Molecular Cloning And Functional Characterization Of An Apple Phytochrome-Interacting Factor Gene MdPIF1

Apple (Malus×domestica B.) is one of the most important fruit tree crops in the world. Assessile organisms, apple trees must subtly integrate various environmental signals foroptimization of growth and development. Among these signals, light plays a prominent rolenot only as an energy source, but also as a key environmental signal regulating variousaspects of the plant life cycle. MdPIF1gene was isolated depending on its differentialexpression in seeds germinated response in apple (Malus×domestic cv.‘Royal Gala’). Tocharacterize the function of MdPIF1gene, MdPIF1gene was introduced into apple callus andArabidopsis with Agrobacterium-mediated transformation.1. Cloning of MdPIF1Within the publicly available apple GenBank (http：//www.ncbi.nlm.nih.gov), there is a ESTshowing high similarity in BLAST searches to Arabidopsis PIF1/PIL5. Subsequently, its fulllength cDNA was cloned and hereafter named MdPIF1(GenBank accession numberMDC011443.241).2. Sequence analysis of MdPIF1MdPIF1ORF was1894bp in length. Sequence analysis showed that8introns were insertedinto the open reading frame (ORF) of MdPIF1. Programs MEGA4.0were used to constructphylogenetic tree for the bHLH proteins. All arabidopsis bHLH proteins and MdPIF1wereincluded and aligned. The result showed that MdPIF1protein was clustered within the sameclade featured with AtbHLH015(AtPIF1/PIL5). In addition, comparison of genomic structureshowed that both MdPIF1and AtPIF1genes contained nine exons, suggesting their similargenomic arrangement.3. Expression of MdPIF1in different tissues and in response to various stressesThe MdPIF1transcript levels were analyzed in different tissues by semi-quantitative RT-PCR.The result showed that MdPIF1transcripts constitutively expressed at different levels invarious tissues tested such as spring shoot, dormant bud, floral bud, flower, young leaf,mature leaf, young fruit and fruit skin. To examine if MdPIF1is induced by some stresses, itsexpression was analyzed with semi-quantitative RT-PCRs under various abiotic stresses. Theresults showed thatMdPIF1expression was not induced by low temperature, high salinity,PEG and ABA treatments.4. Expression of MdPIF1in germinated seeds and dormancy bud of applesMdPIF1transcript levels were analyzed with semi-quantitative RT-PCRs in apple seedsstratified for different times and buds during dormancy releasing. The results showed that MdPIF1expression was downregulated with stratification. However, MdPIF1transcript levelincreased with dormancy breaking.5. Overexpression of MdPIF1in Arabidopsis and apple callusTo further characterize the functions in planta, the construct containing MdPIF1driven by35S promoter was genetically transformed into Arabidopsis and apple callus, respectively.MdPIF1overexpression exhibited the growth and development associated with red-lightphotomorphogenesis both in transgenic Arabidopsis and apple callussuggesting MdPIF1is akey negative regulator for red light-mediated growth and development in plants.