Quantitative Proteomic Study On Anti-chilling Mechanisms In Apple Fruit During Cold Storage And Anthocyanin Degradation In Plants

In recent years,based on the continuous improvement of plant genomics,proteomics has become one of the most important research methods for efficient and rapid screening of key proteins in plant physiological research.Based on the advantages of proteomics,we studied the anti-chilling mechanism in apple(Malus domestica)in response to chilling injury and the degradation of plant anthocyanins.Apple is a typical climacteric fruit,and severe chilling injury often happens during the cold storage and result in significantly reduced fruit quality and taste.To date,apple chilling injury was studied through physiological and biochemical level and the precaution,but the specific antichilling mechanisms remain unclear.In this paper,proteomics techniques were used to analyze the proteomic changes between chilling damaged and non-damaged fruits in terms of storage methods,different cultivars and rootstocks.The possible anti-chilling mechanisms were analyzed at protein level.Many vegetables and fruits including apples as well as many ornamental plants show rapid anthocyanin degradation during development or under pests/diseases attack or the changing of environment,which severely affect the appearance of these horticulture products.So far,there is detailed molecular information available on anthocyanin synthesis,while there is little information on the stability and catabolism of anthocyanins in plants.In this paper,taking advantages of the detailed genomic information in Arabidopsis thaliana(At),we first analyzed the proteome change during anthocyanin degradation in At seedlings after blue light treatment,after which,great amount of anthocyanin in the seedlings was induced.Furthermore,(Brunfelsia calycina Benth.)flower petals and the leaves of Osmanthus fragrans var.Semperflorens were used to study the rapid anthocyanin degradation while(Rosa chinensis Jacques)flower petals and the leaves of(Excoecaria cochinchinensis Lour)were used to study the maintenance of anthocyanin in vivo.The main results were outlined as follows:1.Proteomic changes in ‘Ambrosia’ apple fruit during cold storage in response to delayed cooling treatmentScald susceptible ’Ambrosia’ apples were stored under 20 ℃ as a warm environment for 7 days after harvest and then moved to 0 ~ 1 ℃ for 1 month(1 M)and 3 months(3 M)storage,which was called “Delay cooling treatment(DLT)”.After storage,significantly low scald incidence was observed in the DLT fruits while compared to the control.There were 380 and 425 common proteins of the three biological repeats in 1 M and 3 M fruits respectively,while there were 16 and 36 significantly changed proteins respectively.There were 259 common proteins for the whole study.The expression of anti-chilling related proteins was mainly affected by the DLT and storage time.Membrane proteins such as ABC transporter F family member 4,O-glycosyl hydrolase,aquaporin PIP1-4 etc.were found to be involved in the anti-chilling mechanism of post-harvest apple fruits while late embryogenesis abundant protein 76,nucleoside diphosphate kinase and hsp70-Hsp90 protein were closely related to the chilling injury of the control fruit.2.Comparison of the fruit proteome expression profile between the chilling damaged and non-damaged apple cultivarsIn the Cultivar Evaluation Trial(CET),two biological repeats(CET2014 and CET2015)of fruits with different cultivars were sampled after harvesting,storing and observation.The proteome expression profile of the chilling damaged and non-damaged fruits was suscessfully constructed.2230 different proteins with 375 common proteins and 76 important significantly changed proteins were identified in CET2014 cultivars while 2318 with 228 and 57 in CET2015.Combined the two biological repeats,there were 18 key proteins that were up-regulated in almost all cultivars,and 36 were down-regulated.The main proteins that were significantly up-regulated after storage were caffeic acid Omethyltransferase etc.in secondary metobolism,ras gene-related protein Rab7 in signaling pathway,thaumatin-like protein 1a,acidic endochitinase SE2,polygalacturonase etc.Legumin A proteins,signaling proteins(14-3-3 proteins),S-adenosylmethionine-dependent methyltransferase,polygalacturonase-inhibiting protein,serpin-ZX were the main downregulated proteins.These results suggested that these proteins might play a key role in causing or preventing the chilling injury in many apple cultivars.3.Quantitative proteomic investigations on the anti-chilling mechanisms in ‘Honeycrisp’ apple fruit of different rootstocks after cold storageThe scald incidence in Geneva rootstock ’Honeycrisp’ fruit was much higher than Bud.(Budagovsky)rootstock ’Honeycrisp’,which promoted us to investigate the fruit proteome expression between the two rootstocks.977 different proteins with 555 common proteins and 57 significantly changed proteins were identified in Geneva groups while 1098 with 643 and 64 in Bud.groups.Compared with the lower scald incidence fruit of Bud.70-20-20,there were 9 significantly up-regulated proteins in Geneva groups: aquaporin PIP2-8,auxin transport protein BIG,isoeugenol synthase 1,protein ASPARTIC PROTEASE IN GUARD CELLL 1,guanine deaminase etc.while 11 significantly down-regulated proteins: quinone oxidoreductase,phospholipid hydroperoxide glutathione peroxidase 6(PhGPX6),the nutrition storage related protein,and two function-unknown proteins(desiccationrelated PCC13-62).Compared with non-scald fruit of Bud.9,there were 10 significantly up-regulated proteins in the Bud.groups: pentatricopeptide repeat-containing protein,cinnamoyl-CoA reductase 1,cationic peroxidase 1,disease resistance related proteins,glutathione S-transferase,β-galactoside etc.while 24 significantly down-regulated proteins: seed maturation protein,nutrition storage related protein,70 kDa heat shock protein and other stress-related proteins,quinone oxidoreductase and PhGPX6 and other redox-related proteins,carbohydrate metabolism-related proteins etc.Thus,suggesting that there were more proteins associated with scald resistance in fruit of Bud.rootstocks while there might be more proteins associated with scald occurrence in Geneva rootstocks.4.First study on the anthocyanin degradation mechanism in Arabidopsis thaliana seedlings by proteome expression profileAnthocyanin content was up to the highest level(7.76 Abs/g FW)on the 6th day after blue light treatment(BLT),which was 15.2 times of that before BLT and 33.7 times of the control,and 9.5 times of green seedlings on the 13 th day after BLT.The secondary metabolites of phenols,aromatic acids or phenylpropanols,flavonols also increased significantly after BLT and decreased during recovery.The proteome profiles during anthocyanin synthesis and degradation in At were successfully constructed,in which we got 133 key proteins might be in response to anthocyanin degradation in At seedlings.Significantly up-regulated proteins in cell function and stress-related function were: redox related proteins(dehydroascorbate reductase,glutathione peroxidase 2,glutathione Stransferase 6,etc.);transport-related proteins(clathrin heavy chain,outer plastid envelope protein 16-1,secretion-associated RAS 1B,etc.);abiotic stress-related proteins(arabinogalactan protein 31,MLP-like protein,etc.);signaling proteins(calcium-binding EF-hand family protein,calnexin 1 etc.);Hormone-associated protein(nitrilase 1,lipoxygenase);Proteolysis peptidase-like protein;secondary metabolism protein.There were 517 significantly changed proteins in the whole study,of which 37 were located in vacuoles or vacuolar membranes.In addition,among all the significantly changed proteins,the up-regulated glycosidases were arabidogalactan 31,xyloglucan endotransglucosylase /hydrolase protein 7,beta-glucosidase 23;the up-regulated oxidases were ascorbate peroxidase,acyl-CoA oxidase,ACC oxidase etc.The results showed that a huge number of proteins were up-regulated during the anthocyanin synthesis and degradation in At seedlings,and the related proteins were likely to be hidden in these key proteins.5.Comparing the anthocyanin degradation pattern between ornamental plant petals or leaves with different colour changing mannerAnthocyanin degradation decreases ornamental or nutritional values of horticultural products.To investigate factors that may influence colour change in plants,anthocyanin degradation was compared between the flowers of B.calycina and R.chinensis,the leaves of O.fragrans and E.cochinchinensis,in which the petals and leaves representing the rapid and slow degradation of the anthocyanins respectively.In this study,active gel analysis,high performance liquid chromatography,cell neutral red and vanillin staining,enzymatic determination and anthocyanin degradation related peroxidase(PODs)immune assay were investigated.Much lower POD activity in rose petals and E.cochinchinensis leaves than that of B.calycina petals and O.fragrans leaves,which may be related to high tannin content in petals or leaves.Epicatechin gallate(ECG)and gallocatechin gallate(GCG)were detected in rose as 161.3±12.34 and 273.56±41.23 μg/g FW respectively,while not detected in B.calycina.Catechin gallate(CG),gallocatechin(GC)and gallic acid(GA)were 8.33 ± 0.68,3.48 ± 0.04 and 0.75 ± 0.04 mg/D FW respectively in E.cochinchinensis,while epigallocatechin(EGC)and ECG were 4.13 ± 0.21 and 0.55 ± 0.07 mg/D FW respectively in O.fragrans.ECG,GCG inhibited the B.calycina POD activities with IC50 s as 21.5 and 9.7 μM respectively,and the IC50 s of CG,ECG,GA,GC,EGC on O.fragrans POD were 14.85,9.27,16.72,36.12 and 28.1 μM respectively.Catechins and epicatechin did not affect POD activity,while the Km values used as B.calycina POD substrates were 0.48 and 1.23 mM.The expression of POD associated with anthocyanin degradation was high in all four plants.In summary,high levels of tannins,especially gallated catechins,may inhibit the degradation-related enzymes in rose petals or E.cochinchinensis leaves,leading to the maintenance and color stability of anthocyanins in vivo.