 ± 0.2 cm) were inoculated with BSMV-VIGS- TaCPK34 or BSMV-VIGS-GFP virus on the second fully expanded leaves with 10 μL transcript mixtures each leaf, respectively. - The BSMV-VIGS method (e.g., viral vector construction, viral RNA transcription, viral inoculation, and TaCPK34-silenced wheat plant measurement) were performed as described in our previous studies [54]. - Total proteins from leaves of BSMV-VIGS-TaCPK34-, or BSMV-VIGS-GFP-inoculated wheat seedlings were extracted at 14 days after water stress using the trichloroacetic acid/acetone method. - Samples were labeled with 113–116 for BSMV-VIGS-TaCPK34-inoculated wheat seedlings, and 117–119 and 121 for BSMV-VIGS- GFP-inoculated wheat seedlings. - Proteins whose abundances differed significantly by fold-changes of ≥1.5 or ≤ 0.67 between BSMV-VIGS-TaCPK34- and BSMV-VIGS-GFP-inoculated wheat plants were selected and considered to be used for further study.. - The CDS regions of the genes encoding wheat GST1, Prx113, and peroxidase identified in our proteomic data, were amplified and ligated into the BiFC vector, which contains a yellow fluorescent protein in carboxy-terminal part, to construct a YFP-C- genes (YC-gene). - Likewise, the TaCPK34 gene was also inserted into the amino-terminal part of the yellow fluorescent protein (YFPN) to form YN-TaCPK34. - The location of silenced gene-specific fragment in full-length of the TaCPK34 gene. - BSMV-VIGS-mediated silencing of the TaCPK34 gene. - The promoter sequences of the genes encoding the identified proteins with known functions.. - All peptides of the identified proteins in leaves of BSMV-VIGS-TaCPK34- or BSMV-VIGS-GFP-in- oculated wheat seedlings suffering from drought for 14 days.. - All identified proteins in leaves of BSMV-VIGS-TaCPK34- or BSMV-VIGS-GFP-inoculated wheat seedlings suffering from drought for 14 days in all four biological replicates.. - BSMV-VIGS: Barley stripe mosaic virus-induced silencing;. - CPKs: Calcium-dependent protein kinases;. - And the funding bodies were not involved in the design of the study, in the collection, analysis and interpretation of data or in writing the manuscript.. - Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling and H 2 O 2 homeostasis in stomatal guard cells under drought stress. - Calcium-dependent protein kinases in plants: evolution, expression and function. - subcellular localization and targets of calcium-dependent protein kinases. - AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis. - Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca 2+ -mediated stomatal regulation in response to drought stress. - Transgenic Arabidopsis plants expressing tomato glutathione S-transferase showed enhanced resistance to salt and drought stress. - The evolutionary history and diverse physiological roles of the grapevine calcium-dependent protein kinase gene family. - Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L. - Draft genome of the wheat A-genome progenitor Triticum urartu.. - A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. - Biotic and abiotic stress responses through calcium-dependent protein kinase (CDPK) signaling in wheat (Triticum aestivum L. - Calcium-dependent protein kinase CPK21 functions in abiotic stress response in Arabidopsis thaliana. - A rice calcium-dependent protein kinase OsCPK9 positively regulates drought stress tolerance and spikelet fertility. - Analysis of the bread wheat genome using whole-genome shotgun sequencing. - Functional analysis of the group 4 late embryogenesis abundant proteins reveals their relevance in the adaptive response during water deficit in Arabidopsis. - Low temperature-induced 30 (LTI30) positively regulates drought stress resistance in Arabidopsis: effect on abscisic acid sensitivity and hydrogen peroxide accumulation. - A role for barley calcium-dependent protein kinase CPK2a in the response to drought. - CDPK1 from ginger promotes salinity and drought stress tolerance without yield penalty by improving growth and photosynthesis in Nicotiana tabacum. - Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana. - Identification of the TaBTF3 gene in wheat (Triticum aestivum L.) and the effect of its silencing on wheat chloroplast, mitochondria and mesophyll cell development. - Proteomics reveals the effects of salicylic acid on growth and tolerance to subsequent drought stress in wheat
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt