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