《园艺作物育种学》课程教学资源（学术研究）Comparative transcriptome profiling of chilling stress responsiveness in grafted watermelon seedlings

Plant Physiology and Biochemistry 109(2016)561-570 Contents lists available at ScienceDirect PPB Plant Physiology and Biochemistry ELSEVIER journal homepage:www.elsevier.com/locate/plaphy Research article Comparative transcriptome profiling of chilling stress responsiveness CrossMark in grafted watermelon seedlings Jinhua Xu .5,Man Zhang Guang Liu b,Xingping Yang B,Xilin Hou" State Key Laboratory of Crop Genetics and Germplasm Enhancement.College of Horticulture.Nanjing Agricultural University.Nanjing 210095.Jiangsu. China Institute of Vegetable.Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,Nanjing 210014. Jiangsu,China ARTICLE INFO ABSTRACT Article history: Rootstock grafting may improve the resistance of watermelon plants to low temperatures.However Received 6 April 2016 information regarding the molecular responses of rootstock grafted plants to chilling stress is limited.To Received in revised form elucidate the molecular mechanisms of chilling tolerance in grafted plants,the transcriptomic responses 3 November 2016 Accepted 3 November 2016 of grafted watermelon under chilling stress were analyzed using RNA-seq analysis.Sequencing data were Available online 5 November 2016 used for digital gene expression(DGE)analysis to characterize the transcriptomic responses in grafted watermelon seedlings.A total of 702 differentially-expressed genes(DEGs)were found in rootstock grafted(RG)watermelon relative to self-grafted(SG)watermelon:among these genes,522 genes were Keywords: Watermelon up-regulated and 180 were down-regulated.Additionally.164 and 953 genes were found to specifically Rootstock grafting expressed in RG and SG seedlings under chilling stress,respectively.Functional annotations revealed that Differentially-expressed gene(DEG) up-regulated DEGs are involved in protein processing.plant-pathogen interaction and the spliceosome. Chilling stress whereas down-regulated DEGs are associated with photosynthesis.Moreover,13 DEGs were randomly selected for quantitative real time PCR(gRT-PCR)analysis.The expression profiles of these 13 DEGs were consistent with those detected by the DGE analysis,supporting the reliability of the DGE data.This work provides additional insight into the molecular basis of grafted watermelon responses to chilling stress. 2016 Published by Elsevier Masson SAS 1.Introduction 2010).The root system activity and SOD activity of grafted cu- cumber seedlings were found to be higher than those of ungrafted Low temperature is one of the major environmental factors that cucumbers under chilling stress (Li et al.,2008).Zhou et al.(2009) severely limits plant growth and development,especially for the reported that figleaf gourd grafting significantly alleviated cu- chilling sensitive cultivated watermelon (C.lanatus).Watermelon cumber seedling growth inhibition by chilling at 7 C while also grows best at temperatures ranging from 21 to 29C,with growth increasing light utilization and reducing the accumulation of ceasing at 10C death occurring at temperatures of 1 C(Noh et al., reactive oxygen species after chilling.Inhibition of the light- 2013).Because of its low-temperature sensitivity,it is very difficult saturated rate of CO2 assimilation,the maximum carboxylation to obtain good yields and fruit quality during the cold seasons.To activity,Rubisco content and initial Rubisco activity were all found avoid such difficulties and improve the growth performance. to be weaker in grafted cucumber plants after chilling at 7C(Zhou watermelon seedlings are usually grafted onto rootstocks to confer et al.,2007).The relative growth rate of shoots and root mass ratios resistance to low temperatures (Lee and Oda,2003) increased at 15C when tomato seedlings were grafted onto a cold- Grafting has been attempted in several crops to increase plant tolerant rootstock(Venema et al.,2008).Grafted watermelon(Ding tolerance to low temperatures.Rootstocks alleviated the negative et al,2011)and muskmelon (Justus and Kubota,2010)seedlings effects of low temperatures on scion performance by supplying the were found to have better storability under low-temperature scion with more water,nutrients and hormones (Schwarz et al., storage than non-grafted seedlings.These studies have helped us to understand the physiological basis of increased low-temperature tolerance of rootstock-grafted plants.In addition to the physiolog- Corresponding author. ical responses,novel proteins were identified in pumpkin rootstock E-mail address:hxl@njau.edu.cn (X.Hou). grafted cucumber plants using proteomics techniques,and these http://dx.doi.org/10.1016/j.plaphy.2016.11.002 0981-9428/2016 Published by Elsevier Masson SAS

Research article Comparative transcriptome profiling of chilling stress responsiveness in grafted watermelon seedlings Jinhua Xu a, b , Man Zhang b , Guang Liu b , Xingping Yang b , Xilin Hou a, * a State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China b Institute of Vegetable, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, Jiangsu, China article info Article history: Received 6 April 2016 Received in revised form 3 November 2016 Accepted 3 November 2016 Available online 5 November 2016 Keywords: Watermelon Rootstock grafting Differentially-expressed gene (DEG) Chilling stress abstract Rootstock grafting may improve the resistance of watermelon plants to low temperatures. However, information regarding the molecular responses of rootstock grafted plants to chilling stress is limited. To elucidate the molecular mechanisms of chilling tolerance in grafted plants, the transcriptomic responses of grafted watermelon under chilling stress were analyzed using RNA-seq analysis. Sequencing data were used for digital gene expression (DGE) analysis to characterize the transcriptomic responses in grafted watermelon seedlings. A total of 702 differentially-expressed genes (DEGs) were found in rootstock grafted (RG) watermelon relative to self-grafted (SG) watermelon; among these genes, 522 genes were up-regulated and 180 were down-regulated. Additionally, 164 and 953 genes were found to specifically expressed in RG and SG seedlings under chilling stress, respectively. Functional annotations revealed that up-regulated DEGs are involved in protein processing, plant-pathogen interaction and the spliceosome, whereas down-regulated DEGs are associated with photosynthesis. Moreover, 13 DEGs were randomly selected for quantitative real time PCR (qRT-PCR) analysis. The expression profiles of these 13 DEGs were consistent with those detected by the DGE analysis, supporting the reliability of the DGE data. This work provides additional insight into the molecular basis of grafted watermelon responses to chilling stress. © 2016 Published by Elsevier Masson SAS. 1. Introduction Low temperature is one of the major environmental factors that severely limits plant growth and development, especially for the chilling sensitive cultivated watermelon (C. lanatus). Watermelon grows best at temperatures ranging from 21 to 29
C, with growth ceasing at 10
C death occurring at temperatures of 1
C (Noh et al., 2013). Because of its low-temperature sensitivity, it is very difficult to obtain good yields and fruit quality during the cold seasons. To avoid such difficulties and improve the growth performance, watermelon seedlings are usually grafted onto rootstocks to confer resistance to low temperatures (Lee and Oda, 2003). Grafting has been attempted in several crops to increase plant tolerance to low temperatures. Rootstocks alleviated the negative effects of low temperatures on scion performance by supplying the scion with more water, nutrients and hormones (Schwarz et al., 2010). The root system activity and SOD activity of grafted cu￾cumber seedlings were found to be higher than those of ungrafted cucumbers under chilling stress (Li et al., 2008). Zhou et al. (2009) reported that figleaf gourd grafting significantly alleviated cu￾cumber seedling growth inhibition by chilling at 7
C while also increasing light utilization and reducing the accumulation of reactive oxygen species after chilling. Inhibition of the light￾saturated rate of CO2 assimilation, the maximum carboxylation activity, Rubisco content and initial Rubisco activity were all found to be weaker in grafted cucumber plants after chilling at 7
C (Zhou et al., 2007). The relative growth rate of shoots and root mass ratios increased at 15
C when tomato seedlings were grafted onto a cold￾tolerant rootstock (Venema et al., 2008). Grafted watermelon (Ding et al., 2011) and muskmelon (Justus and Kubota, 2010) seedlings were found to have better storability under low-temperature storage than non-grafted seedlings. These studies have helped us to understand the physiological basis of increased low-temperature tolerance of rootstock-grafted plants. In addition to the physiolog￾ical responses, novel proteins were identified in pumpkin rootstock grafted cucumber plants using proteomics techniques, and these * Corresponding author. E-mail address: hxl@njau.edu.cn (X. Hou). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy http://dx.doi.org/10.1016/j.plaphy.2016.11.002 0981-9428/© 2016 Published by Elsevier Masson SAS. Plant Physiology and Biochemistry 109 (2016) 561e570

562 J.Xu et aL Plant Physiology and Biochemistry 109 (2016)561-570 proteins were further classified into two categories involved in 2.3.RNA-seg library preparation and illumina sequencing stress defense and photosynthesis (Li et al.,2009).Similarly.Yang et al.(2012)found 40 differentially expressed proteins in bottle Leaves from 6 RG or SG watermelon seedlings were pooled as gourd rootstock-grafted watermelon seedlings compared to the one single biological replicate.The experiment was repeated to self-grafted plants under salt exposure.These studies suggested the obtain three biological replicates.These pooled leaf samples were possibility that rootstocks could mediate gene expression patterns used for the RNA-seg analysis.Total RNA was isolated using TRIzol in scions under stress.However,studies on transcriptomic changes reagent (Invitrogen,Carlsbad,CA,USA)and digested using DNase I in rootstock grafted plants responding to chilling stress are lacking at 37 C for 30 min to remove any possible genomic DNA and are urgently required. contamination.The quality and concentration of each sample were In the present work,the squash rootstock-grafted watermelon determined using an Agilent 2100 Bioanalyzer.Six ug aliquots of seedlings (RG)were found to be more tolerant of chilling stress total RNA from each sample were purified using oligo(dT)magnetic than the self-grafted watermelon seedlings (SG).To explore the bead adsorption.Purified mRNAs were fragmented with endonu- differences in the gene expression between RG and SG watermelon clease and ligated with adaptors to generate libraries with unique seedlings under chilling stress,digital gene expression(DGE)based the 5'and 3'tags.After 15 cycles of linear PCR amplification,105 bp on Illumina sequencing was applied to identify differentially- PCR products were quantified and purified.Denatured molecules expressed genes(DEGs)between RG and SG.The transcriptome were then fixed onto an Illumina Sequencing Chip(Flow Cell)for data presented here provide straight forward information sequencing.RNA-seq libraries were sequenced on an Illumina regarding the molecular state of grafted plants challenged by HiSeqTM 2000 System by BGI-Tech(Shenzhen,China) chilling stress,which is important for understanding the tran- scriptomic changes of grafted watermelon plants in response to 2.4.Functional annotation of differentially-expressed genes (DEGs) chilling. Raw sequence reads were filtered through the lllumina pipeline by BGI Shenzhen,China.Gene expression levels were calculated 2.Methods using the RPKM (Reads Per kb per Million reads)method (Mortazavi et al.2008).Differentially expressed genes (DEG) 2.1.Plant material and chilling stress treatment among the samples were identified using the novel NOlseq method (Tarazona et al.,2011)with a probability>0.8 and an absolute value The watermelon line MW022 was used as the scion and the of the log2 Ratio>1 as the threshold to evaluate the significance squash Jingxinzhen NO.4 (JX)was used as the rootstock.An of gene expression differences.In brief,NOISeg method computes "insertion grafting"procedure described by Lee and Oda (2003) differential expression described as bellow:first,gene expression of was used in this study.Watermelon plants grafted onto their own sample in each group was used to calculate log2 (fold change)M roots were used as controls.Grafted seedlings were grown in a and absolute different value D of all pair conditions(gene expres- growth chamber at 28C/18C(16/8 h)day/night temperatures,a sion value will be substituted by 0.001 if it doesn't express in some relative humidity of 70%,and a photon flux destiny of sample).Second,average expression value of each gene standing 400 umol m-2 s-1.After the full development of the third true for replicates will be used to calculate M and D.Two replicates in leaves,the grafted plants were treated with a low temperature at one of the experimental conditions is sufficient to run the algo- 10 C at the same relative humidity and illumination intensity. Leaves from 6 rootstock-grafted (RG)or self-grafted (SG)water- rithm:Mi log2 and Di =Then,all these M/D values melon seedlings were pooled as one biological replicate at 0 d and are pooled together to generate the noise distribution.If gene i 1 d after the chilling treatment,respectively.Three biological rep- differentially expresses between two groups,we set Gi=1,other- licates were collected.All collected samples were immediately wise set Gi =0,and give a definition for probability of gene i frozen in liquid nitrogen and stored at-70C until use. differentially expressing as following formula P(G=1,)-P(G=1M=m,D=d） 2.2.Assessment of chilling damage index(Cl)and measurement of malonyldialdehyde (MDA)content -P(MI<m,D'<d） Eighteen rootstock-grafted(RG)or self-grafted (SG)watermelon When P is greater than threshold value,its corresponding gene seedlings were moved to a climate chamber at a temperature of is thought to differentially express between groups. 10 C with a 16 h light/8 h dark cycle.After 12 d of the chilling Gene Ontology(GO)was used to analyze biological functions by treatment,the chilling damage index was measured according to mapping all DEGs to GO databases (http://www.geneontology.org/ Yang et al.(2008).The degrees of chilling tolerance were measured )GO terms meeting a threshold of corrected p-value<0.05 were with 6 grades as follows:level 0:no symptom;level 1:chlorosis or defined as significantly enriched.The DEGs were also mapped to crinkled at the edge of old leaves;level 2:chlorosis or crinkled at the Kyoto Encyclopedia of Genes and Genomes (KEGG)database the edge of less functional leaves;level 3:chlorosis or crinkled at (Kanehisa et al.,2008)to identify significantly enriched metabolic the edge of functional leaves with good new leaves;level 4:chlo- pathways or signal transduction pathways. rosis or crinkled and wilting of functional leaves with damaged new leaves;level 5:severe damage of new leaves,plants wilting or 2.5.Quantitative real-time PCR (qRT-PCR)analysis death.The chilling indices(CI)of RG and SG seedlings was tabu- lated according to the following formula:Cl =(each Leave samples used for gRT-PCR were the same as those used for level x number of plants with the corresponding level)/total sequencing.Total RNA from watermelon leaves sampled at 0 d and number of measured plants. 1 d after chilling treatment was extracted using the RNApure Plant The MDA content was measured using 2-thiobarbituric acid as Kit with DNase I (CWBiotech,Beijing.P.R.China).The BU- described by Hodges et al.(1999). Superscript RT Kit was used for first-strand cDNA generation with

proteins were further classified into two categories involved in stress defense and photosynthesis (Li et al., 2009). Similarly, Yang et al. (2012) found 40 differentially expressed proteins in bottle gourd rootstock-grafted watermelon seedlings compared to the self-grafted plants under salt exposure. These studies suggested the possibility that rootstocks could mediate gene expression patterns in scions under stress. However, studies on transcriptomic changes in rootstock grafted plants responding to chilling stress are lacking and are urgently required. In the present work, the squash rootstock-grafted watermelon seedlings (RG) were found to be more tolerant of chilling stress than the self-grafted watermelon seedlings (SG). To explore the differences in the gene expression between RG and SG watermelon seedlings under chilling stress, digital gene expression (DGE) based on Illumina sequencing was applied to identify differentially￾expressed genes (DEGs) between RG and SG. The transcriptome data presented here provide straight forward information regarding the molecular state of grafted plants challenged by chilling stress, which is important for understanding the tran￾scriptomic changes of grafted watermelon plants in response to chilling. 2. Methods 2.1. Plant material and chilling stress treatment The watermelon line MW022 was used as the scion and the squash Jingxinzhen NO.4 (JX) was used as the rootstock. An “insertion grafting” procedure described by Lee and Oda (2003) was used in this study. Watermelon plants grafted onto their own roots were used as controls. Grafted seedlings were grown in a growth chamber at 28
C/18
C (16/8 h) day/night temperatures, a relative humidity of 70%, and a photon flux destiny of 400 mmol m2 s 1 . After the full development of the third true leaves, the grafted plants were treated with a low temperature at 10
C at the same relative humidity and illumination intensity. Leaves from 6 rootstock-grafted (RG) or self-grafted (SG) water￾melon seedlings were pooled as one biological replicate at 0 d and 1 d after the chilling treatment, respectively. Three biological rep￾licates were collected. All collected samples were immediately frozen in liquid nitrogen and stored at 70
C until use. 2.2. Assessment of chilling damage index (CI) and measurement of malonyldialdehyde (MDA) content Eighteen rootstock-grafted (RG) or self-grafted (SG) watermelon seedlings were moved to a climate chamber at a temperature of 10
C with a 16 h light/8 h dark cycle. After 12 d of the chilling treatment, the chilling damage index was measured according to Yang et al. (2008). The degrees of chilling tolerance were measured with 6 grades as follows: level 0: no symptom; level 1: chlorosis or crinkled at the edge of old leaves; level 2: chlorosis or crinkled at the edge of less functional leaves; level 3: chlorosis or crinkled at the edge of functional leaves with good new leaves; level 4: chlo￾rosis or crinkled and wilting of functional leaves with damaged new leaves; level 5: severe damage of new leaves, plants wilting or death. The chilling indices (CI) of RG and SG seedlings was tabu￾lated according to the following formula: CI ¼ P(each level  number of plants with the corresponding level)/total number of measured plants. The MDA content was measured using 2-thiobarbituric acid as described by Hodges et al. (1999). 2.3. RNA-seq library preparation and illumina sequencing Leaves from 6 RG or SG watermelon seedlings were pooled as one single biological replicate. The experiment was repeated to obtain three biological replicates. These pooled leaf samples were used for the RNA-seq analysis. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and digested using DNase I at 37
C for 30 min to remove any possible genomic DNA contamination. The quality and concentration of each sample were determined using an Agilent 2100 Bioanalyzer. Six mg aliquots of total RNA from each sample were purified using oligo (dT) magnetic bead adsorption. Purified mRNAs were fragmented with endonu￾clease and ligated with adaptors to generate libraries with unique the 50 and 30 tags. After 15 cycles of linear PCR amplification, 105 bp PCR products were quantified and purified. Denatured molecules were then fixed onto an Illumina Sequencing Chip (Flow Cell) for sequencing. RNA-seq libraries were sequenced on an Illumina HiSeq™ 2000 System by BGI-Tech (Shenzhen, China). 2.4. Functional annotation of differentially-expressed genes (DEGs) Raw sequence reads were filtered through the Illumina pipeline by BGI Shenzhen, China. Gene expression levels were calculated using the RPKM (Reads Per kb per Million reads) method (Mortazavi et al., 2008). Differentially expressed genes (DEG) among the samples were identified using the novel NOIseq method (Tarazona et al., 2011) with a probability0.8 and an absolute value of the jlog2 Ratioj  1 as the threshold to evaluate the significance of gene expression differences. In brief, NOISeq method computes differential expression described as bellow: first, gene expression of sample in each group was used to calculate log2 (fold change) M and absolute different value D of all pair conditions (gene expres￾sion value will be substituted by 0.001 if it doesn't express in some sample). Second, average expression value of each gene standing for replicates will be used to calculate M and D. Two replicates in one of the experimental conditions is sufficient to run the algo￾rithm: Mi ¼ log2 xi 1 xi 2 ! and Di ¼ xi 1 xi 2 . Then, all these M/D values are pooled together to generate the noise distribution. If gene i differentially expresses between two groups, we set Gi ¼ 1, other￾wise set Gi ¼ 0, and give a definition for probability of gene i differentially expressing as following formula: P Gi ¼ 1 xi 1; xi 2  ¼ P Gi ¼ 1 Mi ¼ mi ;Di ¼ di  ¼ P M* < mi ;D* <di  When P is greater than threshold value, its corresponding gene is thought to differentially express between groups. Gene Ontology (GO) was used to analyze biological functions by mapping all DEGs to GO databases (http://www.geneontology.org/ ). GO terms meeting a threshold of corrected p-value0.05 were defined as significantly enriched. The DEGs were also mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al., 2008) to identify significantly enriched metabolic pathways or signal transduction pathways. 2.5. Quantitative real-time PCR (qRT-PCR) analysis Leave samples used for qRT-PCR were the same as those used for sequencing. Total RNA from watermelon leaves sampled at 0 d and 1 d after chilling treatment was extracted using the RNApure Plant Kit with DNase I (CWBiotech, Beijing, P. R. China). The BU￾Superscript RT Kit was used for first-strand cDNA generation with 562 J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570

J.Xu et aL Plant Physiology and Biochemistry 109(2016)561-570 563 the oligo(dT)primer (Biouniquer,Beijing.P.R.China).Analysis of significantly increased compared with their control seedlings after qRT-PCR was carried out using the 1 x SYBR Green PCR Master Mix 1 d chilling treatment(P 0.05).Compared with RG seedlings (PE-Applied Biosystems.USA)and the GeneAmp 7300 Sequence chilled SG seedlings exhibited MDA concentrations nearly 1.2-fold Detection System (PE-Applied Biosystems,USA)according to higher (Fig.1D).These results indicate that RG and SG water- manufacturer's instructions.Expression profile of 13 randomly melon seedlings differ in their response to chilling stress,with selected DEGs were analyzed.The watermelon 18S rRNA gene rootstock grafting apparently enhancing the chilling tolerance of (GenBank accession No.AB490410)was used as a reference gene. watermelon. and relative expression levels of DEGs were normalized to the constitutive expression level of 18S rRNA and calculated using the 3.2.DGE sequencing in grafted watermelon leaves under chilling 2-AACT method (Livak and Schmittgen.2001:Yin et al..2001). stress Three technical replicates were performed for each biological replicate.SAS 9.2(SAS Institute,Cary,NC)was used for all statistical RNAs from RG and SG watermelon seedlings with no chilling analyses. treatment(C)or with chilling stress treatment(LT)were extracted to prepare four cDNA libraries for RNA-seq analysis.11,695,581 3.Results (99.22%).11,806,210(99.38%).12,074,690(99.22%)and12,275.446 (99.35%)clean reads remained in the SG-C,SG-LT,RG-C and RG-LT 3.1.Chilling damage index and the MDA content of SG and RG libraries,respectively,when reads containing adaptors,poly-N,and under chilling stress low quality reads were filtered.This data indicates that the sequencing depth was sufficient for the transcriptome coverage in To identify the effects of grafting on watermelon coffering cold watermelon (Table 1).A total of 10,256,476 (87.7%).10,410,835 tolerance,squash rootstock grafted (RG)watermelon and self- (88.18%).10.614,574(87.91%)and10,839,819(88.3%)clean reads in grafted (SG)watermelon seedlings were evaluated by cold stress SG-C,SG-LT,RG-C and RG-LT libraries were mapped to the refer- treatment.RG seedlings exhibited less severe wilting than SG ence watermelon genome by allowing a 3-bp mismatch.This sug- seedlings after 1d of treatment at 10C(Fig.1A and B).After 12 days gests that the DGE data are reliable and sufficient for subsequent of treatment,the chilling damage index(CI)of RG and SG seedlings bioinformatics analysis. was evaluated.The CI values of SG and RG were 3.0 and 2.1. respectively(Fig.1C).This phenotypic performance showed that RG 3.3.DEGs responding to low temperature in SG and RG exhibited better chilling-stress tolerance than SG.In addition,MDA content was measured to investigate physiological variation in RG The SG and RG transcriptome profiles were analyzed to find and SG seedlings.The MDA contents of RG and SG seedlings differentially expressed genes.Gene expression alterations in SG Self-grafted Rootstock-grafted SG RG D 25 05G ■RG 15 0.5 Fig.1.Phenotypes of grafted watermelon seedlings under chilling stress. A.Comparison of self-grafted and rootstock-grafted watermelon seedlings in control.B.Comparison of self-grafted and rootstock-grafted watermelon seedlings treated at 10C for 1d.C.Chilling damage index of SG and RG watermelon seedlings treated at 10 'C for 12d.D.MDA content of SG and RG watermelon seedlings treated at 10C for 1d.Values are means of 3 replicates.Vertical bars indicate standard error.Three biological replicates were used,with six plants per replicate.Values are presented as mean SE of three biological replicates.Lowercase letters(a,b and c)indicate the significant differences between RG and SG genotypes

the oligo (dT) primer (Biouniquer, Beijing, P. R. China). Analysis of qRT-PCR was carried out using the 1 x SYBR Green PCR Master Mix (PE-Applied Biosystems, USA) and the GeneAmp® 7300 Sequence Detection System (PE-Applied Biosystems, USA) according to manufacturer's instructions. Expression profile of 13 randomly selected DEGs were analyzed. The watermelon 18S rRNA gene (GenBank accession No. AB490410) was used as a reference gene, and relative expression levels of DEGs were normalized to the constitutive expression level of 18S rRNA and calculated using the 2△△CT method (Livak and Schmittgen, 2001; Yin et al., 2001). Three technical replicates were performed for each biological replicate. SAS 9.2 (SAS Institute, Cary, NC) was used for all statistical analyses. 3. Results 3.1. Chilling damage index and the MDA content of SG and RG under chilling stress To identify the effects of grafting on watermelon coffering cold tolerance, squash rootstock grafted (RG) watermelon and self￾grafted (SG) watermelon seedlings were evaluated by cold stress treatment. RG seedlings exhibited less severe wilting than SG seedlings after 1d of treatment at 10
C (Fig. 1A and B). After 12 days of treatment, the chilling damage index (CI) of RG and SG seedlings was evaluated. The CI values of SG and RG were 3.0 and 2.1, respectively (Fig. 1C). This phenotypic performance showed that RG exhibited better chilling-stress tolerance than SG. In addition, MDA content was measured to investigate physiological variation in RG and SG seedlings. The MDA contents of RG and SG seedlings significantly increased compared with their control seedlings after 1 d chilling treatment (P < 0.05). Compared with RG seedlings, chilled SG seedlings exhibited MDA concentrations nearly 1.2-fold higher (Fig. 1D). These results indicate that RG and SG water￾melon seedlings differ in their response to chilling stress, with rootstock grafting apparently enhancing the chilling tolerance of watermelon. 3.2. DGE sequencing in grafted watermelon leaves under chilling stress RNAs from RG and SG watermelon seedlings with no chilling treatment (C) or with chilling stress treatment (LT) were extracted to prepare four cDNA libraries for RNA-seq analysis. 11,695,581 (99.22%), 11,806,210 (99.38%), 12,074,690 (99.22%) and 12,275,446 (99.35%) clean reads remained in the SG-C, SG-LT, RG-C and RG-LT libraries, respectively, when reads containing adaptors, poly-N, and low quality reads were filtered. This data indicates that the sequencing depth was sufficient for the transcriptome coverage in watermelon (Table 1). A total of 10,256,476 (87.7%), 10,410,835 (88.18%), 10,614,574 (87.91%) and 10,839,819 (88.3%) clean reads in SG-C, SG-LT, RG-C and RG-LT libraries were mapped to the refer￾ence watermelon genome by allowing a 3-bp mismatch. This sug￾gests that the DGE data are reliable and sufficient for subsequent bioinformatics analysis. 3.3. DEGs responding to low temperature in SG and RG The SG and RG transcriptome profiles were analyzed to find differentially expressed genes. Gene expression alterations in SG Fig. 1. Phenotypes of grafted watermelon seedlings under chilling stress. A. Comparison of self-grafted and rootstock-grafted watermelon seedlings in control. B. Comparison of self-grafted and rootstock-grafted watermelon seedlings treated at 10
C for 1d. C. Chilling damage index of SG and RG watermelon seedlings treated at 10
C for 12d. D. MDA content of SG and RG watermelon seedlings treated at 10
C for 1d. Values are means of 3 replicates. Vertical bars indicate standard error. Three biological replicates were used, with six plants per replicate. Values are presented as mean ± SE of three biological replicates. Lowercase letters (a, b and c) indicate the significant differences between RG and SG genotypes. J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570 563

564 J.Xu et al Plant Physiology and Biochemistry 109 (2016)561-570 Table 1 Summary of sequencing data. sample Raw tag Clean tag TN 1P(粉) Tags mapped to genome TN TP(粉 SG-T 11.879.434 11.806.210 9938 10.410,835 88.18 RG-T 12,356.129 12.275.446 9935 10.839.819 88.3 SG-C 11,787.043 11.695.581 9922 10.256.476 87.7 RG-C 12.169.968 12.074.690 9922 10.614.574 87.91 and RG after 1 day of chilling stress were compared to their aquaporin(Fig.3.Table S7).Differences between the two methods respective controls.The significance of gene expression difference were on the scale of the fold changes,which may be due to the between SG and RG was assessed using the NOISeq method different sensitivity and algorithms between the two assay (Tarazona et al.,2011).A probability >0.8 and an absolute value of methods. the llog2 Ratio>1 were used as thresholds to define the significant differences in transcript abundance between two libraries.At 3.4.Functional classifications of DEGs control temperature,36 genes were differentially expressed in RG plants as compared to SG plants (Table S1,Fig.2A).After 1d of To determine the similarities and differences in chilling-induced chilling stress,a total of 1491 (973 up-and 518 down-regulated) transcriptomes between RG and SG,DEGs were analyzed for GO (Table S2)and 702(522 up-and 180 down-regulated)(Table S3) classification and enrichment.A total of 2193 DEGs(1491 in SG and chilling-responsive genes were identified in SG and RG,respec- 702 in RG)between control and chilling stress treated SG and RG tively.953 chilling-responsive genes (574 up-and 379 down- watermelon seedlings were categorized into 43 functional groups regulated)were exclusively identified in SG (Table S4).whereas using GO classifications (Fig.4).In the biological process category. 164 chilling-responsive genes (123 up-and 41 down-regulated) metabolic process,single-organism process,cellular process,and were uniquely observed in RG(Table S5).538 genes(399 up-and response to stimulus were the main groups.For the cellular com- 139 down-regulated)were commonly regulated by chilling stress ponents category,cell,cell part and organelle were the main in both SG and RG (Table S6,Fig.2B). groups.Among the molecular function category,binding and cat- To verify the RNA-seq based gene expression levels,13 DEGs alytic activity were the main groups.Additionally,we found that with differential expression patterns were randomly selected from very few differences could be observed between rootstock grated SG or RG DEGs for qRT-PCR analysis.Before the qRT-PCR assay,we watermelon plants and self-grafted plants in either up-regulated testified the stability of the reference gene.Results showed that DEGs or down-regulated DEGs.These results indicate that the expression of 18S rRNA was stable (Fig.S1)and thus was used as majority of DEGs responding to chilling stress were involved in the reference gene for data normalization.As expected,expression of metabolic process,cell and catalytic activity,which suggest that these genes verified by gRT-PCR showed the same expression chilling stress treatment mainly affects physiological metabolism patterns as in DGE analysis except for expansion protein and and cell differentiation in grafted watermelon. To further understand the biological functions of DEGs,pathway enrichment analysis was performed by identifying the metabolic A pathways or signal transduction pathways that were significantly 973 enriched in DEGs.Analysis of DEGs in SG plants assigned 575 DEGs 1050 to 104 KEGG pathways.In RG plants,274 DEGs were assigned to 77 900 KEGG pathways.Based on KEGG analysis,up-regulated DEGs in SG 750 were highly enriched in protein processing in the endoplasmic 600 518 522 reticulum (ko04141).plant-pathogen interaction (ko04626)and 450 the spliceosome(ko03040).Although the number of significantly ■up-regulated enriched terms for up-regulated DEGs in RG was less than that in 300 80 ■down-regulated SG,the main significantly enriched terms of up-regulated DEGs in 150 2115 RG were similar to those in SG.The photosynthesis-antenna pro- 0 teins (ko00196)pathway was the most common significantly SG-C vs RG-C SG-CvsSG-LT RG-Cvs RG-LT enriched term for down-regulated DEGs in SG and RG (Table S8). B 3.5.DEGs encoding transcription factors SG-Cvs SG-LT RG-Cvs RG-LT Among the DEGs of SG.141transcription factors were identified (Table S9),including WRKY(15).C3H(13),C2H2(12).NAC(12). bHLH(9).ERF(8).MYB(2)and MYB-related(9)families,etc.62 574 399 123 transcription factors were identified in the DEGs of RG,including 个 WRKY(6).C3H(4).C2H2(5).NAC(4).bHLH(6).ERF(5).and HSF(5) families,etc.Among the transcription factors that responded to low temperature in SG and RG,48 transcription factors were identified 379 139 41 in both SG and RG.14 transcription factors were found to be uniquely regulated in RG,including the ERF(3)and HSF(4)families. Fig.2.Differentially expressed genes (DEGs)among different libraries 3.6.Differentially expressed genes in RG watermelon leaves relative A.DEG numbers among no treatment(C)and low-temperature treatment(LT)libraries of RG and SG watermelon seedlings.B.Venn diagram of DEGs under the control and to SG leaves under chilling stress low-temperature conditions.The upwards or downwards arrows indicate the up-or down-regulated DEGs,respectively. There were 52 DEGs induced by rootstock when RG seedlings

and RG after 1 day of chilling stress were compared to their respective controls. The significance of gene expression difference between SG and RG was assessed using the NOISeq method (Tarazona et al., 2011). A probability 0.8 and an absolute value of the jlog2 Ratioj  1 were used as thresholds to define the significant differences in transcript abundance between two libraries. At control temperature, 36 genes were differentially expressed in RG plants as compared to SG plants (Table S1, Fig. 2A). After 1d of chilling stress, a total of 1491 (973 up- and 518 down-regulated) (Table S2) and 702 (522 up- and 180 down-regulated) (Table S3) chilling-responsive genes were identified in SG and RG, respec￾tively. 953 chilling-responsive genes (574 up- and 379 down￾regulated) were exclusively identified in SG (Table S4), whereas 164 chilling-responsive genes (123 up- and 41 down- regulated) were uniquely observed in RG (Table S5). 538 genes (399 up- and 139 down-regulated) were commonly regulated by chilling stress in both SG and RG (Table S6, Fig. 2B). To verify the RNA-seq based gene expression levels, 13 DEGs with differential expression patterns were randomly selected from SG or RG DEGs for qRT-PCR analysis. Before the qRT-PCR assay, we testified the stability of the reference gene. Results showed that expression of 18S rRNA was stable (Fig. S1) and thus was used as reference gene for data normalization. As expected, expression of these genes verified by qRT-PCR showed the same expression patterns as in DGE analysis except for expansion protein and aquaporin (Fig. 3, Table S7). Differences between the two methods were on the scale of the fold changes, which may be due to the different sensitivity and algorithms between the two assay methods. 3.4. Functional classifications of DEGs To determine the similarities and differences in chilling-induced transcriptomes between RG and SG, DEGs were analyzed for GO classification and enrichment. A total of 2193 DEGs (1491 in SG and 702 in RG) between control and chilling stress treated SG and RG watermelon seedlings were categorized into 43 functional groups using GO classifications (Fig. 4). In the biological process category, metabolic process, single-organism process, cellular process, and response to stimulus were the main groups. For the cellular com￾ponents category, cell, cell part and organelle were the main groups. Among the molecular function category, binding and cat￾alytic activity were the main groups. Additionally, we found that very few differences could be observed between rootstock grated watermelon plants and self-grafted plants in either up-regulated DEGs or down-regulated DEGs. These results indicate that the majority of DEGs responding to chilling stress were involved in the metabolic process, cell and catalytic activity, which suggest that chilling stress treatment mainly affects physiological metabolism and cell differentiation in grafted watermelon. To further understand the biological functions of DEGs, pathway enrichment analysis was performed by identifying the metabolic pathways or signal transduction pathways that were significantly enriched in DEGs. Analysis of DEGs in SG plants assigned 575 DEGs to 104 KEGG pathways. In RG plants, 274 DEGs were assigned to 77 KEGG pathways. Based on KEGG analysis, up-regulated DEGs in SG were highly enriched in protein processing in the endoplasmic reticulum (ko04141), plant-pathogen interaction (ko04626) and the spliceosome (ko03040). Although the number of significantly enriched terms for up-regulated DEGs in RG was less than that in SG, the main significantly enriched terms of up-regulated DEGs in RG were similar to those in SG. The photosynthesis-antenna pro￾teins (ko00196) pathway was the most common significantly enriched term for down-regulated DEGs in SG and RG (Table S8). 3.5. DEGs encoding transcription factors Among the DEGs of SG, 141transcription factors were identified (Table S9), including WRKY (15), C3H (13), C2H2 (12), NAC (12), bHLH (9), ERF (8), MYB (2) and MYB-related (9) families, etc. 62 transcription factors were identified in the DEGs of RG, including WRKY (6), C3H (4), C2H2 (5), NAC (4), bHLH (6), ERF (5), and HSF (5) families, etc. Among the transcription factors that responded to low temperature in SG and RG, 48 transcription factors were identified in both SG and RG. 14 transcription factors were found to be uniquely regulated in RG, including the ERF (3) and HSF (4) families. 3.6. Differentially expressed genes in RG watermelon leaves relative to SG leaves under chilling stress There were 52 DEGs induced by rootstock when RG seedlings Table 1 Summary of sequencing data. sample Raw tag Clean tag TN TP (%) Tags mapped to genome TN TP (%) SG-T 11,879,434 11,806,210 99.38 10,410,835 88.18 RG-T 12,356,129 12,275,446 99.35 10,839,819 88.3 SG-C 11,787,043 11,695,581 99.22 10,256,476 87.7 RG-C 12,169,968 12,074,690 99.22 10,614,574 87.91 Fig. 2. Differentially expressed genes (DEGs) among different libraries. A. DEG numbers among no treatment (C) and low-temperature treatment (LT) libraries of RG and SG watermelon seedlings. B. Venn diagram of DEGs under the control and low-temperature conditions. The upwards or downwards arrows indicate the up- or down-regulated DEGs, respectively. 564 J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570

J.Xu et aL Plant Physiology and Biochemistry 109(2016)561-570 565 were compared with SG seedlings under chilling stress (Table S10). 4.2.Specific responses to low-temperature stress in RG plants Among these genes,42 were up-regulated,including 3 genes (WMU41743,WMU41479,WMU50993)encoding phloem protein. Differences in DEG numbers and functional annotation in RG 1 gene (WMU49190)encoding LRR serine/threonine-protein ki- and SG libraries under chilling stress indicate that rootstock graft- nase,1 gene (WMU78566)encoding methyltransferase,1 gene ing enhanced the plant response to chilling stress in a different way. (WMU36427)encoding early nodulin-like protein 3-like and 1 gene Further analysis found a subset of DEGs in response to chilling (WMU31369)encoding expansin-A1-like.Two genes(WMU75582. stress that were specifically expressed only in rootstock-grafted WMU62304)showing the greatest increase in expression in RG seedlings (Table S5).Most of these genes were up-regulated un- have no annotation in the NCBI database.Ten genes were down der chilling stress,suggesting they may play roles in enhancing the regulated,including 2 genes(WMU44051,WMU41261)encoding chilling tolerance of rootstock-grafted seedlings. transcription factor,2 genes(WMU45207.WMU16709)encoding HARBI1-like gene,and 1 gene(WMU62127)encoding HSP25.5. 4.2.1.Metabolism Among the DEGs induced by rootstock under control condition, In the present work,27 transcripts related to metabolism were 6 genes in SG and 6 genes in RG were also responsive to low detected in RG seedlings under chilling stress.Of these,19 genes temperature too.Two genes(WMU22881,WMU28938)encoding were up-regulated and 8 genes were down-regulated.Glutathione aquaporin PIP2-1-like were up-regulated by rootstock in RG and S-transferases (GSTs).rate-limiting enzymes of the MAP pathway, up-regulated by low temperature in SG.Two genes (WMU63563, conjugate glutathione to several electrophilic substrates,and WMU11846)encoding HSP were down-regulated by rootstock and therefore,GSTs function in the plant detoxification system.The up-regulated by low temperature in RG. detoxifying activity of GSTs was found to be associated with path- ogen attack,oxidative and heavy-metal stress,and environmental stresses.Overexpression of GST improved the growth rate of trans- 4.Discussion genic tobacco seedlings exposed to chilling stress(Roxas et al.,1997). Expression of ScgstI and GST enzyme activity accumulated in cold 4.1.Rootstock grafted seedlings shown less sensitivity to chilling tolerant potato and in its hybrid with a sensitive line but declined in stress than self-grafted seedlings a freezing sensitive potato (Seppanen et al.,2000).Here,we observed two up-regulated GSTs after chilling stress in RG.sug- Grafting is regarded as a promising tool to increase the resis- gesting that rootstock grafting activates GST accumulation in tance of watermelon seedlings to chilling stress.To better under- response to chilling stress.perhaps through its detoxifying property. stand the molecular mechanisms of how rootstock regulate the 1-aminocyclopropane-1-carboxylate (ACC)oxidase catalyzes scion's low temperature tolerance,we used the Illumina HiSeg the conversion of ACC to ethylene,which is the second step of the 2000 system to investigate changes in transcriptomes in response ethylene biosynthetic pathway(Yang and Hoffman,1984).It has to low-temperature stress between rootstock grafted and self- been reported that ACC oxidase activity could be stimulated when grafted watermelon seedlings.The sequence data of the four apple fruit is exposed to low temperatures (Lelievre et al.,1995). analyzed libraries(SG-LT,SG-C,RG-LT,RG-C)consisted of approx- Down-regulation of ethylene biosynthesis related genes resulted in imately 12 million tags per library (Table 1),and the distribution of uneven ripening in cold-stored tomatoes(Rugkong et al..2011).In total clean tags was uniform across all sequenced libraries in the this report,two genes encoding ACC oxidase protein were up- whole dataset. regulated in RG.This may indicate that the chilling tolerance of To compare the transcriptome variation in RG and SG seedlings rootstock grafted seedlings may be a consequence of chilling- under low-temperatures,DEGs among the four libraries were induced ethylene accumulation caused by activation of ACC oxi- checked with a llog2 Ratio>1.Relatively fewer DEGs(702 DEGs) dase gene,or perhaps due to the protection elicited by the inter- related to chilling response were obtained in RG than that(1491 action of ethylene and methyl jasmonate (MelA)(Yu et al.,2011). DEGs)in SG seedlings.Additionally,52 DEGs were found when Protein disulfide isomerase(PDI)is a member of the thioredoxin comparing RG-LT and SG-LT libraries,which is fewer than the super family and is considered a major catalysts for protein folding number of DEGs found in RG and SG when comparing chilling and in the lumen of the endoplasmic reticulum(ER).It is well estab- control treatments.The number did not support our hypothesis lished that PDI possesses multiple function,including acting as a that rootstock grafting may intensively alter the regulation of the molecular chaperone(Huang et al.,2005).Transgenic rice seedlings scion's transcriptome to respond to chilling stress.A similar result with high Hg tolerance and more effective photosynthesis showed was also reported in previous comparative transcriptome work in a correlation with overexpression of the PDI gene MTH1745 (Chen freezing-sensitivity and cold-hardness for grapes under cold stress et al.,2012).In this experiment,two PDIs were up-regulated in (Xin et al.,2013).In this study,the authors explained that the lower RG seedlings under chilling stress.This may suggest that the number of DEGs was because of poor alignment and annotation of accumulation of PDI may increase the stability of proteins and wild grape genes to the reference genome(43.61%in cold treated therefore enhance chilling tolerance in RG seedling. and 39.4%in control libraries).While this explanation does not Two 9-cis-epoxycarotenoid dioxygenase (NCED)genes were apply to our work,as approximately 88%of the unique tags were found to be up-regulated in the chilling-tolerant rootstock grafted found to be aligned to reference genes in both RG and SG libraries watermelon seedlings.Previous studies have shown that expres- (Table 1).Thus,we suggest that rootstock grafting reduced the sion of NCED genes can be induced by environmental stresses such sensitivity of watermelon seedlings to chilling stress and modified as drought,cold,salt and osmotic stresses (Gomez-Cadenas et al. gene expression patterns at the transcriptome level in a different 2003:Zhang et al,2014).Overexpression of NCED has been found manner during chilling stress.Moreover,it was found that more of to enhance plant tolerance to multiple abiotic stresses(Xian et al., the DEGs in RG libraries (75%)were up-regulated under chilling 2014).NCED indirectly catalyzes the conversion of C40-caroten- treatment compared to SG(60%),which indicates that these genes oids to ABA,which is considered the key regulatory step in the ABA are involved in the chilling stress response and that rootstock biosynthesis.Therefore,it is presumed that enhanced plant toler- grafting could induce the expression of some essential genes ance to chilling stress from rootstock grafting may be associated related to chilling tolerance to enhance plant response to low- with the increase in ABA accumulation which is caused by temperature stress (Gu et al.,2015). expression of NCED genes

were compared with SG seedlings under chilling stress (Table S10). Among these genes, 42 were up-regulated, including 3 genes (WMU41743, WMU41479, WMU50993) encoding phloem protein, 1 gene (WMU49190) encoding LRR serine/threonine-protein ki￾nase, 1 gene (WMU78566) encoding methyltransferase, 1 gene (WMU36427) encoding early nodulin-like protein 3-like and 1 gene (WMU31369) encoding expansin-A1-like. Two genes (WMU75582, WMU62304) showing the greatest increase in expression in RG have no annotation in the NCBI database. Ten genes were down￾regulated, including 2 genes (WMU44051, WMU41261) encoding transcription factor, 2 genes (WMU45207, WMU16709) encoding HARBI1-like gene, and 1 gene (WMU62127) encoding HSP25.5. Among the DEGs induced by rootstock under control condition, 6 genes in SG and 6 genes in RG were also responsive to low temperature too. Two genes (WMU22881, WMU28938) encoding aquaporin PIP2-1-like were up-regulated by rootstock in RG and up-regulated by low temperature in SG. Two genes (WMU63563, WMU11846) encoding HSP were down-regulated by rootstock and up-regulated by low temperature in RG. 4. Discussion 4.1. Rootstock grafted seedlings shown less sensitivity to chilling stress than self-grafted seedlings Grafting is regarded as a promising tool to increase the resis￾tance of watermelon seedlings to chilling stress. To better under￾stand the molecular mechanisms of how rootstock regulate the scion's low temperature tolerance, we used the Illumina HiSeq 2000 system to investigate changes in transcriptomes in response to low-temperature stress between rootstock grafted and self￾grafted watermelon seedlings. The sequence data of the four analyzed libraries (SG-LT, SG-C, RG-LT, RG-C) consisted of approx￾imately 12 million tags per library (Table 1), and the distribution of total clean tags was uniform across all sequenced libraries in the whole dataset. To compare the transcriptome variation in RG and SG seedlings under low-temperatures, DEGs among the four libraries were checked with a jlog2 Ratioj  1. Relatively fewer DEGs (702 DEGs) related to chilling response were obtained in RG than that (1491 DEGs) in SG seedlings. Additionally, 52 DEGs were found when comparing RG-LT and SG-LT libraries, which is fewer than the number of DEGs found in RG and SG when comparing chilling and control treatments. The number did not support our hypothesis that rootstock grafting may intensively alter the regulation of the scion's transcriptome to respond to chilling stress. A similar result was also reported in previous comparative transcriptome work in freezing-sensitivity and cold-hardness for grapes under cold stress (Xin et al., 2013). In this study, the authors explained that the lower number of DEGs was because of poor alignment and annotation of wild grape genes to the reference genome (43.61% in cold treated and 39.4% in control libraries). While this explanation does not apply to our work, as approximately 88% of the unique tags were found to be aligned to reference genes in both RG and SG libraries (Table 1). Thus, we suggest that rootstock grafting reduced the sensitivity of watermelon seedlings to chilling stress and modified gene expression patterns at the transcriptome level in a different manner during chilling stress. Moreover, it was found that more of the DEGs in RG libraries (75%) were up-regulated under chilling treatment compared to SG (60%), which indicates that these genes are involved in the chilling stress response and that rootstock grafting could induce the expression of some essential genes related to chilling tolerance to enhance plant response to low￾temperature stress (Gu et al., 2015). 4.2. Specific responses to low-temperature stress in RG plants Differences in DEG numbers and functional annotation in RG and SG libraries under chilling stress indicate that rootstock graft￾ing enhanced the plant response to chilling stress in a different way. Further analysis found a subset of DEGs in response to chilling stress that were specifically expressed only in rootstock-grafted seedlings (Table S5). Most of these genes were up-regulated un￾der chilling stress, suggesting they may play roles in enhancing the chilling tolerance of rootstock-grafted seedlings. 4.2.1. Metabolism In the present work, 27 transcripts related to metabolism were detected in RG seedlings under chilling stress. Of these, 19 genes were up-regulated and 8 genes were down-regulated. Glutathione S-transferases (GSTs), rate-limiting enzymes of the MAP pathway, conjugate glutathione to several electrophilic substrates, and therefore, GSTs function in the plant detoxification system. The detoxifying activity of GSTs was found to be associated with path￾ogen attack, oxidative and heavy-metal stress, and environmental stresses. Overexpression of GST improved the growth rate of trans￾genic tobacco seedlings exposed to chilling stress (Roxas et al., 1997). Expression of Scgst1 and GST enzyme activity accumulated in cold tolerant potato and in its hybrid with a sensitive line but declined in a freezing sensitive potato (Seppanen et al., 2000 € ). Here, we observed two up-regulated GSTs after chilling stress in RG, sug￾gesting that rootstock grafting activates GST accumulation in response to chilling stress, perhaps through its detoxifying property. 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the conversion of ACC to ethylene, which is the second step of the ethylene biosynthetic pathway (Yang and Hoffman, 1984). It has been reported that ACC oxidase activity could be stimulated when apple fruit is exposed to low temperatures (Lelievre et al., 1995 ). Down-regulation of ethylene biosynthesis related genes resulted in uneven ripening in cold-stored tomatoes (Rugkong et al., 2011). In this report, two genes encoding ACC oxidase protein were up￾regulated in RG. This may indicate that the chilling tolerance of rootstock grafted seedlings may be a consequence of chilling￾induced ethylene accumulation caused by activation of ACC oxi￾dase gene, or perhaps due to the protection elicited by the inter￾action of ethylene and methyl jasmonate (MeJA) (Yu et al., 2011). Protein disulfide isomerase (PDI) is a member of the thioredoxin super family and is considered a major catalysts for protein folding in the lumen of the endoplasmic reticulum (ER). It is well estab￾lished that PDI possesses multiple function, including acting as a molecular chaperone (Huang et al., 2005). Transgenic rice seedlings with high Hg tolerance and more effective photosynthesis showed a correlation with overexpression of the PDI gene MTH1745 (Chen et al., 2012). In this experiment, two PDIs were up-regulated in RG seedlings under chilling stress. This may suggest that the accumulation of PDI may increase the stability of proteins and therefore enhance chilling tolerance in RG seedling. Two 9-cis-epoxycarotenoid dioxygenase (NCED) genes were found to be up-regulated in the chilling-tolerant rootstock grafted watermelon seedlings. Previous studies have shown that expres￾sion of NCED genes can be induced by environmental stresses such as drought, cold, salt and osmotic stresses (Gomez-Cadenas et al., 2003; Zhang et al., 2014). Overexpression of NCED has been found to enhance plant tolerance to multiple abiotic stresses (Xian et al., 2014). NCED indirectly catalyzes the conversion of C40-caroten￾oids to ABA, which is considered the key regulatory step in the ABA biosynthesis. Therefore, it is presumed that enhanced plant toler￾ance to chilling stress from rootstock grafting may be associated with the increase in ABA accumulation which is caused by expression of NCED genes. J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570 565

566 J.Xu et al Plant Physiology and Biochemistry 109 (2016)561-570 chlorophyll a-b binding protein calcium-binding protein 1.20 1500.C0 20000 8 1500.00 50000 00 0.0 nG-C RG-LT 5G-C G-L HSF C1 HSF24 40. RG-C RG-4T s-c G-0 RG-LT 5G-C SG-LT NAC zinc transporter 1200 70a00 ◆ 4500 3000 1500 000 RG-C RG-T 36-c 5G-T RG-C RG-LT SG-C ERF/DERB 1C WRKY transcription factor 600 45.00 40 00 0.00 RG-C RG-LT SG-C SG-T HSP expansin protein 1600m 1200m 0.00 RG-LT SG-C 6-r RG-C RG-LT SG-T Transducin/WD40 repeat-like aquaporin superfamily protein 500 100.0 30.m a00 nc-C G-L RG-LT LRR-family protein ■TPCR G- G-LT

566 J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570

J.Xu et aL Plant Physiology and Biochemistry 109(2016)561-570 567 single-organism process signaling rhythmic process response to stimulus reproductive process reproduction Down-regulated genes in SG regulation of biological process Down-regulated genes in RG positive regulation of biological process negative regulation of biological process Up-regulated genes in SG multicellular organismal process Up-regulated genes in RG multi-organism process metabolic process Process locomotion localization immune system process growth establishment of localization developmental process cellular process cellular component organization or biogenesis biological regulation biological adhesion transporter activity structural molecule activity receptor activity Molecular Function protein binding transcription factor activity nucleic acid binding transcription factor activity molecular transducer activity enzyme regulator activity elevtron carrier activity catalytic activity binding antioxidant activity symplast organelle part organelle nucleoid Cellular membrane-enclosed lumen membrane part membrane Component macromolecular complex extracellular region part extracellular region extracellular matrix part extracellular matrix cell part cell junction cell 50% 40%30%20%10%0% 10%20%30%40%50% Fig.4.Gene ontology functional analysis of DEGs in RG-LT and SG-LT libraries.The x-axis indicates the percentage of up-or down-regulated genes in RG or SG libraries under chilling stress in each GO term.The y-axis shows the names of functional categories. 4.2.2.Transport to maintain structural integrity by using metal ions as scaffolds In this work,four genes related to transport were up-regulated (Mohan et al.,2010).The strong expression of this gene might be in RG seedlings under chilling stress as follows:metal ion binding one of the chilling tolerance mechanisms exhibited by rootstock protein,heavy metal-associated isoprenylated plant protein(HIPP). grafted plants. DnaJ protein and zinc transporter.Metal ion binding proteins not HIPP proteins are a new class of plant proteins with the only play important roles in many biological processes but also help following two conserved domains:a heavy metal associated Fig.3.Confirmation of expression of 13 randomly-selected DEGs by qRT-PCR analysis.The x-axis indicates the four samples.RG:rootstock grafted watermelon:SG:self-grafted watermelon:C:no treatment:LT:chilling stress treatment.The y-axis shows the expression leveL Squares represent expression from DEG analysis.Columns indicate relative expression as measured by qRT-PCR Relative levels of gene expression were calculated using 18sRNA.PCR reactions were performed three times.Error bars indicate standard error of the mean

4.2.2. Transport In this work, four genes related to transport were up-regulated in RG seedlings under chilling stress as follows: metal ion binding protein, heavy metal-associated isoprenylated plant protein (HIPP), DnaJ protein and zinc transporter. Metal ion binding proteins not only play important roles in many biological processes but also help to maintain structural integrity by using metal ions as scaffolds (Mohan et al., 2010). The strong expression of this gene might be one of the chilling tolerance mechanisms exhibited by rootstock grafted plants. HIPP proteins are a new class of plant proteins with the following two conserved domains: a heavy metal associated Fig. 3. Confirmation of expression of 13 randomly-selected DEGs by qRT-PCR analysis. The x-axis indicates the four samples. RG: rootstock grafted watermelon; SG: self-grafted watermelon; C: no treatment; LT: chilling stress treatment. The y-axis shows the expression level. Squares represent expression from DEG analysis. Columns indicate relative expression as measured by qRT-PCR. Relative levels of gene expression were calculated using 18sRNA. PCR reactions were performed three times. Error bars indicate standard error of the mean. Fig. 4. Gene ontology functional analysis of DEGs in RG-LT and SG-LT libraries. The x-axis indicates the percentage of up- or down-regulated genes in RG or SG libraries under chilling stress in each GO term. The y-axis shows the names of functional categories. J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570 567

568 J.Xu et al Plant Physiology and Biochemistry 109(2016)561-570 domain (HAM)and an isoprenylation motif at the C-terminal end. temperature and dehydration(Shinozaki and Yamaguchi-Shinozaki HAM proteins have been reported to play important roles in metal 2000).In this work,one elicitor-responsive protein,one salt toler- transport and metal homeostasis processes.Isoprenylation acts in ance protein and one CASP-like protein were identified specifically post-translational modification of regulatory proteins(Barth et al., up-regulated in RG seedlings under chilling stress,which suggests 2009).Barth et al.(2004)observed the induction of barley HvFP1,a that rootstock grafting take a multiple stress-related networks that group Ill HIPP gene,during cold stress.In the present work,a HIPP can identify and react to elicitors in a more efficient manner,thus gene was up-regulated after 1-d of low temperature treatment in eliciting the defense response of grafted plants. RG seedlings,suggesting that HIPP also plays a role in the chilling stress response. 4.2.6.Transcription factors DnaJ proteins are a family of conserved co-chaperones for Transcription factors(TFs)mediate the gene transcription pro- HSP70s and are involved in responses to various stresses(Rajan and cesses by initiating transcription of defense genes in plant innate D'Silva,2009),chloroplast movement(Suetsugu et al.,2005),and immune systems.Many TFs have been shown to function in plant developmental processes (Shen et al.,2011).Plants with overex- resistance against various stresses,especially low temperature(Luo pressed Dnal in transgenic tobacco exhibited higher tolerance to et al.,2012).In this work,11 stress related TFs were found to be drought stress and resistance to P.solanacearum (Wang et al.,2014) uniquely present in RG libraries under chilling stress,including 5 In the present work,we observed the up-regulation of a Dnal HSFs,4 bHLHs and 2 other TFs.The heat shock transcription factor protein in RG seedlings under chilling stress.This may indicate that (HSF)family has been implicated in various abiotic stresses(Mittal low sensitivity of RG seedlings to chilling stress might be a conse- et al.,2009).Rice Hsf genes showed inducible expression in quence of reduced accumulation of H2O2 and O2 caused by DNAJ response to low temperature stress. gene expression. Basic helix-loop-helix(bHLH)proteins are the second largest transcription factor familiy in plant genomes (Castilhos et al.,2014: 4.2.3.Signal transduction Jin et al.,2014).The bHLH family has been shown to be one of the Four genes function as signal transduction,and two of these major plant regulators involved in various plant physiological and were found to be up-regulated while one was down-regulated.Two developmental processes(Sajeevan and Nataraja,2016)and abiotic (WMU07974,WMU31278)of them were identified as two- stresses,such as drought,low temperature,and osmotic stress component response regulator-like proteins,which are involved (Dong et al,2014:Chinnusamy et al,2003;Feng et al.2012:Liu in the His-Asp phosphorelay signal transduction and are reported et al.,2013).Many studies have shown that the stress tolerance of play important roles in various developmental and environmental plants is a complex stress signaling and response networks of conditions by regulating different stress-responsive genes(Mason plants.It has been reported that HsfA genes exclusively induced the et al.,2010).WMU37022 was predicted to be a receptor-like pro- expression of DREB2A (Liu et al.,2011).HaDREB2 and HaHSFA9 tein kinase(RLK).It is well established that RLKs contribute greatly interact in vitro and mediate synergistic transactivation (Mizoi in plant responses to both biotic and abiotic stresses.Over- et al.,2012).The apple bHLH gene MdClbHLH1 activated MdCBF2 expression of GsLRPK enhanced the resistance of yeast and Arabi- gene expression through the CBF pathway and promoted the cold dopsis to cold stress and increased the expression of a number of tolerance of transgenic apple plants (Feng et al.,2012).Similar re- cold responsive gene markers(Yang et al,2014).The up-regulation sults were also obtained by Feng et al.(2013).where SlICEla of these genes suggests that several signal transduction pathways interacted with CBF/DREB and enhanced the freezing tolerance of are involved in the chilling tolerance of RG seedlings. transgenic tobacco.These works suggest that various transcription factors cross talk with one another for their maximal response to 4.2.4.HSP environmental stresses and play crucial roles in the signal trans- Heat shock proteins(HSPs)constitute a stress-responsive family duction network (Yamaguchi-Shinozaki and Shinozaki,2006). of proteins that are essential for maintaining cellular homeostasis under stressful conditions in plants.The accumulation of HSPs 5.Conclusion protects plants against stress and against any subsequent stressful situation (Aghdam et al..2013).There are five families of HSPs as A number of differentially expressed genes related to rootstock follows:HSP60s,HSP70s,HSP90s,HSP100s and small HSPs(sHSPs) grafting regulated chilling tolerance were identified using the In this work,9 genes were found to encode HSPs,and all of them Illumina sequencing based DGE,and the expression patterns of 13 were up-regulated.In addition to heat shock,other environmental randomly selected DEGs were further validated by gRT-PCR.These stresses including low temperatures that cause protein misfolding DEGs were involved in multiple biological functions,including the and aggregation,may trigger the accumulation of HSPs.Expression induction of protein processing.plant-pathogen interaction,the of CsHSP17.5 was quickly activated when chestnut seedlings were spliceosome and suppression of photosynthesis.We also investi- exposed to cold conditions.Moreover,purified CsHSP17.5 signifi- gated the transcription factors respond to low-temperature stress cantly protected the cold-labile enzyme lactate dehydrogenase in rootstock grafted plants.This work provides comprehensive from freeze-induced inactivation (Lopez-Matas et al.,2004). insight into the molecular basis of the gene expression profiles of Recently,it was reported that sHSPs are essential for maintaining rootstock grafted watermelon seedlings in response to chilling membrane quality attributes under chilling stress (Aghdam et al., stress.Further research will focus on the functional characteriza- 2013).These results suggest that HSPs can play relevant roles in tion of these candidate genes of grafted watermelon seedlings the acquisition of cold tolerance under chilling stress via transgenic approaches. 4.2.5.Stress related genes Contributions Molecular and cellular responses of plants to low temperature stress are complex.A variety of stress-inducible genes were induced, I.X..X.Y.and X.H.conceived the idea and led the study design. which function not only in protecting plant cells by producing J.X.carried out the experiment,performed analyses and wrote the functional proteins but also in regulating genes that are involved in pater.M.Z.assisted with data analysis and writing.G.L assisted signal transduction pathways.Cross-talk has been shown to exist with material preparation.All authors contributed to the editing of between the following two different stress signaling pathways:low the manuscript

domain (HAM) and an isoprenylation motif at the C-terminal end. HAM proteins have been reported to play important roles in metal transport and metal homeostasis processes. Isoprenylation acts in post-translational modification of regulatory proteins (Barth et al., 2009). Barth et al. (2004) observed the induction of barley HvFP1, a group III HIPP gene, during cold stress. In the present work, a HIPP gene was up-regulated after 1-d of low temperature treatment in RG seedlings, suggesting that HIPP also plays a role in the chilling stress response. DnaJ proteins are a family of conserved co-chaperones for HSP70s and are involved in responses to various stresses (Rajan and D'Silva, 2009), chloroplast movement (Suetsugu et al., 2005), and developmental processes (Shen et al., 2011). Plants with overex￾pressed DnaJ in transgenic tobacco exhibited higher tolerance to drought stress and resistance to P. solanacearum (Wang et al., 2014). In the present work, we observed the up-regulation of a DnaJ protein in RG seedlings under chilling stress. This may indicate that low sensitivity of RG seedlings to chilling stress might be a conse￾quence of reduced accumulation of H2O2 and O2 caused by DNAJ gene expression. 4.2.3. Signal transduction Four genes function as signal transduction, and two of these were found to be up-regulated while one was down-regulated. Two (WMU07974, WMU31278) of them were identified as two￾component response regulator-like proteins, which are involved in the His-Asp phosphorelay signal transduction and are reported play important roles in various developmental and environmental conditions by regulating different stress-responsive genes (Mason et al., 2010). WMU37022 was predicted to be a receptor-like pro￾tein kinase (RLK). It is well established that RLKs contribute greatly in plant responses to both biotic and abiotic stresses. Over￾expression of GsLRPK enhanced the resistance of yeast and Arabi￾dopsis to cold stress and increased the expression of a number of cold responsive gene markers (Yang et al., 2014). The up-regulation of these genes suggests that several signal transduction pathways are involved in the chilling tolerance of RG seedlings. 4.2.4. HSP Heat shock proteins (HSPs) constitute a stress-responsive family of proteins that are essential for maintaining cellular homeostasis under stressful conditions in plants. The accumulation of HSPs protects plants against stress and against any subsequent stressful situation (Aghdam et al., 2013). There are five families of HSPs as follows: HSP60s, HSP70s, HSP90s, HSP100s and small HSPs (sHSPs). In this work, 9 genes were found to encode HSPs, and all of them were up-regulated. In addition to heat shock, other environmental stresses including low temperatures that cause protein misfolding and aggregation, may trigger the accumulation of HSPs. Expression of CsHSP17.5 was quickly activated when chestnut seedlings were exposed to cold conditions. Moreover, purified CsHSP17.5 signifi- cantly protected the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (Lopez-Matas et al., 2004). Recently, it was reported that sHSPs are essential for maintaining membrane quality attributes under chilling stress (Aghdam et al., 2013). These results suggest that HSPs can play relevant roles in the acquisition of cold tolerance. 4.2.5. Stress related genes Molecular and cellular responses of plants to low temperature stress are complex. A variety of stress-inducible genes were induced, which function not only in protecting plant cells by producing functional proteins but also in regulating genes that are involved in signal transduction pathways. Cross-talk has been shown to exist between the following two different stress signaling pathways: low temperature and dehydration (Shinozaki and Yamaguchi-Shinozaki, 2000). In this work, one elicitor-responsive protein, one salt toler￾ance protein and one CASP-like protein were identified specifically up-regulated in RG seedlings under chilling stress, which suggests that rootstock grafting take a multiple stress-related networks that can identify and react to elicitors in a more efficient manner, thus eliciting the defense response of grafted plants. 4.2.6. Transcription factors Transcription factors (TFs) mediate the gene transcription pro￾cesses by initiating transcription of defense genes in plant innate immune systems. Many TFs have been shown to function in plant resistance against various stresses, especially low temperature (Luo et al., 2012). In this work, 11 stress related TFs were found to be uniquely present in RG libraries under chilling stress, including 5 HSFs, 4 bHLHs and 2 other TFs. The heat shock transcription factor (HSF) family has been implicated in various abiotic stresses (Mittal et al., 2009). Rice Hsf genes showed inducible expression in response to low temperature stress. Basic helixeloopehelix (bHLH) proteins are the second largest transcription factor familiy in plant genomes (Castilhos et al., 2014; Jin et al., 2014). The bHLH family has been shown to be one of the major plant regulators involved in various plant physiological and developmental processes (Sajeevan and Nataraja, 2016) and abiotic stresses, such as drought, low temperature, and osmotic stress (Dong et al., 2014; Chinnusamy et al., 2003; Feng et al., 2012; Liu et al., 2013). Many studies have shown that the stress tolerance of plants is a complex stress signaling and response networks of plants. It has been reported that HsfA genes exclusively induced the expression of DREB2A (Liu et al., 2011). HaDREB2 and HaHSFA9 interact in vitro and mediate synergistic transactivation (Mizoi et al., 2012). The apple bHLH gene MdClbHLH1 activated MdCBF2 gene expression through the CBF pathway and promoted the cold tolerance of transgenic apple plants (Feng et al., 2012). Similar re￾sults were also obtained by Feng et al. (2013), where SlICE1a interacted with CBF/DREB and enhanced the freezing tolerance of transgenic tobacco. These works suggest that various transcription factors cross talk with one another for their maximal response to environmental stresses and play crucial roles in the signal trans￾duction network (Yamaguchi-Shinozaki and Shinozaki, 2006). 5. Conclusion A number of differentially expressed genes related to rootstock grafting regulated chilling tolerance were identified using the Illumina sequencing based DGE, and the expression patterns of 13 randomly selected DEGs were further validated by qRT-PCR. These DEGs were involved in multiple biological functions, including the induction of protein processing, plant-pathogen interaction, the spliceosome and suppression of photosynthesis. We also investi￾gated the transcription factors respond to low-temperature stress in rootstock grafted plants. This work provides comprehensive insight into the molecular basis of the gene expression profiles of rootstock grafted watermelon seedlings in response to chilling stress. Further research will focus on the functional characteriza￾tion of these candidate genes of grafted watermelon seedlings under chilling stress via transgenic approaches. Contributions J.X., X.Y. and X.H. conceived the idea and led the study design. J.X. carried out the experiment, performed analyses and wrote the pater. M.Z. assisted with data analysis and writing. G.L. assisted with material preparation. All authors contributed to the editing of the manuscript. 568 J. Xu et al. / Plant Physiology and Biochemistry 109 (2016) 561e570

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Acknowledgements This work was supported by National Industrial Technology System for Watermelon & Melon (CARS-26-NO.8) and the Agri￾cultural science and technology innovation funds in Jiangsu Prov￾ince, China (CX(15)1018). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2016.11.002. References Aghdam, M.S., Sevillano, L., Flores, F.B., Bodbodak, S., 2013. Heat shock proteins as biochemical markers for postharvest chilling stress in fruits and vegetables. Sci. Hortic. 160, 54e64. Barth, O., Zschiesche, W., Siersleben, S., Humbeck, K., 2004. Isolation of a novel barley cDNA encoding a nuclear protein involved in stress response and leaf senescence. Physiol. Plant 121, 282e293. Barth, O., Vogt, S., Uhlemann, R., Zschiesche, W., Humbeck, K., 2009. 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