Functional Characterization Of Cotton GhMT3a Gene And Genome-wide Analysis Of Protein Phosphatase PP2C Gene Family In Arabidopsis And Rice

Abiotic stresses such as low temperature,drought and high salt,influence plant growth, productivity and development.To survive these challenges,plants have developed elaborate mechanisms to perceive external signals and to manifest adaptive responses with the proper physiological and morphological changes.One of the most common and crucial consequences is the generation of ROS in plants,which can elicit a potentially damaging oxidative burden on cellular constituents and/or act as signals for engaging mechanisms that ameliorate oxidative stress.Although the deleterious effects of ROS have long been known,knowledge on the molecular mechanisms of ROS-mediated gene regulations is limited and whether they play different roles in plant stress response have remained largely unexplored.Cotton is one of the most important economic crops in our country.People are always interested in identifying and developing new cotton breed to make it can be widely cultivated in saline-alkali soil.Metallothioneins(MTs) are defined as a family of proteins with the characteristics of low molecular weight,high cysteine(Cys) residue content,and metal-binding ability.Plant MT proteins are distinguishable on the basis of the distribution of cysteine residues in their amino acid sequences.In animals,MTs are ubiquitous proteins associated with numerous cellular functions,including the regulation of metal homeostasis in cells and the response to metal toxicity and oxidative stress.In fungi,MTs have been proposed to be primarily involved in the response to metal toxicity or as general stress proteins.Recently,increasing numbers of reports have indicated that plant MTs may play important roles as they do in animals and fungi.In this research,a cDNA library was constructed by using mRNA isolated from salt-induced cotton seedlings and screened by differential hybridization.A cDNA clone,GhMT3a,which encodes a 64-amino acid type 3 plant MT,was isolated and characterized.Protein phosphatases(PPs),by reversing the action of protein kinases,provide modulations of protein phosphoregulation.The protein phosphatase 2Cs(PP2Cs) from various organisms have been implicated involved in diverse environmental stress responses and developmental processes.To date,a genome-wide overview of the PP2C gene family in plants is not yet available.In this study,we performed multiple BLAST algorithms to search against the corresponding data set using the known PP2C proteins got from several database as our query.Then,we eliminated the proteins that did not contain the PP2C domains after detecting by SMART and Pfam.We further compared the rice and Arabidopsis PP2C genes to identify both shared and specific subfamilies.Following the analysis of gene structure and protein motifs,we traced gene duplication events that likely contributed to the expansion of the PP2C family.The expression patterns of the PP2Cs from rice and Arabidopsis were analyzed and compared.The upstream regulatory regions of the subfamily A members were finally analyzed in order to provide insight into the transcriptional regulation machineries that define the temporal and spatial expression and the stimuli responses.The main results are as follows:1.A cDNA clone,was isolated from a NaCl-induced cotton cotyledon cDNA library by differential hybridization screening.This clone showed high homology with type3 metallothein-like genes from various plants species.Our results indicated that this gene encodes a type3 MT protein,which was designated as GhMT3a.2.Northern blot analysis indicated that the mRNA accumulation of GhMT3a was induced by salt stress(NaCl),CuSO₄,ZnSO₄,low temperature(4℃),drought(25%PEG),ABA(abscisic acid),ethylene and H₂O₂ and paraquat(PQ).Interestingly,N-Acetyl-L-cysteine(NAC) as a kind of antioxidant,could effectively reduce not only ROS accumulation but also the GhMT3a transcript level induced by salinity,drought,and cold stresses.These results suggested that GhMT3a might be regulated directly by ROS production under abiotic stress conditions.3.The transgenic tobacco plants overexpressing GhMT3a exhibited enhanced tolerance against high salinity(200 and 300mM NaCl),low temperature(4℃) and drought(25%PEG) compared with WT plants.Moreover,H₂O₂ levels in transgenic tobacco plants were only half of those in WT plants under such stress conditions.The GhMT3a-overexpressing yeast cells were less sensitive to oxidants such as PQ and H₂O₂ than the control cells.These results suggested that GhMT3a could scavenge ROS effectively in eukaryotie cells and improved stress tolerance might be due to the keep of ROS balance in tobacco by overexpressing GhMT3a.4.A recombinant GST-GhMT3a fusion protein was constructed and expressed in E.coli. The purified GST,GhMT3a,and GST-GhMT3a fusion proteins were determined in vitro for their ability to bind metal ions.The results showed that the purified GhMT3a could bind Zn²⁺ ions in vitro and the binding capacity per GhMT3a molecule would be no more than five Zn²⁺.5.We used multiple BLAST algorithms to identify 80 and 78 PP2C family genes in Arabidopsis and rice,respectively.The phylogenetic trees of PP2C gene family were generated with the neighbor-joining method.Based on the statistical support of each branch, we divided the Arabidopsis and rice PP2C family into 13 and 11 subfamilies,respectively, which are supported by the analyses of gene structures and protein motifs.We further investigated the relationship between the Arabidopsis and rice PP2C protein families by the construction of a phylogenetic tree using the full-length protein sequences of 78 OsPP2Cs and 80 AtPP2Cs.The rice and Arabidopsis genes in each subfamily are thus more closely related to each other than to the PP2C genes of the same species from a different subfamily, suggesting that an ancestral set of PP2C genes defining each subfamily already existed before the monocot-eudicot divergence.6.Based on the chromosomal location information on Internet database,we localized Arabidopsis and rice PP2C genes in chromosomes.After analyzing the PP2C genes in duplicated segmental regions,we found whole genome and chromosomal segment duplications mainly contributed to the expansion of both OsPP2Cs and AtPP2Cs.But tandem or local duplication occurred more frequently in rice.7.Eleven motifs were identified shared among related proteins within the PP2C family using the MEME motif search tool.Some motifs are widespread among PP2C proteins and the others are specific to only one or two subfamilies.The different motif distribution in the respective protein sequences may provide an anchor to study the divergence of gene function in different subfamilies.8.We examined the expression of Arabidopsis PP2C genes in different tissure using Genevestigater,massively parallel signature sequencing(MPSS) and EST data.The expression of rice PP2C genes in different tissues were also summarized using MPSS and EST data. According to expression profiles,PP2C genes can be classified into three groups.The largest group is the genes that expressed in all tissues,including 49 genes in Arabidopsis and 27 genes in rice,and the second group contains 28 AtPP2Cs and 45 OsPP2Cs whose expression were detected in the majority,but not all,of the tissues.The third group includes 3 AtPP2Cs and 6 OsPP2Cs with tissue specific expression.9.The expression of all AtPP2Cs in response to ABA treatment and several environmental stimuli were examined using the Genevestigator microarray dataset.Unlike the PP2C genes from other subfamilies,all members from subfamily A were induced by several stimuli and ABA treatment.The expression pattern of the OsPP2C subfamily A genes using RT-PCR is in good agreement with the microarray data for Arabidopsis subfamily A members,suggesting that the members of this subfamily play foremost roles in ABA-mediated processes related to stress responses both in monocots and eudicots.10.The 1000-base-pair(bp) upstream promoter sequences of the 19 PP2C genes in subfamily A in Arabidopsis and rice were analyzed using the PlantCARE database and a frequency distribution approach.The abundance of ABA responsive elements in subfamily A PP2C gene promoters provides additional evidences for their role in ABA signaling.The different motif distributions in Arabidopsis and rice point to evolutionary divergent patterns of PP2C gene regulatory networks.