XU Peng-yue ,XU Li ,XU Hai-feng ,HE Xiao-wen ,HE Ping ,CHANG Yuan-sheng ,WANG Sen,ZHENG Wen-yan,WANG Chuan-zeng,CHEN Xin,Ll Lin-guang#,WANG Hai-bo#

1 Shandong Institute of Pomology, Shandong Academy of Agricultural Sciences, Tai’an 271000, P.R.China

2 Modern Agriculture Research Institute of Yellow River Delta, Shandong Academy of Agricultural Sciences, Dongying 257000,P.R.China

Abstract Cold stress is an important factor that limits apple production.In this study,we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337’ and ‘60-160’,which are resistant and sensitive to cold stress,respectively.The enriched pathways of differentially expressed genes (DEGs) and physiological changes in ‘M9T337’ and ‘60-160’ plantlets were clearly different after cold stress (1°C) treatment for 48 h,suggesting that they have differential responses to cold stress.The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance.When we overexpressed MdWRKY40is and MdWRKY48 in apple calli,the overexpression of MdWRKY48 had no significant effect on the callus,while MdWRKY40is overexpression promoted anthocyanin accumulation,increased callus cold tolerance,and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2.Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter.Yeast twohybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor MdMYB15L through the leucine zipper (LZ).When the LZ of MdWRMY40is was knocked out,MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance,indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L.In summary,MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation,and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2,indirectly promoting MdCBF2 expression,and thereby improving cold tolerance.These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

Keywords: MdWRKY40is,anthocyanin accumulation,MdMYB15L,MdCBF2,cold tolerance

1.lntroduction

Cold stress is an important ecological factor that limits plant growth and geographical distribution,causing serious economic losses every year (Dinget al.2019).Low-temperature stress not only causes changes in the physiology and biochemistry of plant membrane systems,reactive oxygen species,and metabolites,but it also affects gene expression and protein synthesis (Zhuet al.2016).Apples are mainly cultivated in temperate regions,and low-temperature injury is an important environmental factor that restricts their yields.Dwarf rootstock cultivation is the main apple production model promoted in China.Therefore,studying the cold resistance mechanism of apple rootstocks is of practical significance.

Plants can sense and transduce low-temperature signals,which initiate the activity of upstream transcription factors,regulate the expression of downstream coldresponsive genes,and ultimately activate complex and intersectional metabolic pathways,thereby marshaling a series of mechanisms for rapid perception and adaptation to low temperature (Chinnusamyet al.2007).In this process,plants enhance their tolerance to lowtemperature stress by inducing cold-related genes;synthesizing soluble sugars,flavonoids,and other protective substances;enhancing antioxidant capacity;and changing membrane components (Cooket al.2004;Ruellandet al.2009).The C-repeat binding factor/drought-responsive element binding protein (CBF/DREB)-mediated cold signaling pathway has been studied thoroughly.In the inducer of the CBF expression (ICE)-CBF-cold-responsive (COR) pathway,CBF binds to the CRT/DRE element of theCORgene promoter to activate its expression (Maruyamaet al.2004).ArabidopsisCBF1/2/3 can induce approximately 100 downstreamCORgenes (Gilmouret al.2004).ICE can positively regulateCBFin rice,tomato,and apple (Nosenkoet al.2016;Denget al.2017;Anet al.2021).In addition,the transcription factor BZR1 and the calmodulin-binding transcription activator CAMTA of the brassinosteroid signaling pathways actively regulate CBF expression(Kidokoroet al.2017;Liet al.2017).Some negative regulators of CBF have also been identified,including MYB15 in the MYB transcription family (Agarwalet al.2006;Xuet al.2018);PIF3,an important transcription factor in the light signaling pathway (Jianget al.2017);and EIN3,a key factor in the ethylene signaling pathway(Robisonet al.2019).However,a large proportion of the low-temperature signals in plants are independent of the CBF pathway.Transcription data show that fewer than 20% ofCORgenes are regulated by CBF1/2/3 (Jiaet al.2016;Zhaoet al.2016).

The defining features of the WRKY transcription factor family are the WRKY domain,the conserved N-terminal WRKYGQK sequence,and the C2H2 (CX4–5-CX22–23-HXH)-type or C2HC (CX7-CX23-HXC)-type zinc finger structure at the C-terminus (Eulgemet al.2000).Based on the number of WRKY domains and the characteristics of the zinc finger structure,WRKY transcription factors can be divided into three categories;of which category II is further divided into either five subcategories,IIa,IIb,IIc,IId and IIe,or three subcategories,IIa+IIb,IIc,and IId+IIe,due to their different amino acid sequences and phylogenetic relationships (Zhang and Wang 2005).Almost all the promoter regions of target genes regulated by WRKY transcription factors have W-box (TTGACC/T)elements and are highly conserved (Ciolkowskiet al.2008).

WRKY transcription factors are widely involved in plant responses to stress.In rice,Arabidopsis,andBrassica napus,41,20,and 74 WRKY genes are involved in abiotic stress responses,respectively (Ramamoorthyet al.2008;Chenet al.2012).WRKY transcription factors often participate in hormone signaling pathways in response to low-temperature stress.For example,banana MaWRKY26 enhances fruit cold tolerance by binding to and activating key genes in the jasmonic acid signaling pathway (Yeet al.2016),while MaWRKY31/33/60/71 can bind to and activate the abscisic acid (ABA) synthesis genesMaNCED1andMaNCED2,which increases the endogenous ABA content and improves the cold tolerance of the fruits (Luoet al.2017).Similar roles have been found in cucumber CsWRKY46,which can bind to the key gene of ABA signaling,ABI5,to regulate the cold signaling pathway (Zhanget al.2016).AtWRKY34 inArabidopsiscan act on the CBF signaling pathway and affect the cold tolerance of pollen (Zouet al.2010),but there are few studies on the CBF-dependent pathway mediated by WRKY transcription factors,and the mechanism of these effects is still not clear.

Anthocyanins are important secondary metabolites of plants.They are widely present in the vacuoles of stems,leaves,flowers,fruits,roots and other organs.They not only give plants rich colors but also act in protecting plants from environmental stresses such as low temperature and ultraviolet radiation (Fieldet al.2001;Leng and Qi 2003).The anthocyanin biosynthetic pathway and structural genes have been identified.The regulatory mechanism of the MBW complex (MYB–bHLH–WD40) has been studied in depth (Jaakolaet al.2013;Xuet al.2015;Cuiet al.2021).MYB transcription factors,especially MYB10 and its homologous proteins,play a key role in anthocyanin synthesis.For example,MdMYB10andMdMYB110adetermine the synthesis of anthocyanins in two types of the appleMalusniedzwetzkyana(Espleyet al.2009;Chagnéet al.2013).TheMYB10homologous genes in strawberry,peach,and apricot areFaMYB10(Kuiet al.2010),PpMYB10(Ravagliaet al.2013),andPaMYB10(Xiet al.2019),respectively.Other transcription factors,such as the WRKY proteins,also play important roles in the regulation of anthocyanin synthesis.For example,B.napusBnWRKY41-1 andArabidopsisAtWRKY75 negatively regulate anthocyanin biosynthesis (Devaiahet al.2007;Duanet al.2018).Apple MdWRKY11 and pear PyWRKY26 promote the expression of anthocyanin synthesis activatorsMdMYB10andPyMYB114,respectively,thereby promoting the synthesis of anthocyanins (Liuet al.2019;Liet al.2020).Anthocyanin synthesis can be induced by and protect against cold stress (Wanget al.2018),and there is a complex interactive relationship between the two.

In this study,we investigated the cold-resistant apple rootstock ‘60-160’ (CR) and the cold-sensitive rootstock‘M9T337’ (CS),and found that the WRKY transcription factor MdWRKY40is can interact with the cold signaling pathway inhibitor MdMYB15L to interfere with its inhibition ofMdCBF2.It also directly promotes the accumulation of anthocyanins,thereby improving cold tolerance.Our findings provide a new perspective on the molecular mechanisms of anthocyanin synthesis and cold signaling in apples.

2.Materials and methods

2.1.Plant materials and processing

The ‘M9T337’ (dwarfing) and ‘60-160’ (dwarfing)rootstocks were grown at the Tianping Lake Experimental Base,Shandong Institute of Pomology,China.The latter was introduced to our institute from the Michurinsk State Agricultural University (Michurinsk,Russia) according to a mutual agreement.In the early stage,our team successfully induced the formation of tissue-cultured plantlets using the young shoot tips of rootstocks as explants (Sunet al.2014).Tissue-cultured plantlets of the rootstock ‘M9T337’ were cultured on Murashige and Skoog (MS) medium+1 mg L–16-benzyladenine (6-BA)+0.2 mg L–13-indole butyric acid (IBA),and tissuecultured plantlets of ‘60-160’ rootstocks were cultured on Quoirin and Lepoivre (QL)+0.5 mg L–16-BA+0.05 mg L–1IBA.The tissue-cultured plantlets were treated with cold stress at 1°C in a constant-temperature incubator(16 h light/8 h dark) for either 6 or 48 h,and the treated leaves were cryopreserved at–80°C for transcriptome analysis.Two-week-old ‘Orin’ apple callus (cultured on MS+0.5 mg L–16-BA+1 mg L–12,4-D;at 25°C in the dark)were used for subsequent gene transformation.The inner epidermis of onion was plated on MS solid medium and cultured in the dark at 28°C for 12–24 h for the bimolecular fluorescence complementation analysis.

2.2.Transcriptome analysis

Oligo (dT) magnetic beads were used to enrich the mRNA with PolyA tails,and the qualified total RNA was detected for library construction and sequencing.The Illumina NEBNext?UltraTM RNA Library Prep Kit was used to construct the general transcriptome library.The sequencing platform was Illumina NovaSeq 6000,and HISAT2 v2.0.5 Software was used to compare the clean reads at the paired ends against the reference genome.FeatureCounts v1.5.0-p3 Software was used to calculate the reads of each gene in each sample,and then the fragments per kilobase of transcript per million mapped reads (FPKM) value of each gene was calculated based on the length of the gene.

DESeq2 R (1.16.1) Software was used to perform the differential expression analysis between pairwise comparisons.Genes with correctedP-values ofPadj<0.05 and |log2(fold change)|>0 were considered to be significantly differentially expressed genes (DEGs)in this analysis.The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was performed using the clusterProfiler package of R Software,and the gene length bias was corrected.All KEGG terms that satisfiedPadj<0.05 were considered to be the significantly enriched functional items.

2.3.Measurement of malondialdehyde (MDA) and relative conductivity

The MDA measurement was based on the method of Ohyaet al.(1993) with slight modifications.A small amount of quartz sand and 2 mL of 10% trichloroacetic acid (TCA) were added to a 0.1-g sample,the mixture was ground into a slurry,and the volume was adjusted to 5 mL with TCA.The samples were centrifuged at 4 000 r min–1for 10 min.A 2-mL aliquot of the supernatant was aspirated and added to 2 mL of 0.6% thiobarbituric acid for the experimental samples,and 2 mL of distilled water was added to the control.The mixture was mixed and reacted in boiling water for 15 min.After cooling,the mixture was centrifuged at 4 000 r min–1for 10 min,and the supernatant was collected.Its absorbance (A)values at 450,532,and 600 nm were measured using a spectrophotometer.The concentration of MDA was calculated as: 6.45×(A532?A600)?0.56×A450,and the MDA content was calculated as: concentration×volume/mass,expressed in μmol g–1.

The conductivity was measured using the Angersbachet al.(1999) method with slight modifications.Fresh samples (0.1 g) were weighed into a test tube,10 mL of deionized water was added,and a vacuum pump was used to evacuate the sample for 10 min until the leaf was submerged under water.The test tube was maintained at 20°C for 40 min,and then placed in boiling water for 10 min and cooled to 20°C.The conductivity k2was measured at this point,and the relative conductivity was given as: k1/k2.

2.4.Anthocyanin extraction and determination

The method was carried out as described by Jiet al.(2015).Plant material (0.5 g) was weighed and ground into powder in liquid nitrogen,extracted with 20 mL of 1% (v/v) HCl-methanol at 4°C in the dark for 24 h,and centrifuged at 12 000 r min–1for 10 min,and the supernatant was retained.The absorbance of the supernatant at 530 nm was measured using a UV spectrophotometer.

2.5.Apple callus transformation and ectopic expression in Arabidopsis

The method was based on Xuet al.(2020).The coding sequences (CDSs) ofMdWRKY48,MdWRKY40isandMdLLZWRKY40iswere ligated into the pRI-101 vector including the 35S CaMV promoter and the GFP tag,respectively,to establish the 35S:MdWRKY48-GFP,35S:MdWRKY40is-GFP and 35S:MdLLZWRKY40is-GFP recombinant plasmids (Appendix A).These three plasmids were transformed intoAgrobacterium tumefaciensLBA4404,and 2-week-old ‘Orin’ calli were infected by transformedAgrobacterium.These calli were cultured on MS media at 25°C for 48 h in the dark.Subsequently,these calli were transferred onto MS media supplemented with 250 mg L?1carbenicillin and 50 mg L?1kanamycin for transgenic selection.

Ectopic expression was performed according to Huet al.(2013).TheA.tumefacienscontaining candidate genes were used to infectArabidopsis.The T1transgenicArabidopsisplants were then selected by plating onto MS medium containing kanamycin.The kanamycin-resistant seedlings were transferred to soil and grown in a growth chamber (Ningbo-Jiangnan,http://www.nbjnyq.com/).The T2seeds were then collected and grown as described above.

2.6.Protoplast subcellular localization

Protoplasts were extracted as described by Xuet al.(2020).A total of 2 g of transgenic ‘Orin’ apple callus was weighed,and 10 mL of cell wall enzymatic hydrolysis solution was added.The tube was wrapped in aluminum foil in the dark,and placed in a vacuum pump for 30 min and then in the dark room at 24°C for 12 h.The protoplasts were released through gentle shaking by hand,10 mL of W5 solution (5 mol L–1NaCl,1 mol L–1CaCl2,1 mol L–1KCl,0.3 mol L–1MES) was added,and the solution was filtered through 75-μm nylon cloth.The filtrate was centrifuged at 500 r min–1for 10 min,and the supernatant was discarded.The precipitate was dissolved in 10 mL of W5 solution and automatically precipitated on ice for 30 min.Finally,the precipitate was dissolved in 2 mL of MMg solution (0.8 mol L–1mannitol,1 mol L–1MgCl2,0.3 mol L–1MES),and the green fluorescent protein (GFP) signal was detected by fluorescence microscopy.

2.7.Yeast one-hybrid (Y1H)

Y1H analyses were performed using yeast strain Y187 (Clontech,Palo Alto,CA,USA) according to the manufacturer’s protocols.The CDS ofMdWRKY40iswas inserted into the pGADT7 vector,while the promoters ofMdCBF2andMdDFRwere inserted into a pHIS2 vector(Appendix A).Different combinations of the recombinant plasmids were cotransformed into Y187,and their interactions were examined on media that were deficient in Trp,Leu,and His (SD/–Trp–Leu–His) but contained optimal 3-AT concentrations.

2.8.Electrophoretic mobility shift assay (EMSA)

The EMSA was carried out using an EMSA kit (Pierce,Rockford,IL,USA) and its protocols.The promoter ofMdDFRwas used to build the probes labeled with biotin(Sangon Biotech,Shanghai,China).MdWRKY40is-His protein (60 ng) was purified for the binding reactions,which were established in 20-mL reactant mixtures including 0.1 mmol L–1ethylenediaminetetraacetic acid,17% glycerol,4 mg poly(dI-dC),1 mmol L–1DTT,100 mmol L–1KCl,25 mmol L–1HEPES-KOH (pH 7.5),1 pmol labeled probe and competitor DNA (25,50,or 100 pmol).The reactions were incubated at 25°C for 30 min.The samples were subjected to a prerun in 0.5% TBE buffer at 100 V for 1 h,and then analyzed by electrophoresis on 6% acrylamide gels including 0.5%TBE buffer and 3.6% glycerol for 2 h at 4°C.The DNA was transferred onto nylon membranes and the signals were detected by chemiluminescent acid detection.

2.9.Knockout of the conserved sequence in the leucine zipper of MdWRKY40is by overlap PCR

The conserved sequence of the MdWRKY40is leucine zipper (NKKLTEML) was removed through the overlap PCR technique.The principle and procedure of this PCR technique are as follows.It used two pairs of primers: F1: 5′-ATGGACTCAACGTGGGTGAA-3′;R1:5′-AGGGCAGTCTCCACACTTATCCTATTCAAC-3′;F2: 5′-AAGTGTGGAGACTGCCCTCTGCGAGA-3′;R2:5′-TCATGAGTGGTCTGAAATTCTTC-3′.

The upstream and downstream regions of the leucine zipper conserved sequence (NKKLTEML)of MdWRKY40is were named MdWRKY40is-1 and MdWRKY40is-2,respectively.The F1 and R1 primers were used to amplify MdWRKY40-1,carrying eight bases(ACTGCCCT) of the downstream region;and the F2 and R2 primers were used to amplify MdWRKY40-2,carrying 10 bases (AAGTGTGGAG) of the upstream region.The amplified upstream and downstream sequences contained 18 bases of overlapping sequences.The two upstream and downstream sequences were mixed in equal amounts as templates,and the MdWRKY40is that did not contain the leucine zipper conserved sequence(NKKLTEML),was amplified using the F1 and R2 primers.We named this as the loss-of-leucine-zipper MdWRKY40is (MdLLZWRKY40is).

2.10.Yeast two-hybrid (Y2H)

The CDSs ofMdWRKY40isandMdLLZWRKY40iswere inserted into the pGADT7 vector to build the recombinant plasmids MdWRKY40is-AD and MdLLZWRKY40is-AD.The MdMYB15L CDS was inserted into the pGBKT7 vector to set up the recombinant plasmid MdMYB15LBD (Appendix A).These recombinant plasmids were cotransformed into yeast cells according to the protocols of the Yeastmaker? Yeast Transformation System 2 Kit(Clontech).Initially,cells were cultured in selective media lacking Leu and Trp (–Leu/–Trp,Clontech),and putative transformants were then transferred to media lacking adenine (Ade),His,Leu,and Trp (–Ade/–His/–Leu/–Trp,Clontech).The substrate X-α-gal was added to media lacking four amino acids (–Ade/–His/–Leu/–Trp) for the detection of β-galactosidase activity.

2.11.BiFC assay

TheMdWRKY40isCDS was inserted into the pSPYNE-35S plasmid,andMdMYB15Lwas inserted into the pSPYCE-35S plasmid (Appendix A).TheA.tumefaciens(LBA4404) containing these two recombinant plasmids were co-transformed into onion epidermal cells,and then cultured on MS media at 28°C in the dark for two days.The YFP fluorescence was observed by an epifluorescence microscope at an excitation wavelength of 488 nm (Olympus BX53F,Tokyo,Japan).

2.12.Data analysis

All the results presented below are based on the averages of three parallel experiments.The statistical analysis was carried out using Duncan’s new multiple range test.Different lowercase letters on the chart columns,such as a,b,c,etc.,represent significant differences (P<0.01).The phylogenetic tree was constructed using MEGA 5.0,and the related protein sequences were analyzed by Clustal X.The primer list was shown in Appendix B.

3.Results

3.1.The physiological changes and induced pathways in tissue-cultured plantlets of the ‘M9T337’and ‘60-160’ rootstocks under cold stress

Tissue-cultured plantlets of the ‘M9T337’ and ‘60-160’rootstocks showed no significant change in phenotype at the early stage (0 to 6 h) of cold stress (1°C) treatment,and the leaves appeared green.However,at the late stage of cold stress (6 to 48 h),the leaves of ‘M9T337’plantlets wilted,while the ‘60-160’ plantlets showed no significant changes (Fig.1-A).The analyses of interrelated physiological indicators showed that the relative conductivity and malondialdehyde (MDA) content of‘M9T337’ plantlets increased under cold stress treatment,while they first increased and then decreased in the ‘60-160’ plantlets.At the early stage of cold stress treatment,the relative conductivities and MDA contents of ‘M9T337’and ‘60-160’ plantlets did not show significant differences,while at the late stage of cold stress treatment,the relative conductivity and MDA content of M9T337 were significantly higher than those of the ‘60-160’ plantlets (Fig.1-B and C),which is consistent with the results of the plantlets phenotypes described above.These results indicated that ‘M9T337’ is cold sensitive (CS),while ‘60-160’ is cold resistant (CR).

Fig.1 Analysis of related physiological changes and differentially expressed gene (DEG) enriched pathways of tissue-cultured plantlets of the cold-resistant (CR) rootstock ‘60-160’ and cold-sensitive (CS) rootstock ‘M9T337’ under cold stress (1°C).A phenotypes.CT,cold stress treatment.B,relative conductivity.Bars are SD (n=3).Different letters above the columns represent significant differences (P<0.01).C,malondialdehyde (MDA) contents.D and E,KEGG pathway enrichment analysis of DEGs in the ‘60-160’ (D) and ‘M9T337’ (E).1,2,and 3 after CR or CS,treated with cold stress for 0,6,and 48 h,respectively.

The enrichment analysis of the KEGG pathways of the DEGs of the two rootstocks after cold stress treatment showed that the DEGs were enriched in plant hormone signal transduction,starch and sucrose metabolism,alpha-linolenic acid metabolism,and linoleic acid metabolism in the early and late stages of treatment.For some pathways,such as carbon fixation in photosynthetic organisms and arginine and proline metabolism,the DEGs of the two rootstocks were only enriched in the early stage of cold stress treatment.Interestingly,the DEG enriched pathways of the two rootstock plantlets differed in the late stage of cold stress treatment.The DEGs of ‘M9T337’ were enriched in porphyrin and chlorophyll metabolism,while the DEGs of ‘60-160’ were enriched in flavonoid biosynthesis (Fig.1-D and E).These differences may be related to the difference in the cold resistance performance of the two rootstocks late in the cold stress treatment.

3.2.Screening of potential WRKY transcription factors in response to cold stress

The above results showed that after 48 h of cold stress(1°C) treatment,tissue-cultured plantlets of ‘M9T337’(CS3) and ‘60-160’ (CR3) showed significant differences in their phenotypes and related physiological indicators.Therefore,we analyzed the differential WRKY genes in the tissue-cultured plantlets of the two rootstocks at this stage.The first screen (Fig.2-A) showed that eight WRKY genes were upregulated in the CR3.Further analysis of the expression levels of these eight WRKY genes during the cold stress treatment showed that the WRKY genes in the two plantlets induced by cold stress were MD15G1039500 (LOC103442002) and MD16G1151000(LOC103403524) (Fig.2-B),so they were considered to be potential cold tolerance-related genes.Based on the gene descriptions in Fig.2-A,we named MD15G1039500(LOC103442002) as MdWRKY40is and MD16G1151000(LOC103403524) as MdWRKY48.

Fig.2 Screening and identification of the cold stress-related WRKY transcription factors MdWRKY40is and MdWRKY48.A,analysis of differentially expressed WRKY genes in tissue-cultured plantlets of the cold-resistant (CR) rootstock ‘60-160’ and cold-sensitive(CS) rootstock ‘M9T337’ under cold stress (1°C) for 48 h.B,the expression levels of WRKY genes under cold stress (1°C) for 0 to 48 h.The genes indicated by the red underline are induced by cold stress.Colors from blue to red represent low to high levels of gene expression.C,phylogenetic tree of MdWRKY40is and MdWRKY48 along with other WRKY transcription factors.The red font indicates the selected WRKY transcription factors.D,transcription factor domains of class IIa and IIc WRKYs.E,analysis of the amino acid sequences of MdWRKY40is and MdWRKY48.1,2,and 3 after CR or CS,treated with cold stress for 0,6,and 48 h,respectively.

A phylogenetic tree was constructed using MdWRKY40is,MdWRKY48 and related WRKY transcription factors.MdWRKY40is and the known class IIa WRKY transcription factors including AtWRKY60 and OsWRKY71 were in the same region,while MdWRKY48 and the known class IIc WRKY transcription factor AtWRKY23 were in the same area (Fig.2-C and D).Further amino acid sequence analysis showed that the sequence similarity rates of MdWRKY40is with MdWRKY40,AtWRKY60,OsWRKY71 and MdWRKY48 were 74.61,69.87,66.48 and 59.79%,respectively.In addition,there is a leucine zipper at the N-terminus of the MdWRKY40is protein (Fig.2-E),which may be related to the interactions among proteins.

3.3.MdWRKY40is is involved in anthocyanin synthesis and improves cold tolerance in apple callus

The selected candidate genes were transformed into apple callus,and an ‘Orin’ apple callus that overexpressed the empty plasmid was used as the control.All transgenic calli were subcultured at 25 or 4°C for 10 days from an initial weight of 0.2 g,and then they were stored in a phytotron with constant light (photon flux density 80 μmol m–2s–1) for coloration assays.The weights of these transgenic calli were all similar after growth at 25°C.However,the weight of the callus overexpressingMdWRKY40iswas significantly greater than that of the control after growth at 4°C,but the weight of the callus overexpressingMdWRKY48was not different from that of the control (Fig.3-A and C).These findings suggested that the overexpression ofMdWRKY40iscould increase callus cold tolerance.In addition,the overexpression ofMdWRKY40ischanged the callus coloration from yellow to light pink and promoted the accumulation of anthocyanin at 25°C.The callus overexpressingMdWRKY48did not change significantly.Under cold stress treatment at 4°C,no significant color change was observed in the control,while the callus overexpressingMdWRKY40ishad a darker color and higher anthocyanin content (Fig.3-A and C).The same number of wild type and transgenicArabidopsisT2seeds were cultured at 25 and 4°C for 10 d,respectively.The ectopic expression ofMdWRKY40isinArabidopsiswas found to improve the seed survival rate at 4°C (Fig.3-B),suggesting that the overexpression ofMdWRKY40iscould also improve cold tolerance inArabidopsis.

Fig.3 Transgenic and functional analysis of MdWRKY40is.A,phenotypes of three transgenic calli that were subcultured at 25 or 4°C.GFP,‘Orin’ callus overexpressing the empty plasmid;OEWRKY48,‘Orin’ callus overexpressing MdWRKY48;OEWRKY40is,‘Orin’ callus overexpressing MdWRKY40is.B,ectopic expression of MdWRKY40is in Arabidopsis.WRKY40is-WT,overexpression of MdWRKY40is in wild type Arabidopsis; WT,wild type Arabidopsis.C,weights,anthocyanin levels,and related gene expression levels of the three transgenic calli cultured at 25 and 4°C.D,subcellular localization of MdWRKY40is in callus protoplasts.E,analysis of the expression levels of anthocyanin synthesis-and cold signaling pathway-related genes in the three transgenic calli.Bars are SD (n=3).Different letters above the columns represent significant differences (P<0.01).

The subcellular localization of MdWRKY40is in protoplasts showed that it was localized in the nucleus(Fig.3-D).Further quantitative analysis of the related genes showed that low-temperature stress at 4°C induced the expression ofMdWRKY40is(Fig.3-C).Overexpression ofMdWRKY40isin callus significantly promoted the expression of the anthocyanin synthesis structural geneDFRand the cold signaling core geneCBF2.However,it had no effect on the expression levels of some other key transcription factors for anthocyanin synthesis and cold signaling,such asMdMYB10,MdbHLH33,MdMYB15L,andMdICE1(Fig.3-E).

3.4.Analysis of MdWRKY40is binding to the promoters of the anthocyanin synthesis structural gene MdDFR and the cold signaling core gene MdCBF2

The results above indicated that transgenicMdWRKY40iscould promote the expression ofMdDFRandMdCBF2.Therefore,we then determined whether it could bind to the promoters ofMdDFRandMdCBF2.Consulting the PlantCARE database,we found that theMdDFRandMdCBF2promoters contain three and two WRKY transcription factor binding W-box elements,respectively,whose specific positions in the promoters are shown in Fig.4-A.A yeast one-hybrid screen showed that 120 or 150 mmol L–13-AT inhibited the expression of the reporter geneHIS3under the control of either theMdCBF2orMdDFRpromoter (Fig.4-B).At these inhibitory concentrations,further experiments showed that MdWRKY40is could only bind to theMdDFRpromoter but not to theMdCBF2promoter (Fig.4-C).The electrophoretic mobility shift assay (EMSA) analysis showed that MdWRKY40is could only bind to the W-box element at positions–755 to–760 of theMdDFRpromoter(Fig.4-D).In summary,MdWRKY40is can directly bind to theMdDFRpromoter,but it cannot bind to theMdCBF2promoter.

Fig.4 MdWRKY40is can directly bind to the MdDFR promoter.A,analysis of the MdDFR and MdCBF2 promoter W-box elements.B,the screening of different 3-amino-1,2,4-triazole (3-AT) concentrations for inhibition of reporter gene expression under the control of the MdCBF2 or MdDFR promoter.C,yeast one-hybrid assays between MdWRKY40is and the promoters of MdDFR and MdCBF2 with the screened 3-AT concentrations.D,EMSA analysis between MdWRKY40is and the W-box element of the MdDFR promoter.T,Trp;H,His;L,Leu.

3.5.MdWRKY40is participates in the cold signaling pathway through the interaction of its N-terminal leucine zipper with MdMYB15L

MdWRKY40 cannot bind to theMdCBF2promoter,so how does it affect the expression ofMdCBF2? The homologous protein MdMYB15L of theArabidopsiscold signaling repressor AtMYB15 can specifically bind toMdCBF2and inhibit its expression (Xuet al.2018).Does MdWRKY40is interact with MdMYB15L to affect the expression ofMdCBF2? To answer these questions,we used the overlap PCR technique to knock out the conserved sequence of the leucine zipper in MdWRKY40is in order to obtain the newly recombined MdLLZWRKY40is (Fig.5-A).Yeast two-hybrid analysis showed that MdWRKY40is could interact with MdMYB15L,while MdLLZWRKY40is could not interact with MdMYB15L (Fig.5-B),suggesting that MdWRKY40is interacts with MdMYB15L through its N-terminal leucine zipper.Bimolecular fluorescence complementation(BiFC) analyses of onion epidermis further verified the interaction between MdWRKY40is and MdMYB15L(Fig.5-C).The ‘Orin’ callus that overexpressed the empty plasmid was used as the control.Overexpression ofMdLLZWRKY40isstill changed the callus from yellow to red,and the anthocyanin content was similar to that of the callus overexpressingMdWRKY40is.However,the weight of callus overexpressingMdLLZWRKY40iswas significantly lower than that of callus overexpressingMdWRKY40isafter growth at 4°C,but it was similar to the control (Fig.5-D),suggesting that MdLLZWRKY40is could not improve the cold tolerance of the callus.Further analysis of the expression level ofMdCBF2showed thatMdLLZWRKY40isoverexpression in callus did not increase the expression level ofMdCBF2(Fig.5-D).This finding indicates that MdWRKY40is interacts with MdMYB15L to affect the expression ofMdCBF2.

Fig.5 MdWRKY40is affects the expression of MdCBF2 by interacting with MdMYB15L,thereby improving the cold tolerance of apple callus.A,the process of knocking out the conserved leucine zipper sequence in MdWRKY40is using overlap PCR.B,yeast two-hybrid analyses between MdWRKY40is or MdLLZWRKY40is and MdMYB15L.T,Trp;H,His;L,Leu;A,Ade;X-α-gal,X-α-Dgalactoside.C,BiFC analyses between MdWRKY40is and MdMYB15L.D,transgenic analysis of MdLLZWRKY40is in apple callus.GFP,‘Orin’ callus overexpressing empty plasmid;OEWRKY40is,‘Orin’ callus overexpressing MdWRKY40is;OELLZWRKY40is,‘Orin’ callus overexpressing MdLLZWRKY40is.Bars are SD (n=3).Different letters above the columns represent significant differences (P<0.01).

4.Discussion

4.1.MdWRKY40 can directly bind the MdDFR promoter and promote its expression,thereby initiating anthocyanin accumulation

Anthocyanin synthesis is mainly regulated by the MBW complex,in which MYB transcription factors,especially MYB10 and its homologs,play a key role (Xuet al.2015,2020;Cuiet al.2021).Many other transcription factors,such as bZIP and BBX,mediate light-induced anthocyanin accumulation (Fanget al.2019;Qiuet al.2019);while ERF is involved in ethylene regulation of anthocyanin synthesis (Zhanget al.2018).In recent years,some new progress has been made regarding the involvement of WRKY transcription factors in anthocyanin synthesis.A WRKY transcription factor in Petunia,PhPH3,plays a role downstream of the MBW complex,and its expression is associated with changes in petal color (Verweijet al.2016).ThePhPH3homologous gene in grapes,VvWRKY26,has also been reported to induce the accumulation of anthocyanins (Alessandraet al.2017).In this study in apple,we identified a nuclear-localized WRKY transcription factor,MdWRKY40is,and homologous overexpression ofMdWRKY40isinduced anthocyanin accumulation in apple callus (Fig.3-A,C,and D).DFR,ANSandUFGT,the late structural genes of anthocyanin synthesis,are target genes of transcription factors such as MYB and bHLH.The binding between these specific proteins and their promoters can initiate anthocyanin synthesis (Wanget al.2018;Xuet al.2020).However,the currently reported WRKY transcription factors are all indirectly involved in anthocyanin synthesis.For example,apple MdWRKY11 can promote the expression of the anthocyanin synthesis activatorMdMYB10,thereby indirectly promoting the expression of anthocyanin synthesis structural genes,leading to anthocyanin accumulation (Liuet al.2019).Alternatively,WRKY transcription factors can act on the MBW complex to affect anthocyanin synthesis.For example,MdWRKY40 can interact with MdMYB1 to participate in woundinginduced anthocyanin biosynthesis (Anet al.2019).In addition,PyWRKY26 of blush pear not only interacts with PybHLH3 but also promotes the expression of the anthocyanin synthesis activatorPyMYB114,thereby promoting the accumulation of anthocyanins (Liet al.2020).

No direct binding relationships between WRKY transcription factors and anthocyanin biosynthesis structural genes have been reported to date.In this study,we found that MdWRKY40is could significantly promote the expression of theMdDFRpromoter,but it had no significant effect on the anthocyanin biosynthesis activatorsMdMYB10andMdbHLH3(Fig.3-C).Yeast one-hybrid and EMSA revealed that MdWRKY40is can bind the promoter ofMdDFR(Fig.4),indicating that MdWRKY40is can directly promote anthocyanin accumulation by regulating the expression of the anthocyanin synthesis structural geneMdDFR.The anthocyanin synthesis process is a protective mechanism for adapting to cold stress,so this induced accumulation of anthocyanin can also be used as a potential protective factor against cold stress.

4.2.Cold stress-induced metabolism and the molecular mechanism of MdWRKY40is involvement in the cold signaling pathway

Low temperature is one of the important environmental factors affecting apple production in commercial apple cultivation areas,and apples are susceptible to impairment by early spring cold and late spring frost(Farajzadehet al.2010).The breeding of cold-resistant apple cultivars and rootstocks is an effective way to adapt to low-temperature stress.Therefore,it is necessary to fully explore cold resistance genes and understand the molecular mechanisms involved in cold signaling pathways.The plant cell membrane is the primary site of low-temperature injury (Kratsch and Wise 2000).Cold stress can cause changes in membrane structure and function.The greater the change in cell membrane permeability,the more severe the damage;and the degree of cell membrane damage is generally expressed as relative conductivity.Meanwhile,cold stress-induced reactive oxygen species and free radicals can lead to an increase in MDA,and its accumulation is an important indicator of lipid peroxidation (Mittleret al.2004).In this study,the MDA content and relative conductivity of‘M9T337’ and ‘60-160’ plantlets increased in the early stage (0 to 6 h) of the 1°C cold stress treatment.In the late stage (6 to 48 h),the ‘60-160’ plantlets showed higher cold tolerance and a decreasing trend in MDA content and relative conductivity,which reached levels significantly lower than those of ‘M9T337’ plantlets (Fig.1-A–C),indicating that their damage was mild.

In addition to changes in the membrane system and reactive oxygen species,plants can undergo corresponding physiological metabolic changes to adapt to a low-temperature environment.For example,sucrose and proline can act as osmotic regulators and reactive oxygen scavengers to stabilize proteins and plasma membranes by maintaining the osmotic pressure balance in order to protect plants from low-temperature damage(Brugièreet al.1999;Chen and Murata 2002;Stitt and Hurry 2002).In addition,various plant hormones can interact to mediate the cold stress response (Wanget al.2016).In this study,after ‘M9T337’ and ‘60-160’ plantlets were subjected to cold stress,their DEGs were enriched in plant hormone signal transduction,starch and sucrose metabolism,and arginine and proline metabolism,which are associated with cold stress.At the late stage of cold stress treatment,the DEGs of ‘M9T337’ plantlets were enriched in porphyrin and chlorophyll metabolism,which may be related to the leaf wilting of the plantlets,while the DEGs of ‘60-160’ plantlets were enriched in the flavonoid biosynthesis pathway (Fig.1-D and E),and the metabolites in that pathway are known protective factors against cold stress.They may contribute to the strong cold tolerance of the ‘60-160’ plantlets.

In recent years,research progress on the involvement of WRKY transcription factors in the cold stress response has indicated their role in plant cold tolerance.Wanget al.(2014) and Liet al.(2018) conducted genomewide analyses of WRKY family genes in grape and watermelon,respectively,and found that theWRKYgenes have different expression patterns in response to low-temperature signals,and that there are multiple regulatory pathways participating in cold resistance.The heterologous expression of Amur grapeVaWRKY33inArabidopsisand the homologous expression ofVbWRKY32inVerbena bonariensissignificantly improved the cold tolerance of the transgenic plants(Sunet al.2019;Wanget al.2020).In this study,we screened MdWRKY40is and MdWRKY48 as potential cold tolerance-related factors (Fig.2-A and B).The homologous transformation of apple callus showed that only MdWRKY40is could improve the cold tolerance of the callus and promote the expression of the core gene of cold signaling,MdCBF2(Fig.3-A,C and E).The role of MdWRKY40is in enhancing cold tolerance was also confirmed by heterologous expression inArabidopsis(Fig.3-B).There are only a few studies on the regulation ofCBFgenes by WRKY transcription factors in cold tolerance.For example,TaWRKY19 in wheat acts onCBF/DREBexpression to regulate plant cold tolerance (Niuet al.2012),while OsWRKY76 in rice can interact with OsbHLH148 to activate the expression ofOsDREB1Bto enhance cold tolerance (Zhanget al.2022).In our study,although MdWRKY40is promoted the expression ofMdCBF2,it could not directly bind to theMdCBF2promoter (Fig.4-B and C).We speculated that MdWRKY40is interacts with MdMYB15L,an inhibitor ofMdCBF2(Xuet al.2018).MdWRKY40is is a class IIa WRKY transcription factor whose N-terminus has a leucine zipper that interacts with other proteins (Chiet al.2013).Based on this knowledge,we first obtained MdLLZWRKY40is by overlap PCR,which lacked its leucine zipper in MdWRKY40is (Fig.5-A).MdWRKY40is interacted with MdMYB15L,while MdLLZWRKY40is did not (Fig.5-B and C),indicating that MdWRKY40is interacts with MdMYB15L through its N-terminal leucine zipper,which is consistent with the function of the leucine zipper domain.Further analysis of apple callus transformation showed that MdLLZWRKY40is still promoted the anthocyanin accumulation,but it did not improve the cold tolerance orMdCBF2expression in the callus (Fig.5-D).This suggests that MdWRKY40is promotes the expression ofMdCBF2and participates in the cold signaling pathway by interacting with MdMYB15L.In summary,MdWRKY40is interferes with the inhibitory effect of MdMYB15L onMdCBF2through the interaction between its own leucine zipper and MdMYB15L,thereby promoting the expression ofMdCBF2and improving cold tolerance.

5.Conclusion

The mechanism through which MdWRKY40is regulates anthocyanin synthesis and participates in the cold signaling pathway is shown in Fig.6.In this study,we used two rootstocks with different levels of cold resistance as test materials,and identified the nuclear-localized WRKY transcription factor MdWRKY40is in apple,which can bind to the promoter of the anthocyanin synthesis structural geneMdDFRand promote its expression,thereby initiating anthocyanin accumulation.Meanwhile,MdWRKY40is can also interact with MdMYB15L,an inhibitor of the cold signaling core geneMdCBF2,through its leucine zipper,thereby protecting the expression ofMdCBF2and improving the cold tolerance of apple callus.These results provide a new perspective for the study of the molecular mechanisms of anthocyanin biosynthesis and the cold signaling pathway,and they provide a molecular basis for the screening of cold-resistant rootstocks.

Fig.6 The regulatory model of MdWRKY40is involvement in anthocyanin biosynthesis and the cold signaling pathway.

Acknowledgements

The authors would like to thank AJE (www.aje.com) for its linguistic assistance during the preparation of this manuscript.This work was supported by the Natural Science Foundation of Shandong Province,China(ZR2021MC045),the Key Research &Development Plan(Major Scientific and Technological Innovation Project)of Shandong Province,China (2021LZGC024) and the earmarked fund for China Agriculture Research System(CARS-27).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.04.033