Functional Characterization Of Wheat TaGF14b And BdGF14a Genes In Response To Drought And Salt Stresses

Environmental stresses such as drought,salinity and extreme temperatures adversely affect plant growth and development processes,especially in cultivating staple cereal crops,and result in crop yield loss.Identification and functional characterizations of stress-related genes aiming at clarifying the molecular mechanisms plants against abiotic stresses are of great value in crop breeding and genetic improvemen.The 14-3-3 protein,a kind of crucial adapter protein,is well known as one of the phosphopeptide-binding proteins and plays significant roles in light signal transduction,plant hormone regulation and plant growth and development.While,there is few reports about functional analyses of 14-3-3 in allopolyploid wheat(Triticum aestivum L.),a worldwide staple crop,of which the yield sufers great loss due to various environmental stresses;and Brachypodium distachyon,an emerging monocotyledonous plant model due to its closer relationship with common wheat and completion of its genome sequencing.In the present study,we identified and cloned the 14-3-3 family genes from wheat.Based on the gene expressions of Ta14-3-3s,Ta GF14 b was selected for further functional analysis.Then,Bd GF14 a a homolog of Ta GF14 b in B.distachyon,was characterized.The main results are as follows:(1)A total of 13 Ta14-3-3s(1 belongs to ? group;12 belong to no-? group)were identified from wheat genome.Phylogenetic analysis indicated that the Ta14-3-3s were highly conserved during evolution.The different expression patterns of these Ta14-3-3s indicate that the Ta14-3-3s might participate in different abiotic stresses and signal pathways.(2)Ta GF14 b was expressed in all assayed organs at varying levels.Ta GF14 b could be induced both in leaves and roots by Na Cl,PEG6000,H2O2 as well as ABA treatments,and the relatively higher upregulated expressions were observed in roots than in leaves.While under Na Cl and PEG treatments with addition of furidone(an ABA biosynthesis inhibitor)or DPI(a H2O2 scavenger),upregulations of Ta GF14 b was suppressed,implying that the response of Ta GF14 b to salt and drought treatments involved ABA and H2O2 signaling.(3)Ectopic expression of Ta GF14 b in tobacco conferred enhanced tolerance to drought and salt stresses.Transgenic tobacco had longer root,better growth status,and higher relative water content,survival rate,photosynthetic rate,and water use efciency than control plants under drought and salt stresses.Ta GF14 b expression could signifcantly enhance the reactive oxygen species(ROS)scavenging system to ameliorate oxidative damage to cells.In addition,Ta GF14 b increased tolerance to osmotic stress evoked by drought and salinity through modifying water conservation and compatible osmolytes in plants.Moreover,the contribution of Ta GF14 b to drought and salt tolerance also relies on the regulations of ABA biosynthesis and ABA signaling,as well as stomatal closure and stress-related gene expressions.The above results indicate that Ta GF14 b enhances tolerance to multiple abiotic stresses through the ABA signaling pathway in transgenic tobaccos by altering physiological and biochemical processes.(4)Bd GF14 a is a homologous gene of Ta GF14 b in B.distachyon,designated for its highest homology with Hv GF14a(99%).The transcript level of Bd GF14 a could be upregulated by Na Cl,H2O2 and ABA treatments.Transient expression of GFP in onion epidermal cells illustrated that Bd GF14 a was located in the whole onion cell,which was the same as that of Ta GF14 b.(5)Expression of Bd GF14 a improved abiotic stress(salt and drought)tolerances in tobaccos as proved with higher survival rate and longer root length under these stresses.Physiological indexes analyses showed that expression of Bd GF14 a enhanced cell membrane stability with the lower ion leakage and MDA,more accumulation of soluble sugar and proline,as well as the regulation of ion transporters;and strengthened the tolerance to oxidative stress(induced by salt and drought stresses)with lower H2O2 content,lighter DAB and NBT staining,higher antioxidant enzyme activities and chlorophyll content under stress conditions.Moreover,addition of exogenous ABA resulted in lower stomatal aperture and shorter root of Bd GF14a-transgenic lines,illustrating that Bd GF14a-expressing tobaccos were more sensitive to exogenous ABA comparing to control lines.The expressions of crucial ABA biosynthesis gene and ABA signaling-related genes were enhanced under abiotic stresses.Additionally,the expressions of stress-related genes were also higher than that of WT,and most of the upregulations could be suppressed when ABA biosynthesis inhibitor was added under drought and salt conditions.Our studies suggest that Bd GF14 a functions as a positive regulator of salt and drought stresses response via ABA dependent signaling pathway.