| Various abiotic stresses including drought,salt and extreme temperatures exert harmful effects on plants during their growth and development processes,and result in the growth retardation and crop yield reduction.Identification and functional characterizations of stress resistance-related genes and analyses of the physiological mechanisms of these genes are of great importance in determing the abiotic stress-related signaling pathways and genetic breeding.Brachypodium distachyon is emerging as a monocotyledonous plant model due to its closer relationship with common wheat(Triticum aestivum)and completion of its genome sequencing in 2010.In the present study,a total of eight Bd14-3-3 genes were cloned from B.distachyon.To investigate the biological functions of Bd14-3-3s,the expression profiles of these Bd14-3-3s in different organs of B.distachyon plants were analyzed.The qRT-PCR analyses were performed to determine the expression patterns of the Bd14-3-3 gene family under different abiotic stress conditions and the signaling molecules.The AREB/ABF genes involved in ABA signaling were cloned to verify the interactions between BdAREB/ABF TFs and the Bd14-3-3 proteins.BdGF14d and BdGF14g were selected for abiotic stress tolerance study by overexpressing them in transgenic tobacco(Nicotiana tabacum)plants,respectively and the functional mechanisms of the genes were analyzed.To determine the subcellular localizations of the BdGF14d and BdGF14g proteins,the leaf epidermal cells of tobacco plants were injected with Agrobacterium tumefaciens that contained the recombinant plasmids pBI121-BdGF14d-GFP and pBI121-BdGF14g-GFP,respectively.The contents and the main results of the present study are as follows:1)A total of eight Bd14-3-3 genes were identified and cloned from B.distachyon through bioinformatics analysis,they were designated as BdF14a,BdGF14b,BdGF14c1,BdGF14c2,BdGF14d,BdGF14e,BdGF14f,and BdGF14g,respectively.A phylogenetic analysis against Arabidopsis and rice was performed,and the results indicated that the Bd14-3-3 family members were highly conserved during the evolutionary process.To study the biological functions of Bd14-3-3s,the expression profiles of these Bd14-3-3s in the roots,stems,leaves,and spikelets of 3-month-old plants of B.distachyon were investigated.The results indicated that most members of this gene family except BdGF14g had relatively lower expression levels in the roots than in the leaves.Besides these differences,all the Bd14-3-3 genes exhibited global expression in the stems,leaves and spikelets.The qRT-PCR analyses were performed to determine the expression patterns of the 14-3-3 gene family under PEG6000,NaCl and H2O2 stresses,and the signaling molecules ABA treatments.The results revealed that only BdGF14d showed distinct up-regulation in response to the NaCl treatment,whereas the other genes exhibited different degrees of down-regulation.Under the PEG treatment,BdGF14e was up-regulated;however,BdGF14f and BdGF14g were up-regulated at 3 h after 20%PEG6000 was added.In response to H2O2,BdGF14b showed evidence of up-regulation,whereas BdGF14cl and BdGF14d exhibited down-regulation;the other genes showed moderate variation.Under the ABA treatment,BdGF14e and BdGF14f showed evidence of up-regulation,and BdF14b and BdGF14g were up-regulated rapidly at 1 h after treatment,but were then down-regulated at 12 h;the other genes exhibited no significant variation in response to ABA treatment.2)To determine the subcellular localizations of the BdGF14d and BdGF14g,the leaf epidermal cells of tobacco plants were injected with A.tumefaciens that contained the recombinant plasmid pBI121-BdGF14d-GFP and pBI121-BdGF14g-GFP,respectively.The results showed that both of the BdGF14d and BdGF14g proteins were localized throughout the cells.3)The AREB/ABF genes involved in ABA signaling were cloned to verify the interactions between BdAREB/ABF TFs and the Bd14-3-3 proteins.The results demonstrated that all the Bd14-3-3s showed interactions with BdAREB/ABF TFs,except for BdGF14c2 which was self-activated.Among these,all the Bd14-3-3 family members were able to interact with BdbZIP62,but they interacted with neither BdbZIP56-2 nor BdFDL36.BdGF14c1 and BdGF14e exhibited the same interaction mode,in that both of them interacted with BdAREB/ABF TFs BdbZIP56-1,BdbZIP71,BdbZIP16,BdbZIP62,BdbZIP41 and BdFDL2;BdGF14d interacted with only two BdAREB/ABF TF members BdbZIP71,BdbZIP62;The BdGF14a interacted with BdbZIP56-1,BdbZIP16,BdbZIP62 and BdbZIP41 BdAREB/ABF TFs;the BdGF14b protein interacted with BdbZIP56-1,BdbZIP71,BdbZIP62 BdAREB/ABF TFs;the BdGF14f interacted with BdbZIP71,BdbZIP 16,BdbZIP62,BdFDL2 and BdbZIP41 proteins;the BdGF14g protein showed interactions with BdbZIP16,BdbZIP62,BdbZIP41 BdAREB/ABF TFs.As signal transduction in plants always occurs via protein-protein interactions,these results suggested that the Bd14-3-3 proteins likely participated in the ABA-mediated signaling pathway by interacting with the BdAREB/ABF TF members.4)BdGF14d was selected for a salt tolerance study by overexpressing it in transgenic tobacco plants.The BdGF14d-oevexpression lines developed distinctly longer roots than WT under NaCl treatment.The 5-week-old seedlings of the three BdGF14d-overexpression,WT and VC lines were treated with NaCl for 35 days in soil and significantly higher survival rates than WT and VC were observed.Moreover,the leaves of transgenic seedlings maintained their growing status,whereas those of the WT and VC plants had already wilted or died.To investigate the salt tolerance mechanism in the BdGF14d-overexpression seedlings in physiological terms,the related indices of MDA,Rec%,H2O2,CAT,POD and Na+ were determined in transgenic lines and control plants after NaCl treatment or under normal growing conditions.The results showed that the BdGF14d-overexpression plants had obviously lower contents of MDA,H2O2,Na+,lower Rec%and higher activities of CAT and POD after the NaCl treatment.Under NaCl and ABA treatments,the degree of stomatal closure in leaves was significantly greater in the BdGF14d-overexpression plants than in the WT plants.Three ABA signaling pathway-related genes,NtNCEDl,NtABF2,TobLTP1,the ROS-scavenging genes NtCAT,NtPOX2,the plasma membrane Na+/H+ antiporter gene NtSOSl and tonoplast Na+/H+ antiporters genes,NtNHX2,NtNHX4 were all significantly up-regulated under salt stress in transgenic lines,relative to WT.5)BdGF14g was selected for a drought tolerance study by overexpressing it in transgenic tobacco plants.The BdGF14g-overexpression lines developed distinctly longer roots than WT under mannitol treatments.The 5-week-old seedlings of the three BdGF14g-overexpression and WT,VC lines were treated with dehydration for 25 days and recovery for 7 days in soil and significantly higher survival rates than WT and VC were observed.Moreover,the leaves of transgenic seedlings maintained their growing status,whereas those of the WT and VC plants had already wilted or died.To investigate the drought tolerance mechanism in the BdGF14g-overexpression seedlings in physiological terms,the related indices of MDA,IL,H2O2,CAT,POD,SOD,RWC and the content of endogenous ABA were determined in transgenic lines and control plants after dehydration treatment or under normal growing conditions.The results showed that the BdGF14g-overexpression plants had obviously lower contents of MDA,H2O2,lower IL,higher contents of RWC,endogenous ABA and higher activities of CAT,POD,and SOD after the dehydration treatment.Under dehydration and ABA treatments,the degree of stomatal closure in leaves was significantly greater in the BdGF14g-overexpression plants than in the WT plants.The ABA signaling pathway-related genes,NtNCEDl and NtABF2 were both significantly up-regulated under dehydration stress in transgenic lines than in WT.The activities of antioxidant enzyme system CAT and SOD of BdGF14g-overexpression lines showed no distinct differences with WT under the mannitol treatment adding endogenous ABA inhibitor. |
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