Functional Analysis Of The Cystatin Genes In Response To Abiotic Stresses In Malus

Cystatins are a well-characterized class of naturally occurring protease inhibitors that function by preventing the catalysis of cysteine proteases.They have been identified in numerous plants,including Arabidopsis,rice,barley,soybean,potato,and wheat.However,information about the cystatin gene family is limited in apple.Here,we performed the first genome-wide identification of the cystatin gene family within Malus.Then,we investigated their gene classification,chromosomal locations,multiple sequence alignment,phylogeny,and exon/intron organization.Transcription patterns for eight selected genes were monitored in several tissues of M.prunifolia and in response to drought,low temperature,heat,abscisic acid(ABA)and oxidative stress.Then,we focused on evaluation of the biological functions of MpCYS2,MpCYS4 and MpCYS5.The main results obtained are listed as follows:1.We identified 26 cystatin genes within the entire apple genome.They were clustered into three distinct groups distributed across several chromosomes.All of their putative proteins contained one or two typical cystatin domains,which shared the characteristic motifs of cystatin.Phylogenetic analysis revealed that all tested species could cluster into four distinct groups--A,B,C,and D.The 26 apple cystatin genes were distributed over Groups A,B,and C.Eight selected genes displayed differential expression patterns in various tissues.Moreover,their transcript levels were also up-regulated significantly in leaves during maturation,senescence or in response to treatment with one or more abiotic stresses.Our results indicated that members of this family may function in tissue development,leaf senescence,and adaptation to adverse environments in apple.Then,we designed specific primers to clone five of them using the mature leaves of M.prunifolia(MpCYS1,MpCYS2,MpCYS3,MpCYS4 and MpCYS5).All cloned sequences shared the highly conserved characteristic motifs of the cystatins.2.MpCYS2 expression canbe induced by water-deficit,methyl viologen,or exogenous ABA treatments.This gene localized to the nucleus,cytoplasm,and plasma membranein onion epidermal cells.Controlled by the 35 S promoter,its ectopic expression in transgenic Arabidopsis linescaused accelerated seed germination and greater seedlinggrowth when plants were exposed to osmotic or oxidativestress.Expression by this gene was also associated withenhanced drought tolerance in those transgenics.Thispositive response was manifested by changes measured inelectrolyte leakage,the chlorophyll concentration,andmalondialdehyde accumulations.Production of reactiveoxygen species(ROS)was appreciably decreased in thedehydration-treated transgenic lines.This gene also influencedroot hair development under osmotic-stress conditions.Our findings indicate that MpCYS2 affects the growthand tolerance of drought-stressed Arabidopsis plants possiblybecause of its influence on ROS accumulation androot hair formation.3.MpCYS4 wasactivated under water deficit,heat,exogenous ABA,or methyl viologen.It alsoup-regulated significantly in leaves during senescence.At cellular level,MpCYS4 protein was found tobe localized in the nucleus,cytoplasm,and plasma membrane of onion epidermalcells.Recombinant MpCYS4 cystatin expressed in Escherichia coli was purified and it exhibited cysteine protease inhibitor activity.Transgenic overexpression of MpCYS4 in Arabidopsis and apple led to ABA hypersensitivity and series of ABA-associated phenotypes,such as enhanced ABA-induced stomatal closing,altered expression of many ABA/stress-responsive genes,and enhanced drought tolerance.These results demonstrate that MpCYS4 is involved in ABA-mediated stress signal transduction and confers droughttolerance at least in part by enhancing stomatal closure and up-regulating thetranscriptional levels of ABA-and drought-related genes.4.MpCYS4 alsoeffects on leaf photosynthesis and leaf protein contents andcompositions when plants encounter natural or stress-induced senescence.Overexpression of this gene in apple rootstock M26 effectively slowed the senescence-related declines in photosynthetic activity and chlorophyll concentrations and prevented cysteine proteinases action during the process of protein degradation(e.g.Rubisco)in senescing leaves.Moreover,MpCYS4 alleviated the associated oxidative injury and enhanced the capacity of reactive oxygen species elimination via activating antioxidant enzymes such asascorbate peroxidase,peroxidase,and catalase,thereby protecting plant cells against free radical damage during leaf senescence.Thus,based on these results,we conclude that MpCYS4 functions both in improving stress tolerance of plants and in delaying natural and stress-induced senescence of apple leaves.5.A yeast two-hybrid assay to screen proteins demonstrated that MpCYS4 can interact with MpFER,a putative apple ortholog of the ArabidopsisFERONIA(FER)receptor-like kinase.This interaction was confirmed by bimolecular fluorescence complementation assays(BiFC).Furthermore,MpCYS4 suppressed the kinase activity of MpFER in vitro.At cellular level,MpFER protein was found to be localized in the plasma membrane of onion epidermal cells.Its gene expression was also effected by water deficit and exogenous ABA treatment.MpFER in the fer-4 mutant background led to a strong level of rescue with the ABA-hypersensitive phenotype of those mutants.Thus,we hypothesize that MpCYS4 functions in apple drought stress responses at least in part by its interaction with MpFER.6.MpCYS5 expression was typically inducedby salt stress treatment.Using biochemical analysis with recombinantMpCYS5 protein,we found that the proteinase activity of papain was inhibited.MpCYS5 ectopicexpression inArabidopsisenhanced salt tolerance.Physiological parametersconfirmedthis phenotype,with the transgenicshaving remarkably lower electrolyte leakagevalues,higher chlorophyll concentrations,and lowerlevels of malondialdehyde upon salt treatment.Furthermore,the accumulation of ROS was markedly regulated by MpCYS5 under stress conditions,as shown by fluctuations in the concentrations ofhydrogen peroxide and superoxide radicals and the activitiesof antioxidant enzymes.We also noted that thisgene modulated tunicamycin-induced endoplasmicreticulum stress tolerance and functioned in theunfolded protein response(UPR)-signaling pathway inArabidopsis.This was confirmed by the expression of eight UPR-responsive genes.All marker genes examined were strongly induced in the wild type,while most of them maintained relatively stable over time in the transgenics.These results demonstrated that ectopic expression of MpCYS5 gene is associated with salt-tolerant and tunicamycin-tolerant phenotypes.