Isolation, Location And Functional Analysis Of Drought-response Gene TaPP2Ac/a From Wheat (Triticum Aestivum L.)

Drought stress is one of environmental factors that greatly impact plant distribution as well as crop production. An effective approach to increase the crop production is to utilize drought-tolerant crops. Using molecular genetics and genetic engineering to obtain the drought-tolerant crops has been one of the most important approaches in modem agriculture. Therefore, understand of the cellular and molecular basis of plant responses to drought stress will be greatly important for genetically improving the drought tolerance of crops.To further understand how plant responds to drought stress, we have constructed the cDNA library responsed to drought-stress from wheat seedlings using suppressive subtractive hybridization (SSH), with the most drought tolerant wheat cultivar Hanxuanl0. Two ESTs encoding protein phosphatase 2A(PP2A) subunits were screened from the cDNA library. Based on the EST sequences, we cloned the catalytic subunit genes and structural subunit genes of wheat protein phosphatase 2A(PP2A) and characterized their functions in drought stress response. Our results are mainly as follows:1. Two cDNAs encoding catalytic subunit of PP2A, were cloned from wheat and designated as TaPP2Ac-1-2 and TaPP2Ac-1-3, respectively. Both TaPP2Ac-1-2 and TaPP2Ac-1-3 contain 314 amino acids, a molecular mass of about 36 kDa and pI value of about 5.03, in which there is an okadaic acid binding motif of PP2Ac. Alignment and phylogenetic analyses indicated that TaPP2Ac-1 was highly homologous to rice OsPP2A-4. The TaPP2Ac-1 mRNAs were induced by high salt, low temperature and exogenous ABA treatments, suggesting that TaPP2Ac-1 is involved in the drought response in wheat. Southern hybridization demonstrated that there is single copy of TaPP2Ac-1 in each of the three wheat genomes, and three loci (TaPP2Ac-1-1, TaPP2Ac-1-2 and TaPP2Ac-1-3) were mapped on chromosome 4A, 4B and 4D with nullisomics-tetrasomics, respectively, among which TaPP2Ac-1-3 was located between Xbcdl5-4D and Xfbb178-4D on the 4BL, 6.9 cM and 6.2 cM from them. TaPP2Ac-1-3 was chosen as a representation for further functional analysis. Transient expression of TaPP2Ac-1-3: GFP fusion protein was mainly found in the nucleus and cytoplasm of onion epidermis cells, and the recombinant TaPP2Ac-I-3 in Escherichia coli is a functional serine/threonine phosphatase. Compared to the wild type, transgenic tobacco plants over-expressing TaPP2Ac-1-3 exhibited stronger drought tolerance, as also shown higher capacity to maintain leaf relative water content, leaf cell membrane stability index, water retention ability and water use efficiency under stress condition. Homozygous transgenic Arabidopsis plants also displayed more tolerant to abiotic stress, including drought, salt, mannitol and other osmotic stresses, than wild type at different developmental stages.2. Two PP2A structural subunit genes, TaPP2Aa-1-1 and TaPP2Aa-1-2 were then cloned and characterized. There was only 12 bp insertion/deletion difference between TaPP2Aa-1-1 and TaPP2Aa-1-2 cDNA. TaPP2Aa-1 protein, which is high identity to rice OsPP2Aa (Q9S7CS), contained a motif of 15 imperfect repeat units with 39 amino acids each. The expression of TaPP2Aa-1 was up-regulated by PEC, NaCl, cold and ABA treatments, implying that TaPP2Aa-1 may mediates different signals. TaPP2Aa-1 was also mainly localized in the nucleus and cytoplasm in onion epidermics cells. The over-expression of recombinant TaPP2Aa-1 in E. coli conferred the cell an improved tolerance to various osmotic stresses. Transgenic tobacco plants over-expressing TaPP2Aa-1 grew better and exhibited less wilting than wild type plants under drought stress condition, and RWC, MSI, WRA and WUE of the leaves of transgenic TaPP2Aa-1 plants were apparently increased.All these results about the catalytic and structural subunit of PP2A demonstrated that the TaPP2A play a role in plant drought and osmotic response, and their expression level could influence the plant tolerance to such stresses. Therefore, our findings might provide the valuable application potentials of PP2A genes to improve the crop tolerance to drought stress.