Changes In Polyamine Contents In Citrus And Its Closely Related Species Under Abiotic Stresses And Isolation, Characterization Of Two Polyamine

Citrus industry usually encounters a series of biotic and abiotic stresses.There are some superior rootstocks germplasms in citrus relative species.Polyamines are a type of plant growth regulators,which have important effects on stress response and morphogenesis.It is important to improve the rootstocks,obtain potential genes that can be used in genetic transformation and produce stress-tolerant germplasms.In this study, the effects of exogenous putrescine on cold tolerance of Poncirus trifoliata were examined and the polyamine contents in different tolerant(cold and salinity) cultivars were investigated using HPLC.Two polyamine biosythetic genes,S-adenosylmethionine decarboxylase(SAMDC) and arginine decarboxylase(ADC),were isolated and identified by molecular biology and biochemical techniques,such as Southern blot,RT-PCR,Real time RT-PCR,Northern blot and enzyme assay.By gene manipulation and genetic transformation,arginine decarboxylase gene was transformed into the mutant of Arabidopsis and citrus to confirm its function.The results were as follows:ADC pathway was involved in the putrescine biosynthesis in Poncirus trifoliata under low temperature stress,low doses of exogenous putrescine alleviated the stress-derived damage but the high doses of exogenous putrescine produced toxicity.Our result revealed that less than 1 mM putrescine could be used to protect citrus from freezing.Total polyamines contents,especially putrescine,were accumulated in tolerant cultivar and decreased in susceptible one under low temperature.Total polyamines were increased under cold acclimation,in particular,putrescine and spermine.Spermidine was enhanced in salt tolerant cultivar and total polyamines were reduced,while spermidine and spermine exhibited the most profound decrement in the susceptible cultivar.RT-PCR in combination with bioinformatic approach produced a SAMDC cDNA (CsSAMDC) from Newhall navel orange(Citrus sinensis Osbeck) which contained an intact open reading frame of 1095 bp.The deduced amino acids shared high sequence homology with SAMDCs in other plants and possessed several highly conserved amino acid residues or motifs.Phylogenetic analysis indicated the gene function was close to those in grape and apple,Prokaryotic expression of CsSAMDC in E.coli gave rise to protein of expected size,which had biochemical activity via enzyme analysis.The gene may be an allelic gene with little variation in the locus in citrus by Southern blot,Real time PCR assay revealed spatial difference of CsSAMDC mRNA in various tissues. Expression pattern of CsSAMDC in Newhall callus largely matched the growth curve and fluctuation of endogenous polyamines.Transcription levels of CsSAMDC in Newhall shoots were enhanced by 10%polyethylene glycol.In addition,steady state mRNA of CsSAMDC was disparate in genotypes with contrasting tolerance to salinity or low temperature under corresponding stresses,as revealed by earlier induction at the initial stage of stress treatment in the tolerant ones.All of these suggest that CsSAMDC is a stress-responsive gene and that the temporally different expression profile may be one of the mechanisms responsible for genotypic variation of stress tolerance.An ADC gene was isolated from Poncirus trifoliata(PtADC).The deduced amino acids possessed two highly conserved motifs.Phylogenetic tree showed PtADC was closely related to Solanaceae ADCs.PtADC was a single copy gene in Poncirus trifoliata. PtADC was expressed in almost all of tested organs(leaf,flower bud and fruit).PtADC expression was correlated with cell growth and stress responses to chilling and dehydration.Upon exposure to freezing and salinity stresses,the levels of PtADC transcripts were induced in the resistant cultivar but inhibited in the susceptible one. Transformantion of PtADC in adc1-1 Arabidopsis mutant gave rise to transgenic plants (adc1-1-OE),adc1-1-OE line was more tolerant to osmotic stress in seed germination and exhibited enhanced tolerance to freezing and drought stresses in plant growth.Root length of the adc1-1-OE line was increased,and leaf stomatal density was reduced.In addition, the adc1-1-OE line showed a late flowering phenotype compared with the adc1-1 mutation.Overexpression PtADC in citrus leaded to dwarfism,reduced leaf stomatal density and showed potential resistance to citrus canker.