Function Analysis Of PePYLs And PeCBF4a Genes From Populus Euphratica

Plants are exposed to different abiotic stress types during the life cycle.These abiotic stresses could cause water deficit to plants.And it can affect plants development and growth through gene expression and metabolism alterations.Abscisic acid(ABA)plays an important role in the plant resistance,and ABA receptor PYLs(Pyrabactin Resistance-Like)is the core element of ABA signaling.The C-repeat binding factors(CBFs)play a key role in abiotic stresses.And the gene expression of CBF4 in Arabidopsis belongs to ABA-dependent.However,little is known about the important component PYLs and CBFs of ABA signaling pathways in P.euphratica.In this paper,through the research of expression features and gene functions of P.euphratica PePYLs and PeCBF4a under abiotic stresses,and by the genetic transformation of PePYLs and PeCBF4a to Arabidopsis thaliana and poplars(Populus tomentosa 'YiXianCiZhu B385')respectively,we measured the physiological and biochemical characteristics of transgenic plants after abiotic stresses and analysis their resistance.And we could know the role of P.euphratica PePYLs and PeCBF4a in the molecular mechanism of plant stress response which is regulated by ABA signaling.The main research results are as follows:(1)The 12 of 14 Arabidopsis thaliana PYL orthologues from P.euphratica(PePYL1/3/4/6/7/8/9/10/11/12/13/14)were isolated.The GFP-PePYL3,GFP-PePYL6,GFP-PePYL9 and GFP-PePYL11 localized to both nucleus and plasma membrane while GFP-PePYL4 in addition localized to cell membrane compartments.We found that the expression of PePYL1/3/4/6/9/11 were strongly induced by ABA although only PePYL6 increased with cold stress duration and PePYL3/6/9 induced by dehydration.These gene expressions were not stimulated significantly by salt stress for PePYL1/3/6 and declining for PePYL4/9/11.The results showed that the different parts of the expression of PePYLs were regulated by ABA and related with adversity stress response.(2)Transgenic Arabidopsis overexpressing PePYL4,PePYL6,PePYL9(PePYL4-OE,PePYL6-OE,PePYL9-OE)and their complementation lines of mutants were generated.After growing in medium with 1?M ABA,the PePYL4-OE seeds were mostly inhibited for only 14.08%germnination.And the PePYL6-OE and PePYL9-OE seeds germination decreased to similar level.After growing in 1/2 medium with 5/1 0/15/20 ?M ABA,the overexpression transgenic lines showed significant reduced root length.The results showed that constitutive expression of these three PePYLs conferred hypersensitivity of ABA inhibition in seeds germination and roots growth.(3)We compared the germination rate of transgenic A.thaliana which were grown in medium with different concentrations of mannitol.The results indicated that PePYL4,PePYL6,PePYL9-expressing seeds were improved osmotic tolerant in germination assay at low but not high concentrations of mannitol.In addition,through the water control of drought stress treatments,we got the results as follows:? The mean RWC(relative water content)of overexpression transgenic lines declined 9.43%,while the Col-0 and mutants lines declined 18.47%and 18.14%,as the complementation lines declined 13.29%;? When compared to the Col-0 and mutant plants,the mean Pn(photosynthetic rate)of PePYLs-OE and complementation lines increased 33.29%,the mean iWUE(instantaneous water use efficiency)increased 92.77%under normal conditions.But under drought stress condition,the mean Pn increased 75.74%and the mean iWUE increased 217.21%;? Proline contents in transgenic lines were almost 1.43-fold higher than the mean of the mutants and complementary lines,and 1.27-fold higher than the Col-0 plants.These results confirmed that PePYL4-OE,PePYL6-OE,PePYL9-OE exhibited tolerance to stress,indicating that PePYL4,PePYL6,PePYL9 may play a positive role in ABA mediated stress responses.(4)The PeCBF4a was isolated and its expression was induced by ABA,dehydration,low temperature and salinity.In order to study the founction of PeCBF4a gene,we generated transgenic poplars overexpressing PeCBF4a(PeCBF4a-OE)under the control of the CaMV 35S promoter or transformed with empty vector.The wild-type(WT)and empty vector lines were used as controls.After the dehydration,salinity and low temperature stress treatments,we got the results as follows:? the Pn of PeCBF4a-OE lines increased 34.7-165.7%and the iWUE increased 48.9-103.7%over controls;?The maximum quantum efficiency of PSII photochemistry(Fv/Fm)values in PeCBF4a-overexpressing lines did not change significantly before and after the stresses treatments,and were 2.14-5.89%higher.The non-photochemical quenching coefficient(NPQ)mean of PeCBF4a-OE lines decreased by 12.02-23.64%while the photochemical quenching(qP)value was 8.75-22.31%higher than controls;? PeCBF4a-OE lines also displayed higher superoxide dismutase(SOD)activities and markedly lower malondialdehyde(MDA)levels compared to controls;? Higher levels of proline and sugars accumulated in transgenic plants;? Overexpression of PeCBF4a induced strong expression of the stress-responsive downstream target genes of PeCBF4a,PtRCI2A(rare-cold-inducible 2A)and PtDI21(drought-induced 21).The PtRCI2A and PtDI21 expression levels were elevated markedly for 3.5-25.2 fold and 4.6-61.2 fold,respectively.In the normal culture condition,the PeCBF4a caused dwarfed phenotypes of transgenic poplar.And by the results of P-V curve measurements,we found that PeCBF4a-OE plants showed lower water contents and osmotic water contents at the turgor loss point than control lines,the osmotic adjustment capability of PeCBF4a-OE plants was enhanced.These results confirmed that PeCBF4a-OE poplars exhibited higher tolerance to stress,indicating that PeCBF4a may play a positive role in stress toleranceThrough the genetic transformation and stress tolerance analysis of Arabidopsis and poplars in this article,the PePYLs orthologues of Arabidopsis thaliana ABA receptor and the PeCBF4a transcription factor,which is downstream of the stress response by ABA signal regulation,had been researched.We found that the different parts of the expression of PePYLs were regulated by ABA and PePYLs-OE plants exhibited tolerance to drought stress.The expression of the PeCBF4a was induced by abiotic stress and PeCBF4a-OE poplars exhibited higher tolerance to dehydration,low temperature and salinity stress.The PePYL4/6/9 and PeCBF4a genes may play positive roles in stress tolerance that is adjusted by the ABA transduction pathway.These results provided the foundation for the usefulness of PePYLs and PeCBF4a,and for the research of plant stress response molecular mechanism which is regulated by the ABA signaling.