PpMAPK6,regulates,peach,bud,endodormancy,release,through,interactions,with,PpDAM6

ZHANG Yu-zheng ,XU Chen ,LU Wen-li,WANG Xiao-zhe,WANG Ning,MENG Xiangguang,FANG Yu-hui,TAN Qiu-ping,CHEN Xiu-de,FU Xi-ling,LI Ling

1 College of Horticultural Science and Engineering,Shandong Agricultural University,Tai’an 271018,P.R.China

2 State Key Laboratory of Crop Biology,Shandong Agricultural University,Tai’an 271018,P.R.China

3 Shandong Collaborative Innovation Center for Fruit &Vegetable Production with High Quality and Efficiency,Tai’an 271018,P.R.China

Abstract The MADS-box (DAM) gene PpDAM6,which is related to dormancy,plays a key role in bud endodormancy release,and the expression of PpDAM6 decreases during endodormancy release.However,the interaction network that governs its regulation of the endodormancy release of flower buds in peach remains unclear.In this study,we used yeast twohybrid (Y2H) assays to identify a mitogen-activated protein kinase,PpMAPK6,that interacts with PpDAM6 in a peach dormancy-associated SSHcDNA library.PpMAPK6 is primarily located in the nucleus,and Y2H and bimolecular fluorescence complementation (BiFC) assays verified that PpMAPK6 interacts with PpDAM6 by binding to the MADSbox domain of PpDAM6.Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of PpMAPK6 was opposite that of PpDAM6 in the endodormancy release of three cultivars with different chilling requirements (Prunus persica ‘Chunjie’,Prunus persica var.nectarina ‘Zhongyou 5’,Prunus persica ‘Qingzhou peach’).In addition,abscisic acid (ABA) inhibited the expression of PpMAPK6 and promoted the expression of PpDAM6 in flower buds.The results indicated that PpMAPK6 might phosphorylate PpDAM6 to accelerate its degradation by interacting with PpDAM6.The expression of PpMAPK6 increased with decreasing ABA content during endodormancy release in peach flower buds,which in turn decreased the expression of PpDAM6 and promoted endodormancy release.

Keywords: peach,endodormancy release,PpDAM6,PpMAPK6,ABA

Bud endodormancy is a key process employed by perennial deciduous fruit trees to avoid adverse winter weather conditions in temperate and northern regions(Joséet al.2011;Wanget al.2019).Endodormancy is a complex mechanism regulated by multiple internal and external physiological factors (Singhet al.2018),and it cannot be released until certain chilling requirements are met.Endodormancy release indicates the beginning of the flowering process.The endodormancy release of peach,which is among the major species grown in greenhouses,determines its flowering time and affects the time at which it enters the market.On the other hand,under global warming,the endodormancy of fruit tree buds is starting to break irregularly as the specific chilling requirements are not being met,which results in reduced output and affects the economic benefits.Therefore,understanding the molecular mechanism of bud endodormancy release is extremely important.

The dormancy-related MADS-Box (DAM) gene is essential for peach growth and development (Jiménezet al.2009;Rioset al.2014;Wellset al.2015).Bielenberget al.(2008) found that six tandem MADS-box transcription factor family genes (DAM1–6) were not expressed in nondormant peach mutants.Among these six DAM genes,two MIKC-type MADS-box transcription factor-encoding genes,DAM5andDAM6,may be related to terminal bud dormancy (Hisayoet al.2011).The expression ofDAM5andDAM6was shown to be upregulated in response to low temperature in autumn and downregulated in response to long-term low temperature in winter.An artificially prolonged low-temperature treatment reduced the expression ofPpDAM5andPpDAM6but increased the bud burst percentage,which was negatively correlated with the expression ofPpDAM5andPpDAM6(Yamaneet al.2011a).Baloghet al.(2019) found thatParDAM6delayed dormancy release and flowering time,while Yamaneet al.(2019) found thatPmDAM6could inhibit bud break by regulating the abscisic acid (ABA) and cytokinin(CTK) contents in dormant buds.Wanget al.(2019) found that the expression ofDAM6decreased with the release of endodormancy,which shows that the DAM gene is an important internal factor regulating the dormancy of peach.

PpDAM6plays an important role in the endodormancy release of perennial deciduous fruit tree buds,but the interaction network through which it regulates the endodormancy release of peach buds is still not very clear.The MAPK cascade pathway is an important signal transduction mechanism in cells and consists of three serine/threonine phosphoprotein kinases: MAPKKK(MAP3K/MEKK/MKKK),MAPKK (MKK/MEK) and MAPK(MPK).These kinases are stimulated specifically by upstream signaling molecules that activate them stepby-step through phosphorylation and dephosphorylation,which amplifies the stress signal and leads to its transmission to the appropriate terminal of the target protein.In recent years,many studies have found that the MAPK cascade also plays an important role in bud endodormancy release in deciduous fruit trees (Duet al.2016;Chenget al.2020).In many well-studied plant species,the A subfamily memberMAPK6typically phosphorylates a substrate to regulate development and the responses to biotic and abiotic stresses (Gallettiet al.2011;Sorenssonet al.2012;Liet al.2017).InArabidopsis,MAPK6participates in the regulation of freezing tolerance,pathogen response and anther development (Heet al.2006;Wanget al.2007;Liet al.2017).However,its function in peach is still poorly understood.

In this study,using Y2H assays,quantitative real-time PCR (qRT-PCR) and other experimental methods,we found thatPpMAPK6interacts withPpDAM6and that it may phosphorylatePpDAM6to accelerate its degradation in peach bud endodormancy release.In addition,PpMAPK6was downregulated by ABA in the regulatory process of endodormancy release.The results of this study revealed a new molecular mechanism of peach bud endodormancy release and provide a new theoretical basis for further research on this mechanism.

2.1.Plant materials and treatments

The peach cultivars ‘Chunjie’ (Prunus persica‘Chunjie’),‘Zhongyou 5’ (Prunus persicavar.nectarina ‘Zhongyou 5’)and ‘Qingzhou peach’ (Prunus persica‘Qingzhou peach’) were grown in the Horticultural Experiment Station of Shandong Agricultural University in Tai’an,Shandong Province,China.‘Chunjie’ is considered a low-chilling-requiring cultivar,‘Zhongyou 5’ is considered a moderate-chilling-requiring cultivar,and‘Qingzhou peach’ is considered a high-chilling-requiring cultivar.Annual shoots of each cultivar were collected approximately every 15 d from 15 October 2018 to 30 January 2019.

To determine the flower bud breaking rate of each cultivar throughout the dormancy period,at each sampling time,25 of the shoots were placed in tap water under 200 μmol m–2s–1light for 16 h at 25°C and a relative humidity of 75%.After 25 d,the percentage of flower buds that had broken dormancy was determined.If the bud break was less than 50%,the flower buds were considered to be in the endodormancy stage (Langet al.1987).

The moderate-chilling-requiring cultivar ‘Zhongyou 5’was selected as the experimental material for the chemical treatments.Sixteen similarly growing ‘Zhongyou 5’trees were divided into four groups,which were treated with deionized water,100 μmol L–1ABA,200 μmol L–1ABA or 300 μmol L–1ABA on 15 November 2018.For the hormone treatments,ABA was dissolved in 4-mL ethanol,then deionized water and 2.5-mL Triton 100 were added to a total volume of 500 mL;for the control,the same volume of ethanol and Triton 100 were added to the deionized water,and all of the solutions were evenly sprayed onto the branches.The shoots of each treatment group were collected every 7 d from 15 November 2018 to 20 December 2018.All the flower buds were removed,immediately placed in liquid nitrogen,and then stored at-80°C for subsequent experiments.

2.2.RNA isolation and quantitative PCR (qPCR)

Total RNA was extracted from 0.1 g of bud tissue using an RNAprep Pure Plant Kit (TianGen,Beijing,China)according to the manufacturer’s instructions.First-strand cDNA was then produced using HiScript Q RT SuperMix for qPCR (+gDNA-wiper) (Vazyme,Nanjing,China)according to the manufacturer’s instructions.qPCR was performed on a CFX96 real-time PCR detection system(Bio-Rad) together with SYBR premixEx Taq(TaKaRa,Dalian,China).Three biological replicates were included for each analysis.The relative expression levels were calculated using the 2-ΔΔCTmethod (Livak and Schmittgen 2001),with thePpUBQgene used as an internal control.The primers used are shown in Appendix A.The data were analyzed using SPSS Statistics v.20.

2.3.Gene cloning and vector construction

The full-length open reading frames (ORFs) ofPpDAM6andPpMAPK6were amplified using flower bud cDNA(sampled on 15 October 2018)via2× Phanta Max Master Mix (P515,Vazyme) according to the manufacturer’s instructions.The primers used were designed using CE Design v1.04 (Vazyme,Nanjing,China) and are shown in Appendix A.The vectors were constructed using a ClonExpress Ultra One Step Cloning Kit (C115,Vazyme)according to the manufacturer’s instructions.

2.4.Y2H assays

A peach dormancy-associated SSHcDNA library was constructed from peach buds collected from dormancy until bud break.The full-length ORFs ofPpDAM6andPpMAPK6were recombined into a pGBKT7 bait vector for verification of self-activation (Appendix B),and the full-length ORF,0–240 bp ORF and 246–717 bp ORF ofPpDAM6were cloned into a pGADT7 vector using the primers listed in Appendix A.The two recombinant plasmids were co-transformed into yeast Y2H-Gold,and the transformants were cultured on selective media (SD/-Trp/-Leu) at 30°C for 3 d.After the yeast cells had grown,the putative transformants (OD600=0.002) were transferred to selective media (SD/-Leu/-Trp/-His/-Ade/X-α-gal/AbA).

2.5.Bimolecular fluorescence complementation(BiFC) assays

The full-length ORF ofPpDAM6was cloned into a pNYFP vector yielding aPpDAM6-NYFP plasmid,and the fulllength ORFs ofPpMAPK6were recombined into a pCYFP vector yielding aPpMAPK6-CYFP plasmid.All the recombinant plasmids were individually transformed intoAgrobacterium tumefaciensstrain GV3101.Equal concentrations ofA.tumefaciensstrain GV3101 containing the plasmids of interest were transiently coexpressed in onion epidermal cells.After incubation at 25°C for 24–72 h,fluorescent and differential interference contrast (DIC) images were observed with a microscope(Zeiss LSM880,Oberkochen,Germany),and the images were analyzed using Zen Lite Software (Zeiss) and Adobe Photoshop 7.0.YFP was visualized by excitation with an argon laser at 514 nm.

2.6.Subcellular localization of PpMAPK6

The ORF sequence ofPpMAPK6without the stop codon was amplified and ligated into a PRI-GFP (35S::GFP)vector for detecting its subcellular localization (Huet al.2016).The primers used are listed in Appendix A.PpMAPK6-GFP and a control GFP construct were infiltrated into onion epidermal cellsvia A.tumefaciensstrain GV3101,as described previously (Chenet al.2018).After 3 d of incubation,the GFP fluorescence signals in the transformed onion cells were observed using a Zeiss LSM880 microscope,and the images were analyzed using Zen Lite Software (Zeiss,Oberkochen,Germany).

3.1.Interactions between PpDAM6 and PpMAPK6

PpDAM6is regulated by a variety of posttranslational modifications that are critical to its stability and transcriptional activity (Hisayoet al.2011).To further explore the regulatory mechanism ofPpDAM6,the fulllength ORF ofPpDAM6was recombined into a pGBKT7 vector for Y2H experiments,ultimately to identify the proteins with which it interacts.The full-length PpDAM6 protein had self-activation activity,but 100 ng mL–1aureobasidin A (AbA) can inhibit the self-activation activity ofPpDAM6(Appendix B-a),so BD-PpDAM6was used as the bait in our assay.A number of proteins that may interact with PpDAM6 were screened from the peach dormancy-associated SSHcDNA library.In this experiment,PpMAPK6was identified as an interacting protein,and subjected to further research.It is most closely related toAtMAPK6in theArabidopsisMAPK gene family,so we named itPpMAPK6(Appendix C).Phylogenetic tree analysis showed thatPpMAPK6is similar to the corresponding sequences in apricot and plum (Appendix D).PpMAPK6was clonedviacDNA reverse transcribed from total RNA extracted from flower buds,which was used as a template.The clonedPpMAPK6ORF was inserted into a pGBKT7 vector.We first characterized the self-activation activity of PpMAPK6.The data in Appendix B-b shows that thePpMAPK6protein has self-activation activity and that 200 ng mL–1AbA can inhibit the self-activation activity ofPpMAPK6,so BD-PpMAPK6was used as the bait vector andPpDAM6-AD as the target vector.The fusion plasmid was then transformed into yeast receptor cells in pairs for point-topoint verification.The results showed that the PpMAPK6 and PpDAM6 proteins interacted in yeast (Fig.1-A).

We performed a BiFC assay to confirm the interaction between PpDAM6 and PpMAPK6 proteinsin vivo.PpDAM6was fused to the N-terminus of enhanced yellow fluorescent protein (NYFP),andPpMAPK6was fused to the C-terminus of enhanced yellow fluorescent protein (CYFP).These structures were transformed into onion epidermal cells and expressed transiently.Nuclear fluorescence was detected whenPpDAM6was coexpressed withPpMAPK6,but in control experiments,no YFP fluorescence was detected.Taken together,these results indicated thatPpDAM6interacts with the PpMAPK6 proteinin vivo.

To determine the functional domains ofPpDAM6that are required for interaction withPpMAPK6,two regions ofPpDAM6were used in a Y2H assay.PpDAM6contains a type II MADS-box domain and a K-box domain (Fig.1-C).ThePpDAM6MADS-box domain strongly interacted withPpMAPK6(Fig.1-E),whereas deletion of the MADS-box domain reduced these interactions,indicating that thePpDAM6MADS-box domain is required for interaction withPpMAPK6in yeast.

Fig.1 PpMAPK6 interacts with PpDAM6.A,PpMAPK6 interacts with PpDAM6 in yeast.Because the PpMAPK6 protein has self-activation activity and 200 ng mL–1 aureobasidin A (AbA) can inhibit the self-activation activity of PpMAPK6,the putative transformants were transferred to selective media (SD/-Leu/-Trp/-His/-Ade/AbA200) and indicated an interaction.Yeast cells transformed with BD-PpMAPK6+AD were included as negative controls.B,PpMAPK6 and PpDAM6 bimolecular fluorescence complementation (BiFC) verification.C,PpDAM6 conserved domain analysis.PpDAM6 contains a type II MADS-box domain and a K-box domain.D,PpDAM6 is segmented into two sections: the 0–240 bp ORF of PpDAM6 contains a MADS-box domain and the 246–717 bp ORF of PpDAM6 contains a K-box domain.E,PpMAPK6 interacts with the MADS-box domain of PpDAM6 in yeast.

3.2.Subcellular localization of PpMAPK6

To determine the subcellular localization ofPpMAPK6,we transiently expressedPpMAPK6-GFP fusion proteins into onion epidermal cells (Fig.2).Confocal microscopy revealed thatPpMAPK6was mainly located in the nucleus.

Fig.2 Cloning and subcellular localization of PpMAPK6.A,PCR-amplified agarose gel electrophoresis of PpMAPK6.B,subcellular localization of PpMAPK6.

3.3.PpMAPK6 and PpDAM6 are involved in peach bud endodormancy release

We next identified the dormancy stages of the three cultivars according to the bud break rate (Fig.3-A).For‘Chunjie’,we determined that the endodormancy stage occurs from 15 October to 30 November,in which the endodormancy release period (transition stage) spanned 30 October to 30 November,and the ecodormancy stage spanned 30 November to 30 December.For ‘Zhongyou 5’,the endodormancy stage was from 15 October to 15 December,the transition stage was from 15 November to 15 December,and the ecodormancy stage was from 15 December to 15 January.For ‘Qingzhou peach’,the endodormancy stage was from 15 October to 30 December,the transition stage was from 30 November to 30 December,and the ecodormancy stage was from 30 December to 30 January.

The expression ofPpMAPK6in ‘Zhongyou 5’and ‘Qingzhou peach’ showed a similar trend in the endodormant flower buds (Fig.3-B).The expression ofPpMAPK6increased rapidly at the beginning of the transition stage and then decreased gradually,which was opposite that ofPpDAM6(Fig.3-C).However,the expression ofPpMAPK6did not change significantly during the whole resting period in ‘Chunjie’.Taken together,these results suggest thatPpMAPK6promotes flower bud endodormancy release in peach,whilePpDAM6maintains flower bud endodormancy.

Fig.3 Expression of PpMAPK6 and PpDAM6 during bud dormancy in ‘Chunjie’,‘Zhongyou 5’ and ‘Qingzhou peach’.A,definitions of the dormancy stages of the peach cultivars.B,expression of PpMAPK6 during bud dormancy.C,expression of PpDAM6 during bud dormancy.The values represent the means±SD of three replicates,and the different letters above the bars represent significant differences at P＜0.05.

3.4.PpDAM6 and PpMAPK6 expression is regulated by ABA

To identify the relationship between the expression ofPpDAM6andPpMAPK6and the ABA content,we chose the moderate-chilling-requiring cultivar ‘Zhongyou 5’ as the plant material and treated the plants with 100 μmol L–1ABA,200 μmol L–1ABA and 300 μmol L–1ABA on November 15.Deionized water was used as a blank control.

The results showed that,compared with the peach flower buds treated with deionized water,the expression ofPpMAPK6in peach flower buds treated with 100 μmol L–1ABA did not change significantly.However,the expression ofPpMAPK6in peach flower buds treated with either 200 μmol L–1ABA or 300 μmol L–1ABA was inhibited at the initial stage,and the upward trend was delayed (Fig.4-A).Moreover,the downward trend ofPpDAM6expression was significantly inhibited under ABA treatment,among which the 200 μmol L–1ABA and 300 μmol L–1ABA treatments had the most significant inhibitory effects on the downregulation ofPpDAM6expression in the flower buds.These results indicated that ABA,which is an important hormone in the peach dormancy process,could inhibit the downregulation ofPpDAM6expression in endodormancy release(Fig.4-B).These results suggest that ABA may inhibit the expression ofPpMAPK6and promote the expression ofPpDAM6.

Fig.4 PpMAPK6 (A) and PpDAM6 (B) expression is regulated by abscisic acid (ABA) in flower buds of ‘Zhongyou 5’ during endodormancy release.The values represent the mean±SD of three replicates,and the different letters above the bars represent significant differences at P＜0.05.

4.1.Interactions between PpMAPK6 and the MADSbox region of PpDAM6

DAM can bind to the GArG motif of the flowering locus T (FT) promoter,which inhibits the expression of FT and contributes to endodormancy release (Hao 2015;Niuet al.2016).C-repeat binding factor (CBF) proteins can bind to the promoters ofPpDAM1andPpDAM3in pear andPmDAM6in plum,which in turn activates the expression of the DAM genes (Niuet al.2016;Zhaoet al.2018).MAPK6 is a Ser/Thr protein kinase which can phosphorylate substrate proteins and eventually cause the physiological and biochemical responses of plant cells to internal and external stimuli (Jonaket al.2002;Wanget al.2013;Zhouet al.2020).InArabidopsis,AtMPK6is involved in ABA and sugar-regulated seed germination and H2O2production (Xinget al.2009,2010).Previous studies have proven that ABA,sugar and H2O2are related to bud dormancy (Gaoet al.2002;Wanget al.2009,2011,2015).In this study,we used Y2H assays to identify the interacting protein ofPpDAM6:mitogenactivated protein kinase,PpMAPK6,which was confirmed

by BiFC analysis (Fig.1-B).PpDAM6is a transcription factor,andPpMAPK6is located in the nucleus,indicating thatPpMAPK6interacts withPpDAM6in the nucleus.DAM genes usually contain a MADS-box region at the N-terminus and a K-box region in the middle region of the protein,and these domains are also found in multiple other MIKCCS-type MADS-box proteins (Sasakiet al.2011).Further Y2H experiments confirmed thatPpMAPK6interacts with the MADS-box region ofPpDAM6(Fig.1-E).Although our results cannot fully verify the target domain ofPpMAPK6in peach,the MADS-box domain is nonetheless an important target.

4.2.PpMAPK6 may phosphorylate and accelerate the ubiquitin-mediated degradation of PpDAM6

MAPK proteins are important signaling enzymes in cascade signaling pathways,and they function in several cell signaling pathways by phosphorylating substrate proteins in response to hormones and biotic and abiotic stresses.MAPKs phosphorylate substrate proteins by interacting with specific substrate proteins (Jinet al.2017;Goyalet al.2018),which can regulate the stability of those proteins (Menget al.2013;Zhaoet al.2017).InArabidopsis thaliana,MAPK6can co-phosphorylateICE1to reduce its stability and accelerate its degradation,and then theICE1is degraded by ubiquitination (Liet al.2017).Previous studies have suggested thatPpDAM6plays an important role in peach flower bud dormancy(Yamaneet al.2011a,b;Wanget al.2020).In this study,we found that in dormant peach cultivarsPpDAM6was maintained at a high expression level during the early endodormancy stage,and that the expression level rapidly decreased during the flower-bud endodormancy release period (transition stage) (Fig.3-C),which contrasts with the results we observed forPpMAPK6(Fig.3-B).The trend among the cultivars with an increased demand for chilling unit accumulation was more significant.We hypothesize thatPpMAPK6may reduce the protein stability ofPpDAM6by phosphorylation of the MADSbox protein domain ofPpDAM6and may accelerate its ubiquitin-mediated degradation.

4.3.A decrease in the ABA content activates the mitogen-activated protein kinase PpMAPK6 in peach flower buds

ABA is generally considered to be a key hormone involved in regulating bud dormancy (Cookeet al.2012;Vergaraet al.2017).ABA can delay the germination of seeds,delay germination until more suitable growing conditions occur and increase survival rates (Richardsonet al.2019).Hydrogen cyanamide (HCN) treatment can accelerate both the reduction in ABA content in grape buds and the release of bud dormancy (Zhenget al.2015).In pear,the ABA response element (ABRE)-binding transcription factorPpAREB1binds toPpDAM1and negatively regulates its activity,andPpDAM1binds to the promoter ofPpNCED3,the key rate-limiting gene for ABA synthesis and positively regulates its expression (Tuanet al.2017).In peach,the ABA content gradually decreases during flower bud endodormancy,and the ABA response genePpABF2can interact withPpTCP20,which inhibits the expression ofPpDAM5/PpDAM6and regulates the dormancy process(Wanget al.2020).In addition to DAM,previous studies found that ABA can regulate the transcription and the activity of MAPK (Liu 2012;Danquahet al.2015).InArabidopsis,AtMPK6is involved in ABA signaling in seed germination and H2O2production (Xinget al.2009,2010).In cotton,GI1MPK6may play an important role in ABAinduced CAT1 expression and H2O2production (Luoet al.2011).In this study,we found that ABA may inhibit the expression ofPpMAPK6and promote the expression ofPpDAM6(Fig.5).Therefore,we speculate that the expression ofPpMAPK6increases with decreasing ABA content during endodormancy release in peach flower buds,which decreases the expression ofPpDAM6and promotes endodormancy release.

ABA negatively regulates bud break in peach.In this study,we found that ABA may inhibit the expression ofPpMAPK6.The expression ofPpMAPK6increased,andPpMAPK6may interact with the MADS-box domain to phosphorylatePpDAM6,which reduces its stability and accelerates the ubiquitin-mediated degradation ofPpDAM6through the ubiquitin/proteasome pathway with a decrease in the ABA content during the endodormancy release of peach flower buds (Fig.5).

Fig.5 Model for PpDAM6 and PpMAPK6 regulation of bud endodormancy release in peach flower buds.Abscisic acid(ABA) may inhibit the expression of PpMAPK6 and promote the expression of PpDAM6. PpMAPK6 may interact with the MADS-box domain to phosphorylate PpDAM6,which reduces its stability and accelerates the ubiquitin-mediated degradation of PpDAM6 with a decrease in ABA content during the endodormancy release of peach flower buds.Induction of targets is represented by solid arrows and inhibition is represented by blocked lines.

Acknowledgements

This work was partially supported by the National Key Research and Development Plan (2018YFD1000104),the National Natural Science Foundation of China(318720415) and the Agricultural Improved Seed Project Grant of Shandong,China (2020LZGC007 and 2020LZGC00702) and the Fruit Industry Technology System Project of Shandong,China (SDAIT-06-04).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm