PpCBF3 From Cold-tolerant Kentucky Bluegrass Involved In Freezing Tolerance In Transgenic Arabidopsis Thaliana

Kentucky bluegrass(Poa pratensis L.),as a common cool season turfgrass in temperate area,is one of the most cold-tolerant perennial grass species.Many researches suggest that there are several cold signaling pathways in plant.Dehydration-Responsive Element Binding proteins(DREB)1/C-repeat(CRT)Binding Factors(CBF)pathway is supposed to be the most important one.To day,DREB1/CBF is identified to response to cold stress as transcriptional activators in plant.DREB1/CBFs reguLate the downstream expression of cold-inducible genes,including cold-reguLated genes(COR)to enhance cold tolerance of plant.In this pathway,the expression of DREB1/CBF is reguLated by its upstream and downstream genes as well as itself to ensure the process to be accurate and instant.The objective of this study was to determine the physiological roles of a CBF gene isolated from a cold tolerant perennial grass species,Kentucky bluegrass(Poa pratensis L.),which designated as PpCBF3,in reguLating plant tolerance to freezing stress in the transgenic Arabidopsis.(1)A fuLl-length sequence consisted of 860 bp nucleotides with a 732 bp ORF encoding a 243-residue polypeptide was obtained from the cold-treated(4?)Kentucky bluegrass.Sequence analysis by Blastx programme showed that the gene had high similarity to DREB1/CBF in other plant species;about 81%similarity to LpCBF3 in perennial ryegrass and 64%to OsDREB1A/CBF3 in rice.A phylogenetic tree revealed that the gene is a putative ortholog of LpCBF3.In addition to an AP2 conserved domain serving a DNA-binding role,two signature sequences(PKK/RPAGRxKFxETRHP and DSAEL)also presented in PpCBF3.(2)Transient transformation of Arabidopsis thaliana mesophyll protoplast with PpCBF3-eGFP fused protein showed that PpCBF3 was localized to the nucleus.(3)qRT-PCR analysis and RT-PCR analysis demonstrated that PpCBF3 exhibited a rapid response to cold stress(4 ?),as the expression level increased within 15 min,reached to the highest level by 1 h of treatment,and maintained high expression even after 24 h of cold treatment compared to the initial non-stress level.However,PpCBF3 was not induced by drought stress[induced by 20%polyethylene glycol 6000 solution(PEG-6000)]or salt stress(150 mM NaCl).(4)To characterize the physiological functions of PpCBF3 involved in freezing tolerance,13 transgenic lines of Arabidopsis were obtained(confirmed by selecting and PCR detection)and 5 transgenic lines exhibiting desirable phenotypes and improved freezing tolerance were selected for further experiments.The percentage of chlorotic leaves was significantly lower in the transgenic lines than in WT following freezing stress(-20?).qRT-PCR and Western blot analysis showed that PpCBF3 was highly expressed in all 5 transgenic lines but not in WT.(5)Transgenic Arabidopsis overexpressing PpCBF3 showed significant improvement in freezing(-20?)tolerance demonstrated by a lower percentage of chlorotic leaves,lower celluLar electrolyte leakage(EL)and H2O2 and O2-content,and higher chlorophyll content and photochemical efficiency compared to the wild type.Relative mRNA expression level analysis by qRT-PCR indicated that the improved freezing tolerance of transgenic Arabidopsis plants overexpressing PpCBF3 was conferred by sustained activation of downstream cold responsive(COR)genes.(6)Some interesting phenotypic changes in the PpCBF3-transgenic Arabidopsis plants included late flowering and slow growth or 'dwarfism',both of which are desirable phenotypic traits for perennial turfgrasses.Therefore,PpCBF3 has potential to be used in genetic engineering for improvement of turfgrass freezing tolerance and other desirable traits.