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