Screening And Functional Analysis Of Key Genes Involved In Ethylene Biosynthesis And Signaling Pathways During Grape Bud Dormancy Release

Grape(Vitis vinifera L.) buds demand chilling to overcome dormancy before the recovery of growth,which is generally not fulfilled in warm-winter regions.Bud dormancy release poses a major obstacle to commercial viticulture in warm-winter regions.Artificial substitutes for chilling are thus mandatory in these regions to avoid prolonged bud dormancy,thereby allowing coordinated and early production of economically viable yields.The only practical means currently available for effective artificial dormancy release in vineyards involves hydrogen cyanamide(HC)application.Unfortunately,due to its toxicity,both to the vines and within the environment,it creates an urgent need for the development of safe and effective alternatives for artificial induction of bud dormancy release.A much better strategy is to search for targets which are affected by the bud release induction of HC.The ability to carry out such tasks is absolutely dependent on comprehensive molecular understanding of the cascade of biochemical changes that is induced by various dormancy release stimuli and that leads to dormancy release.Surprisingly,the network of biochemical pathways involved in grape bud dormancy release,until recently remained largely undiscovered.HC induce for such cascade,suggests that initial perturbation of cytochrome pathway activity in the mitochondria leads to respiratory and oxidative stress,decreased activity of the tricarboxylic acid cycle and decreased production of ATP.To address this energy crisis,the alternative oxidase pathway,glycolysis,pyruvate metabolism and anaerobic respiration are induced,in an order that has yet to be defined.In parallel,changes resulting from the above reprogramming may affect the interplay between ethylene and abscisic acid(ABA) in a way that allows removal of ABA repression of meristem activity and growth resumption.The potential involvement of ethylene biosynthesis and signaling in regulation of dormancy release has received little attention so far.In this research,we will use the dormancy release stimuli such as HC and azide(AZ),ethylene,the ethylene signaling inhibitor NBD,and hypoxia to screen and analyze ethylene biosynthesis genes,members of the ethylene signaling pathways and target genes in downstream of ethylene signaling which involve in the regulation of grape bud dormancy release based on physiological and biochemical technology.The main results were outlined as follows:1.Ethylene biosynthesis transiently increased between endodormancy induction and endodormancy maintenance,and was induced by HC and AZ treatmentThe bud break percentages of Vitis vinifera cv.Early sweet at 21 days were used to prepare seasonal dormancy curve,which showed that grape buds were in deep dormancy statues in December.The ethylene biosynthesis increased at the transition point(Nov.21^th) between endodormancy induction and endodormancy maintenance,but significantly decreased later.The content of 1-aminocyclopropane-1-carboxylic acid(ACC),a precursor of ethylene,were slightly reduced(approximately 1.2-fold)at maximal dormancy depth and were markedly increased(approximately 1.8-fold)during dormancy release.In addition,compared with the control,the ethylene level in grape dormant buds treated with AZ increased 1.7,15.4 and 5.3 folds at 6,24 and 48 h,respectively.While treated with HC,ethylene content increased 2.9,9.5 and 5.6 folds,respectively.Compared with control treatment,the ethylene production in grape dormant buds treated with hypoxia was reduced2.5 and 1.4 folds at 24 and 48 h,respectively.The results showed that both HC and AZ treatment could significantly promote ethylene production in grape dormancy buds,but hypoxia inhibited the synthesis of ethylene.2.Ethylene serves as a stimulus of dormancy release,induces bud break in grape dormancy budsFirstly,a significant enhancement of bud break was seen in response to application of gaseous ethylene(100 ppm),as compared to its control(10,5,2,and 1.3-fold at 12,14,18and 21 days from application).In agreement,our results indicate that application of Ethrel,resulted in significantly increased levels of bud break in the open vase system(2.4-fold at 24 days from application)and in the vineyard(1.3-fold at 42 days from application),as compared to the appropriate control.On the other hand,incubation in 2% silver thiosulfate solution(STS),which inhibits ethylene signaling,significantly downregulated 3 folds of bud break percentage of HC treated buds.Application of CoCl₂,a known inhibitor of ethylene biosynthesis,led to a complete inhibition of endodormancy release of HC-treated buds and untreated buds.In addition,combined treatment with 2,5-norbornadiene(NBD)and HC(NBD-HC) or AZ(NBD-AZ),significantly reduced the effects of HC and AZ on advancing bud endodormancy release over the 28 days monitoring period.In conclusion,ethylene participates in regulation of bud endodormancy release.3.Two subsets of ethylene biosynthesis genes are specially expressed during grapevine bud dormancy release.There are five members of the VvACS family and three members of the VvACO family are expressed in mature grapevine buds.Based on the dichotomous transcriptional behavior responded to HC,AZ,hypoxia,ethylene and NBD treatments,the bud-expressed ACO and ACS genes were divided into two subgroups:subgroup ? includes VvACS1,Vv ACS6,VvACO2 and VvACO4,and subgroup ? includes VvACS2,VvACS9 and VvACO1.The subgroup ? genes whose expression was significantly induced by HC were not affected by hypoxia and ethylene.However,the subgroup ? genes whose transcription was inhibited by HC were upregulated by hypoxia and ethylene.On the other hand,the levels of all three bud-expressed VvACO transcripts are lowest during endodormancy release(from Dec 18th to Jan 8th).This decrease,accompanied by accumulation of ACC,suggests that ethylene biosynthesis during the dormancy cycle may be primarily regulated by the ACC oxidation capacity and VvACO2 may serve as the primary regulator.4.Transcriptome profile(RNA-seq) was used to analyze the key genes that respond to ethylene signaling and are involved in dormancy release of grape budsThere are 180 genes that were upregulated at both time points(24 and 48 h) by all the dormancy release stimuli analyzed(AZ,HC,hypoxia and ethylene),compared to control,and was oppositely regulated by HCNBD,when compared to HC treatment.These genes encoded protein including ABA-responsive element binding protein 3,Auxin/indole-3-acetic acid,Ethylene receptor,Lipoxygenase and Jasmonate amino synthetase,and so on.On the other hand,only 183 genes that were downregulated at both time points(24 and 48 h) by all the dormancy release stimuli analyzed,and upregulated by HCNBD,when compared to HC treatment.These genes encoded protein including 9-cis-epoxycarotenoid dioxygenase,cytochrome P450 monooxygenase and 12-oxophytodienoate reductase,and so on.In addition,the comparative RNAseq analysis identified transcripts of 106 VvAP2/ERF genes in untreated bud and/or buds treated with the tested stimuli in at least one time point.A group of 38 genes out of the 106 are significantly regulated by ethylene,including all members of ERF.? family(VvERF057,VvERF058 and Vv ERF059).5.The expression of VvERF057 was dependent on oxygen concentration,while the expression of Vv ERF059 was induced by ethylene during grape bud dormancy releaseAgrobacterium-mediated transient transformations of tobacco leaf epidermal cells with constructs bearing GFP-tagged version of VvERF031,Vv ERF057,VvERF058 and VvERF059 showed that all of their protein are indeed localized in nucleus.Furthermore,prokaryotic expression technology was used to induce the expression of the four proteins in vitro and purify them through nickel column.Combined with qRT-PCR and western blot analysis,it was found that VvERF057 protein considerably accumulated in response to decrease in oxygen level,and its level gradually decreased upon reoxygenation.Exposure to ethylene had no effect on Vv ERF057 protein accumulation,compared to the KMnO₄ control.Moreover,VvERF059,accumulated as well following hypoxia,also to a lower magnitude,but no degradation was evident following reoxygenation.In the case of VvERF059 protein,ethylene application resulted in 1.5-fold at 48 h.From the above,the gene and protein expression of VvERF057were dependent on oxygen,while the expression of VvERF059 were not dependent on oxygen,but were upregulated by ethylene.6.Plasmodesmata(PD) re-opened in the cell wall between two cells of dormancy buds treated with HC,and ?-1,3-glucanase,an enzyme that was involved in PD re-opening,was regulated by ethylene during grape bud dormancy release.Compared with the closed PD with control treatment,PD re-opened in the cell wall between two cells of dormancy buds treated with HC at 4 days.In both gene and protein detection,?-1,3-glucanase was observed in the highest expression with HC treatment at 4 days,using q RT-PCR and western blot analysis,respectively.Meanwhile,immunogold localization showed that it was mainly distributed in vacuoles of cell with HC treatment at 4 days.In addition,?-1,3-glucanase was upregulated by ethylene at 24 and 48 h,compared to the KMnO₄ control.Therefore,it is believed that ?-1,3-glucanase is regulated by ethylene signaling during grape bud dormancy release.