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Research Article

Differential Expression Screening of Defense Related Genes in Dormant Buds of Cold-Treated Grapevines

Seon Ae Kim1, Soon-Young Ahn1, Jae Hyun Han1, Seung Heui Kim2, Jung Ho Noh2, Hae Keun Yun1,*
Plant Breeding and Biotechnology 2013;1(1):14-23.
Published online: March 31, 2013

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*Corresponding author Hae Keun Yun, haekeun@ynu.ac.kr, Tel: +82-53-810-2942, Fax: +82-53-810-4659
• Received: March 3, 2013   • Revised: March 23, 2013   • Accepted: March 25, 2013

Copyright © 2013 The Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Grape (Vitis spp.), which is an important economic fruit crop worldwide, is the fourth most common fruit produced in Korea. Low temperature is one of the most important environmental factors that affect the successful cultivation of grapes. To screen for specific expression of abiotic stress-related genes against cold treatment in ‘Campbell Early’ and ‘Muscat Bailey A’ grapevines, the expression of various defense-related genes was investigated by real-time PCR and semi-quantitative RT-PCR. Among 67 genes analyzed by real-time PCR and RT-PCR, 43 and 10 were found to be up-regulated, while 4 and 37 were down-regulated in low temperature treated ‘Campbell Early’ and ‘Muscat Baily A’ grapevines, respectively. Genes encoding cold induced protein (CIP), lipoxygenase (LOX), glutathione S-transferase (GST), polygalacturonase-inhibiting protein (PGIP), and cold acclimation specific protein (CAS15A) were up-regulated, while those encoding cinnamyl alcohol dehydrogenase 2 (CAD2) and cold acclimation protein (WCS120) were down-regulated by low temperature treatment in both ‘Campbell Early’ and ‘Muscat Bailey A’. These changes in gene expression showed that defense response-related genes for cold acclimation were induced and wheat cold-specific gene decreased in grapevines. Among tested genes, the transcript levels of CLP, cell wall protein (CW), TIP, TLP, and WRKY transcription factor 10 (WRKY10) increased considerably in response to low temperature in ‘Campbell Early’ grapevine. These findings suggest that differentially expressed genes could be utilized as candidates for useful genetic resources and molecular markers after additional confirmation in various grapevine cultivars treated with low temperature in breeding programs for grapes with cold-tolerance.
  • Key words: Cold stress, Freezing tolerance, Grape breeding, Vitis
Grapes (Vitis spp.) are an important economic fruit crop cultivated globally; however, they often encounter cold stresses due to changes in environmental conditions. Accordingly, cold damage caused by extremely low temperature is a serious problem associated with grape production. As grapevines are deciduous perennial plants, they acquire tolerance to freezing in advance of annual freezes to enable to overwinter in canes and buds (Sreekantan et al. 2010).
Crops respond and adapt to cold stress by changing their patterns of gene expression (Yang et al. 2011). Many cold responsive genes and their gene products are considered to function in cold tolerance at transcriptional and biochemical levels (Sanghera et al. 2011).
Consistent with the expected effects of a calcium signal, cold-acclimation causes phosphorylation of a number of proteins (Momroy and Dhindsa 1995; Monroy et al. 1993). When plants are exposed to cold or chilling stress at temperatures of 0 to −15°C, they attempt to maintain homeostasis to acquire freezing tolerance, which involves extensive reprogramming of gene expression and metabolism and changes in structural components (Hughes and Dunn 1990). Both cold acclimation and chilling tolerance are involved in an array of biochemical, molecular and metabolic processes (Cook et al. 2004; Kotak et al. 2007; Larkindale et al. 2005; Thomashow 1999; Zhu et al. 2007). Therefore, understanding the molecular basis of cold stress responses in plants will help us develop cold-tolerant grape cultivars. Recently, damage by extremely low temperatures during winter has become a serious problem in viticulture in Korea (Ahn et al. 2012); therefore, programs for selection of new grape cultivars tolerant to coldness are required. This study was conducted to obtain a better understanding of the general differential expression and provide a basis for further analysis. The specific goals of this study were to detect differentially expressed genes in grapevines following low temperature treatment and provide useful genetic resources for breeding programs of cold-tolerant grapes.
Plant materials and cold treatment
One-year old vine cane with latent buds of ‘Campbell Early’(moderately tolerant to cold stress) and ‘Muscat Bailey A’ (MBA, sensitive to cold stress) grapevines were harvested in February 2011, cut to small cuttings with 1 bud, and used for cold treatment. Cold treatment of buds was carried out at −20°C in the dark for 0, 6, 12, and 24 h, respectively. The buds treated with coldness were collected at the indicated times, frozen in liquid nitrogen and stored at −80°C until further use. Untreated buds were collected for use as a control (0 h).
RNA isolation and cDNA synthesis
Total RNA was extracted from grapevine buds using a modified version of the method described by Yang et al. (2008). Fifty buds from each treatment were used for RNA extraction. The yield and quality of total RNA were measured based on the absorbance at 230, 260, and 280 nm using a NanoDrop spectrophotometer (ND-1000, Technologies Inc., USA). First-strand cDNA was synthesized from the total RNA (500 ng) using a PrimeScriptTM 1st strand cDNA synthesis kit (Takara Bio Inc., Japan) and subsequently used as a template for PCR.
Real-time PCR and semi-quantitative RT-PCR analysis
The various defense-related genes showing changes of their expression against foreign stresses were selected to analyze their expression patterns to cold treatment in grapevines. The expression of selected genes was tested by real-time PCR and semi-quantitative RT-PCR using gene specific primer pairs (Table 1). Real-time PCR was performed on a C1000TM Thermal Cycler (BioRad, USA) using SYBR Premix Ex (TaKaRa Bio Inc., Japan) as the fluorescent dye. Amplification was conducted by subjecting the samples to one cycle at 95°C for 5 min, 40 cycles of 95°C for 20 s, 60°C for 30 s and 72°C for 30 s. All reactions were performed in triplicate to ensure consistency of the results. Each real-time assay was tested to ensure that there was onlya single PCR product using a high resolution melting profiling technique. Real-time PCR results were analyzed as previously described (Wu et al. 2010). Semi-quantitative RT-PCR was performed by subjecting the samples to the following conditions: initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 1 min and then final extension for 7 min at 72°C. The actin gene primers were used as an internal control. The PCR products were identified by 1.5% (W/V) agarose gel electrophoresis with 0.5X TBE running buffer. After taking the gel picture using a transilluminator (UVP, CA, USA), the expression levels were measured by analysis of the images using a public domain image analysis system (NIH ImageJ, NIH Image, Bethesda, USA).
The differential expression of genes was evaluated by real-time PCR and semi-quantitative RT-PCR with primer pairs (Table 1). The expression patterns of 67 genes involved in defense responses to abiotic stress were evaluated following low temperature treatment of ‘Campbell Early’ and ‘MBA’ grapevines using quantitative real-time PCR (Fig. 1, 2, and 3). Among the tested genes, 43 and 10 were up-regulated, while 4 and 37 genes were down-regulated by low temperature treatment in ‘Campbell Early’ and ‘MBA’, respectively.
Among the tested genes, the transcript levels of cold-tolerance such as CBF like transcription factor (CBF1), ATP dependent Clp protease (ClpP), cell wall protein (CW), sirtuin (SIRT), tonoplast intrinsic protein (TIP), pathogenesis-related (PR) genes such as chitinase (CLP), β-1,3-glucanase (Glu), and thaumatin (TLP), lipoxygenase (LOX), polygalacturonase-inhibiting protein (PGIP), and WRKY transcription factor 10 (WRKY10) increased considerably in response to low temperature at 24 h after treatment. Various genes related with defense-response and cold acclimation were up-regulated in cold-treated grapevines. In ‘MBA’, CLP and glutathione S-transferase (GST) were highly up-regulated by low temperature 24 h after treatment (Fig. 1). The expression of cold acclimation-specific protein (CAS15A), DIP, LOX and PGIP increased in response to low temperature treatment for 12 h when compared with untreated controls in ‘MBA’ (Fig. 2).
However, all genes which were up-regulated by low temperature in ‘Campbell Early’ showed decreases in gene expression values in ‘MBA’. The greatest decreases were observed in PR4a and stilbene synthase (SYST) gene expression in ‘MBA’ grapevines treated with low temperature (Fig. 3). The expression of genes related with defense responses such as PR-proteins decreased in grapevines treated with low-temperature.
Among the screened genes, those encoding CAS15A, cold induced protein (CIP), glutathione S-transferase (GST), hypersensitive induced response protein (HIR), and late embryogenesis abundant protein (LEA), were up-regulated, while genes encoding cinnamyl alcohol dehydrogenase2 (CAD2) and cold acclimation protein (WCS120) were down-regulated by low temperature treatment in both ‘Campbell Early’ and ‘MBA’.
The Venn diagram (Fig. 4) presents the distribution of significantly induced gene expression patterns in response to the low-temperature treatment in both ‘Campbell Early’ and ‘MBA’. Overall, 35 genes encoding ascorbate peroxidase (APX), CBF1, chalcone synthesis related (CHS and CHI), ClpP, CW, cytochrome B5 (CYB5), Glu, glycerol-3-phosphate acyltransferase (GPAT), leucine rich repeat (LRR), lipid transfer protein (LTP), O-methyltransferase (MT), MYB transcription factor (MYB), phenylalanine ammonia lyase (PAL), pathogen related proteins (PR4a and PR6), small heat shock protein (sHSP), SIRT, STSY, TIP, TLP, WRKY10, and regulator of cellular molecules (14-3-3) were up-regulated in ‘Campbell Early’ and down-regulated in ‘MBA’ in response to low-temperature treatment. These changes in gene expression showed that defense response-related genes for cold acclimation were induced and wheat cold-specific gene decreased in grapevines. However, DIP and cold regulated protein 15a (COR15a), which were up-regulated in ‘MBA’, were down-regulated in ‘Campbell Early’ in response to low-temperature treatment. It is worth noting that the expression of the 35 genes following cold treatment differed between ‘MBA’ (sensitive) and ‘Campbell Early’ (moderately tolerant) grapevines. The differential expression of the described genes likely resulted from the difference in responses to cold and cold-acclimation capabilities. Therefore, these candidate sequences with different expression patterns in response to cold could be useful as functional molecular markers to assist in selecting cold-tolerant vines for grape breeding programs. The significant differences in the expression of the aforementioned genes were confirmed by RT-PCR. Specifically, 33 of the aforementioned genes were analyzed by RT-PCR and their expression patterns were found to be consistent with the results of real-time PCR (Fig. 5 and 6).
Low temperature affects virtually all aspects of cellular function in plants. Lipid molecules are also very important in signal transduction during cold stress (Yadav 2010). COR15a expression resulted in a decrease in the propensity of the membranes to form hexagonal II phase lipids in response to freezing (Uemura and Steponkus 1997). WCS120 protein acts as an important protective agent of many vital cellular proteins in cold-acclimated plant tissue (Houde et al. 1995; Nikolai et al. 2008; Sarhan et al. 1997). Although genes encoding the WCS120 protein were reported to be induced in wheat and barley exposed to low temperature, they showed decrease of expression in 2 grapevine cultivars treated with freezing temperature in this study. Their expression and roles in defense responses to low temperature in plants including grapevines should be studied in the near future. The relationship between WRKY and abiotic stress responses has been reported in some plant species. For example, at least 4 WRKY genes were shown to be involved in the drought, cold, or salinity responses in Arabidopsis (Seki et al. 2002).
Generally, expression of late embryogenesis abundant proteins (LEA) responsible for the stabilization of membranes and proteins through detergent-like or chaperone activities (Close 1997) and heat shock proteins (HSPs) responsible for the functional conformations of the protein (Wang et al. 2004) are induced in response to different abiotic stresses in plants. Various stress-responsive genes were induced by drought, high-salinity, cold and ABA in the abiotic-stress signal transduction pathway systems (Ishitani et al. 1997; Xiong and Zhu 2002).
TIL gene expression was up-regulated during cold acclimation and heat-shock treatment from wheat and Arabidopsis (Charron et al. 2002). In addition, the accumulation of compatible osmolytes and antioxidants was stimulated by many factors including cold, drought, and salt stress in plants, and such changes caused by cold stress adversely affected the growth and development of plants (Hasegawa et al. 2000).
Zhang et al. (2005) reported that transcript levels of CHI, which is related to anthocyanin synthesis, differed among interspecies, but were elevated to much higher levels in Poncirus rootstock during cold acclimation. PAL and CHS mRNAs were accumulated at similar levels in response to low temperatures (Leyva et al. 1995).
In plants, cold, drought, and salt stresses stimulated the accumulation of compatible osmolytes and antioxidants (Hasegawa et al. 2000). Xiong and Zhu (2001) reported that the signal transduction networks for cold, drought, and salt stress could be divided into three major signaling types, and that among them, Type I signaling may contribute to the production of compatible osmolytes and antioxidants, and may also be related to cell cycle regulation under osmotic stress.
Dehydrin proteins have been reported to be accumulated in plants exposed to environmental stress factors such as water stress (Caruso et al. 2004) or cold stress (Caruso et al. 2004; Hara et al. 2004). In Arabidopsis, some of the cold-inducible genes encode proteins that share similarity with the response to the ABA/late embryogenesis abundant/dehydration-induced (RAB/LEA/DHN) class of polypeptides (Gilmour et al. 1992; Guo et al. 1992; Lang and Palva 1992).
Seki et al. (2001) reported that, in Arabidopsis, induction of β-amylase occurred as early as 2 h after exposure to cold stress. Kaplan and Guy (2004) reported that BMY8 expression increased dramatically as early as 6 h and peaked at 24 h, then decreased gradually, but remained higher under cold shock at 5°C than control levels at 192 h.
The accumulation of PGIP transcripts after storage in the cold has been reported in apple (Yao et al. 1999), and cold induced the expression of AtPGIP1, but not AtPGIP2, has been observed in Arabidopsis (Ferrari et al. 2003). These findings suggested that low temperatures can increase susceptibility to diseases, and that cold induction of defensive proteins might provide protection from pathogen infections. Finally, antimicrobial proteins such as β-glucanase and chitinase, which are highly homologous with PR proteins, were accumulated in the leaf apoplast of winter rye after cold exposure (Hon et al. 1995).
Plant temperature-induced lipocalin and HSP are thought to participate in plant responses to heat and cold stress (Chi et al. 2009), but it has been suggested that they function in different protection systems. Expression of TIL was induced during treatment of heat shock and cold acclimation in wheat and Arabidopsis (Charron et al. 2002). Additionally, the quantity of Arabidopsis TIL1 was significantly increased upon cold acclimation in Arabidopsis leaf (Kawamura and Uemura 2003).
In this study, 67 genes responding to biotic or abiotic stress were selected for screening for differential expression in ‘Campbell Early’ and ‘Muscat Bailey A’ by low temperature treatment. Most of genes tested in this study showed differential expression patterns in transcription level in response to cold stress in grapevines.
Analysis of the differential expression of defense related genes in two grapevines could provide useful resources to breeding programs for development of low temperature tolerant grapevines. A number of valuable genes specifically expressed in cold tolerant grapevines should be selected through further studies such as transcriptome analysis. Sequences of genes with specific expression to low temperature could be useful in development of molecular markers based on SNP/Indels in tolerant grapevine breeding programs.
This work was supported by a grant (PJ008224) from the Agricultural R&D Project, Rural Development Administration, Republic of Korea.
Fig. 1
Quantitative real-time PCR analysis of up-regulated gene expression in ‘Campbell Early’ (left) and ‘MBA’ (right) in response to low temperature. Each bar indicates the SEs (n=3).
pbb-01-14f1.jpg
Fig. 2
Quantitative real-time PCR analysis of differential gene expression in ‘Muscat Bailey A’ in response to low temperature. Each bar indicates the SEs (n=3).
pbb-01-14f2.jpg
Fig. 3
Quantitative real-time PCR analysis of down-regulated gene expression in ‘Campbell Early’ (left) and ‘Muscat Bailey A’ (right) in response to low temperature. Each bar indicates the SEs (n=3).
pbb-01-14f3.jpg
Fig. 4
Venn diagrams of differentially expressed genes in dormant buds of ‘Campbell Early’ and ‘Muscat Bailey A’ grapevines in response to low temperature. A, up-regulated genes; B, down-regulated genes.
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Fig. 5
Semi-quantitative RT-PCR analysis to screen genes induced in dormant buds of ‘Campbell Early’ grapevines in response to low temperature. A, RT-PCR gel image; B, quantitative data for gel images from ImageJ analysis.
pbb-01-14f5.jpg
Fig. 6
Semi-quantitative RT-PCR analysis to screen genes induced in dormant buds of ‘Muscat Bailey A’ grapevines in response to low temperature. A, RT-PCR gel image; B, quantitative data for gel images from ImageJ analysis.
pbb-01-14f6.jpg
Table 1
Sequences of gene specific primers used for real-time PCR analysis.
Table 1
Name Primer sequences
β-amylase (BMYl) 5′-TCCACTCCCAGAATGGGTTAGA-3′
(XM0022745762) 5′-TTTTAAAACCCGCTCCTTGTCA-3′
Ascorbate peroxidase (APX) 5′-GGTCCGTTTGGGACAATGAA-3′
(EU280159.1) 5′-CGGAAATTGCTCCTTGATCG-3′
ATP dependent Clp protease (ClpP) 5′-TCACCCGACCAAATTGAAGC-3′
(XM00227683.1) 5′-AACTCCCCGATCCTCAGAGC-3′
CBF like transcription factor (CBF1) 5′-GGGTTTGCTCCTTACTCCACCT-3′
(AY390372.1) 5′-CATCATCCCAGCTGAATCCTTT-3′
Cell wall protein (CW) 5′-TTGACAAGAAGCAGCCTGAGTG-3′
(XM_002277798.1) 5′-TGCTGCCATCATAGGGAGTGTA-3′
Chalcone isomerase (CHI) 5′-TACACTGACGCAGAAGCCAAAG-3′
(XM002282072.2) 5′-GACCCATCTTTGGAGAAGCTCA-3′
Chalcone synthesis (CHS) 5′-AGTTCAAGCGCATGTGTGAAAA-3′
(EF192464.1) 5′-CTTCAACCACCACCATGTCTTG-3′
Chitinase-like protein (CLP) 5′-CCGATTTCTTCCAGACCTACCA-3′
(XM002269123.1) 5′-CAAATCCGTGAGGCTGGTAAAC-3′
Cinnarmyl alcohol dehydrogenase2 (CAD2) 5′-GGGCGTCCTCTCTCCATTTC-3′
(NM127743.3) 5′-TCCCCAATGGTTCTTGATGG-3′
Cold acclimation protein WCS120 (WCS120) 5′-CATGGACACGCTGGAGTGAT-3′
(M93342.2) 5′-TGTCCCAGTGCCAGTCGTT-3′
Cold acclimation-specific (CAS15A) 5′-CATGCTGGTGACCACAAAGG-3′
(L12462.1) 5′-TTCCCCATGGTATCCCTCCT-3′
Cold induced protein (CIP) 5′-AAGGGTGCAAAGCAAAGAGATG-3′
(XM002283501.1) 5′-ATGCTGATGATCCATGGGAAGT-3′
Cold regulated protein 15a (COR15a) 5′-ACCTCAACGAGGCCACAAAG-3′
(NM_129815.4) 5′-CCGCAGCTTTCTCAGCTTCT-3′
Cytochrome B5 (CYB5) 5′-AGAGGAGCATCCTGGTGGTG-3′
(XM002283578.1) 5′-GCATGCTGTGGCCCAAAA-3′
Cytochrome p450 (CYP) 5′-TGCAAGCAGTGGTGAAAGAGAC-3′
(CAB85635.1) 5′-GACCTATAGCCCATGCGTTCAC-3′
Dehydrin1 (DHN1) 5′-GGGAGAAGGAAGAAAGGGATGA-3′
(JF896552.1) 5′-GTACTTGTGGCGCTGGTATGC-3′
Dihydroflavonol-4-reductase (DFR) 5′-TGTCTCCAAGACACTGGCTGAG-3′
(JQ308621.1) 5′-TCGGGGAAAGAGCAGTTATGAG-3′
Draught induced protein (R1G1A) (DIP) 5′-CGTCGGTGACATGGATGAAA-3′
(AF503585.1) 5′-TCCTGTTCACGGAAGCCAAT-3′
Endo-β-glucanase precursor (Glu) 5′-GGGGTTATTTGGATCCCATCAT-3′
(AB601116.1) 5′-CAGAAGCGGCGACTTATTGTCT-3′
Flavonol synthase (FLS) 5′-TGCTGAGCAATGGGAAGTACAA-3′
(XM002285805.1) 5′-GAGGTCCGATCATTGCCTTATG-3′
Glutathione peroxidase (GPX) 5′-GAGCACAGGAACCTGGGAGTAA-3′
(XM003631370.1) 5′-AGCACTATCGCCATTCACATCA-3′
Glutathion-S-transferase (GST) 5′-TATAATGTGTGGGCAGCAAACG-3′
(AY156048) 5′-CCAATGTCCAGAAAACCCAAAG-3′
Glycerol-3-phosphate acyltransferase (GPAT) 5′-GAAGCTTCAGCAGGGTCACAAT-3′
(XM002276065.1) 5′-GCTGGGTCTGCTTCTGTTTGAT-3′
Hypersensitive induced response protein (HIR) 5′-TGCATCCTCAAAGGCTTCGT-3′
(AY159555.1) 5′-TGCTGTCTCAGCCTGAAGGA-3′
Late embryogenesis abundant protein (LEA) 5′-CTGACGTGGCAGAAGCAATG-3′
(NM129540.4) 5′-TCGGACGCCATTTTCTTAGC-3′
Lipid transfer protein (LTP) 5′-TGGAAGCAACCATAACATGTGG-3′
(LOC100256718) 5′-TTGAGGCTCTTAATCCCACTGC-3′
Lipoxygenase (LOX) 5′-AACCTTGCGAGGTAATGGTCAA-3′
(XM_002285538.2) 5′-TACCACCAAGTACCGGTCGAGT-3′
Leucine-rich repeats (LRR) 5′-GCCGATTTGGATCTCTCTCTGA-3′
(XM002285517.2) 5′-GTATGCTCACCGCCGAGTTAAT-3′
Manganese superoxide dismutase (Mn-SOD) 5′-GTTCATGAGGGAGGTGGTGAAC-3′
(EU280161.1) 5′-TCTATAGCCCAACCCAGTGAGC-3′
Mitogen-activated protein kinase (MAPK) 5′-CCCTCCTGATGAGGATTCATTG-3′
(AY395740.1) 5′-ATTCGAACTCCATCCCTGTTGA-3′
MYB Transcripton factor (MYB) 5′-CACAAAGTCCATCTCCCCAAAC-3′
(XM_002265978.1) 5′-TCTCCATTTCCTCCTGAACTCG-3′
O-methyltransferase (MT) 5′-CCAGACACCATCCACAACCA-3′
(JF808016.1) 5′-GCAGCAAGGAAGCCAGAATG-3′
Osmotin like protein (OSM) 5′-ACTGCAACTTCGATGCGTCA-3′
(Y10992.1) 5′-TGCGAATTCGGCTAAGGTGT-3′
Pathogen related protein 4a (PR4a) 5′-GCTGCCCAGAGCGCTAGTAA-3′
(AF061329.1) 5′-TCCCAAGTGGAGCAGTAGGC-3′
Pathogen related protein 6 (PR6) 5′-CTGGTGGGAGTTCAGGGAGA-3′
(AY156047.1) 5′-CCAAACACGGACCCTAGTGC-3′
Phenylalanine ammonia lyase (PAL) 5′-TGAACAATGGCGAAAGTGAGAA-3′
(X75967.1) 5′-TCTCTTGCGCTCTCAACCTCTT-3′
Polygalacturonase-inhibiting protein (PGIP) 5′-GTCTTACTCGCCACTCGTCCAT-3′
(AF305093.1) 5′-GTTGGGATTCCACGAAGCTAGA-3′
Proline rich protein2 (PRP2) 5′-CCACCTAAGATTAAACCGCGACT-3′
(XR078193.2) 5′-CGAGGAATCGGAGGAAGCTTAT-3′
Pyrroline-5-carboxylate synthase (P5CS) 5′-AGCCAATGCACTGGAAACAAAT-3′
(AJ005686.1) 5′-CAGGCTTTAGAACCAGCCTTGA-3′
Regulator of cellular molecules (14-3-3) 5′-TCAGGATATTGCAAACGCAGAA-3′
(FJ824803.1) 5′-AAATGCCTGTTTCGCAAGGTTA-3′
Sirtuin (SIRT) 5′-TGATGGCCTCCATCTTCGTT-3′
(JN252254.1) 5′-CACCTCAAAATCCCGCAAGT-3′
Small heat shock protein (sHSP) 5′-CATCTCGAGGTATGGGAACTGG-3′
(XM003634002.1) 5′-GTGTTCTGCTCCACGCTTACCT-3′
Stilbene (STSY) 5′-GGTGCCATTGCAGGAAACTTAC-3′
(X76892.1) 5′-CAAGTGGGTCAAAAGCCTGAGT-3′
Temperature induced lipocalin (TIL) 5′-TCAACGAGACTTGGAGTGATGG-3′
(DQ222993.1) 5′-AACAGGGATAATGGGGAGGAAA-3′
Thaumatin-like protein (TLP) 5′-TTCGCACTTAACCAATTCAGCA-3′
(XM002282928.2) 5′-TGCACCCATTGGAAGTAGGATT-3′
Tonoplast intrinsic protein (TIP) 5′-GGCACCCTAATTTTTGTGTTCG-3′
(XM003632211.1) 5′-ACACTGCCACAAACAGTCCAAG-3′
WRKY transcription facter 10 (WRKY10) 5′-CAGGTGTTCAGTTGATGGATGC-3′
(XM002279371.2) 5′-AAGGGCTTTCGTGGTTATGGAT-3′
β-actin 5′-ACGAGAAATCGTGAGGGATG-3′
(AB372563.1) 5′-ATTCTGCCTTTGCAATCCAC-3′
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Differential Expression Screening of Defense Related Genes in Dormant Buds of Cold-Treated Grapevines
Plant Breed. Biotech.. 2013;1(1):14-23.   Published online March 31, 2013
DOI: https://doi.org/10.9787/PBB.2013.1.1.014
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Differential Expression Screening of Defense Related Genes in Dormant Buds of Cold-Treated Grapevines
Plant Breed. Biotech.. 2013;1(1):14-23.   Published online March 31, 2013
DOI: https://doi.org/10.9787/PBB.2013.1.1.014
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Differential Expression Screening of Defense Related Genes in Dormant Buds of Cold-Treated Grapevines
Image Image Image Image Image Image
Fig. 1 Quantitative real-time PCR analysis of up-regulated gene expression in ‘Campbell Early’ (left) and ‘MBA’ (right) in response to low temperature. Each bar indicates the SEs (n=3).
Fig. 2 Quantitative real-time PCR analysis of differential gene expression in ‘Muscat Bailey A’ in response to low temperature. Each bar indicates the SEs (n=3).
Fig. 3 Quantitative real-time PCR analysis of down-regulated gene expression in ‘Campbell Early’ (left) and ‘Muscat Bailey A’ (right) in response to low temperature. Each bar indicates the SEs (n=3).
Fig. 4 Venn diagrams of differentially expressed genes in dormant buds of ‘Campbell Early’ and ‘Muscat Bailey A’ grapevines in response to low temperature. A, up-regulated genes; B, down-regulated genes.
Fig. 5 Semi-quantitative RT-PCR analysis to screen genes induced in dormant buds of ‘Campbell Early’ grapevines in response to low temperature. A, RT-PCR gel image; B, quantitative data for gel images from ImageJ analysis.
Fig. 6 Semi-quantitative RT-PCR analysis to screen genes induced in dormant buds of ‘Muscat Bailey A’ grapevines in response to low temperature. A, RT-PCR gel image; B, quantitative data for gel images from ImageJ analysis.

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Differential Expression Screening of Defense Related Genes in Dormant Buds of Cold-Treated Grapevines

Sequences of gene specific primers used for real-time PCR analysis.

Name Primer sequences
β-amylase (BMYl) 5′-TCCACTCCCAGAATGGGTTAGA-3′
(XM0022745762) 5′-TTTTAAAACCCGCTCCTTGTCA-3′
Ascorbate peroxidase (APX) 5′-GGTCCGTTTGGGACAATGAA-3′
(EU280159.1) 5′-CGGAAATTGCTCCTTGATCG-3′
ATP dependent Clp protease (ClpP) 5′-TCACCCGACCAAATTGAAGC-3′
(XM00227683.1) 5′-AACTCCCCGATCCTCAGAGC-3′
CBF like transcription factor (CBF1) 5′-GGGTTTGCTCCTTACTCCACCT-3′
(AY390372.1) 5′-CATCATCCCAGCTGAATCCTTT-3′
Cell wall protein (CW) 5′-TTGACAAGAAGCAGCCTGAGTG-3′
(XM_002277798.1) 5′-TGCTGCCATCATAGGGAGTGTA-3′
Chalcone isomerase (CHI) 5′-TACACTGACGCAGAAGCCAAAG-3′
(XM002282072.2) 5′-GACCCATCTTTGGAGAAGCTCA-3′
Chalcone synthesis (CHS) 5′-AGTTCAAGCGCATGTGTGAAAA-3′
(EF192464.1) 5′-CTTCAACCACCACCATGTCTTG-3′
Chitinase-like protein (CLP) 5′-CCGATTTCTTCCAGACCTACCA-3′
(XM002269123.1) 5′-CAAATCCGTGAGGCTGGTAAAC-3′
Cinnarmyl alcohol dehydrogenase2 (CAD2) 5′-GGGCGTCCTCTCTCCATTTC-3′
(NM127743.3) 5′-TCCCCAATGGTTCTTGATGG-3′
Cold acclimation protein WCS120 (WCS120) 5′-CATGGACACGCTGGAGTGAT-3′
(M93342.2) 5′-TGTCCCAGTGCCAGTCGTT-3′
Cold acclimation-specific (CAS15A) 5′-CATGCTGGTGACCACAAAGG-3′
(L12462.1) 5′-TTCCCCATGGTATCCCTCCT-3′
Cold induced protein (CIP) 5′-AAGGGTGCAAAGCAAAGAGATG-3′
(XM002283501.1) 5′-ATGCTGATGATCCATGGGAAGT-3′
Cold regulated protein 15a (COR15a) 5′-ACCTCAACGAGGCCACAAAG-3′
(NM_129815.4) 5′-CCGCAGCTTTCTCAGCTTCT-3′
Cytochrome B5 (CYB5) 5′-AGAGGAGCATCCTGGTGGTG-3′
(XM002283578.1) 5′-GCATGCTGTGGCCCAAAA-3′
Cytochrome p450 (CYP) 5′-TGCAAGCAGTGGTGAAAGAGAC-3′
(CAB85635.1) 5′-GACCTATAGCCCATGCGTTCAC-3′
Dehydrin1 (DHN1) 5′-GGGAGAAGGAAGAAAGGGATGA-3′
(JF896552.1) 5′-GTACTTGTGGCGCTGGTATGC-3′
Dihydroflavonol-4-reductase (DFR) 5′-TGTCTCCAAGACACTGGCTGAG-3′
(JQ308621.1) 5′-TCGGGGAAAGAGCAGTTATGAG-3′
Draught induced protein (R1G1A) (DIP) 5′-CGTCGGTGACATGGATGAAA-3′
(AF503585.1) 5′-TCCTGTTCACGGAAGCCAAT-3′
Endo-β-glucanase precursor (Glu) 5′-GGGGTTATTTGGATCCCATCAT-3′
(AB601116.1) 5′-CAGAAGCGGCGACTTATTGTCT-3′
Flavonol synthase (FLS) 5′-TGCTGAGCAATGGGAAGTACAA-3′
(XM002285805.1) 5′-GAGGTCCGATCATTGCCTTATG-3′
Glutathione peroxidase (GPX) 5′-GAGCACAGGAACCTGGGAGTAA-3′
(XM003631370.1) 5′-AGCACTATCGCCATTCACATCA-3′
Glutathion-S-transferase (GST) 5′-TATAATGTGTGGGCAGCAAACG-3′
(AY156048) 5′-CCAATGTCCAGAAAACCCAAAG-3′
Glycerol-3-phosphate acyltransferase (GPAT) 5′-GAAGCTTCAGCAGGGTCACAAT-3′
(XM002276065.1) 5′-GCTGGGTCTGCTTCTGTTTGAT-3′
Hypersensitive induced response protein (HIR) 5′-TGCATCCTCAAAGGCTTCGT-3′
(AY159555.1) 5′-TGCTGTCTCAGCCTGAAGGA-3′
Late embryogenesis abundant protein (LEA) 5′-CTGACGTGGCAGAAGCAATG-3′
(NM129540.4) 5′-TCGGACGCCATTTTCTTAGC-3′
Lipid transfer protein (LTP) 5′-TGGAAGCAACCATAACATGTGG-3′
(LOC100256718) 5′-TTGAGGCTCTTAATCCCACTGC-3′
Lipoxygenase (LOX) 5′-AACCTTGCGAGGTAATGGTCAA-3′
(XM_002285538.2) 5′-TACCACCAAGTACCGGTCGAGT-3′
Leucine-rich repeats (LRR) 5′-GCCGATTTGGATCTCTCTCTGA-3′
(XM002285517.2) 5′-GTATGCTCACCGCCGAGTTAAT-3′
Manganese superoxide dismutase (Mn-SOD) 5′-GTTCATGAGGGAGGTGGTGAAC-3′
(EU280161.1) 5′-TCTATAGCCCAACCCAGTGAGC-3′
Mitogen-activated protein kinase (MAPK) 5′-CCCTCCTGATGAGGATTCATTG-3′
(AY395740.1) 5′-ATTCGAACTCCATCCCTGTTGA-3′
MYB Transcripton factor (MYB) 5′-CACAAAGTCCATCTCCCCAAAC-3′
(XM_002265978.1) 5′-TCTCCATTTCCTCCTGAACTCG-3′
O-methyltransferase (MT) 5′-CCAGACACCATCCACAACCA-3′
(JF808016.1) 5′-GCAGCAAGGAAGCCAGAATG-3′
Osmotin like protein (OSM) 5′-ACTGCAACTTCGATGCGTCA-3′
(Y10992.1) 5′-TGCGAATTCGGCTAAGGTGT-3′
Pathogen related protein 4a (PR4a) 5′-GCTGCCCAGAGCGCTAGTAA-3′
(AF061329.1) 5′-TCCCAAGTGGAGCAGTAGGC-3′
Pathogen related protein 6 (PR6) 5′-CTGGTGGGAGTTCAGGGAGA-3′
(AY156047.1) 5′-CCAAACACGGACCCTAGTGC-3′
Phenylalanine ammonia lyase (PAL) 5′-TGAACAATGGCGAAAGTGAGAA-3′
(X75967.1) 5′-TCTCTTGCGCTCTCAACCTCTT-3′
Polygalacturonase-inhibiting protein (PGIP) 5′-GTCTTACTCGCCACTCGTCCAT-3′
(AF305093.1) 5′-GTTGGGATTCCACGAAGCTAGA-3′
Proline rich protein2 (PRP2) 5′-CCACCTAAGATTAAACCGCGACT-3′
(XR078193.2) 5′-CGAGGAATCGGAGGAAGCTTAT-3′
Pyrroline-5-carboxylate synthase (P5CS) 5′-AGCCAATGCACTGGAAACAAAT-3′
(AJ005686.1) 5′-CAGGCTTTAGAACCAGCCTTGA-3′
Regulator of cellular molecules (14-3-3) 5′-TCAGGATATTGCAAACGCAGAA-3′
(FJ824803.1) 5′-AAATGCCTGTTTCGCAAGGTTA-3′
Sirtuin (SIRT) 5′-TGATGGCCTCCATCTTCGTT-3′
(JN252254.1) 5′-CACCTCAAAATCCCGCAAGT-3′
Small heat shock protein (sHSP) 5′-CATCTCGAGGTATGGGAACTGG-3′
(XM003634002.1) 5′-GTGTTCTGCTCCACGCTTACCT-3′
Stilbene (STSY) 5′-GGTGCCATTGCAGGAAACTTAC-3′
(X76892.1) 5′-CAAGTGGGTCAAAAGCCTGAGT-3′
Temperature induced lipocalin (TIL) 5′-TCAACGAGACTTGGAGTGATGG-3′
(DQ222993.1) 5′-AACAGGGATAATGGGGAGGAAA-3′
Thaumatin-like protein (TLP) 5′-TTCGCACTTAACCAATTCAGCA-3′
(XM002282928.2) 5′-TGCACCCATTGGAAGTAGGATT-3′
Tonoplast intrinsic protein (TIP) 5′-GGCACCCTAATTTTTGTGTTCG-3′
(XM003632211.1) 5′-ACACTGCCACAAACAGTCCAAG-3′
WRKY transcription facter 10 (WRKY10) 5′-CAGGTGTTCAGTTGATGGATGC-3′
(XM002279371.2) 5′-AAGGGCTTTCGTGGTTATGGAT-3′
β-actin 5′-ACGAGAAATCGTGAGGGATG-3′
(AB372563.1) 5′-ATTCTGCCTTTGCAATCCAC-3′
Table 1 Sequences of gene specific primers used for real-time PCR analysis.

Table 1

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