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First published online February 9, 2007; 10.1104/pp.106.094532 Plant Physiology 143:1739-1751 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Overexpression of an R1R2R3 MYB Gene, OsMYB3R-2, Increases Tolerance to Freezing, Drought, and Salt Stress in Transgenic Arabidopsis1,[C],[W],[OA]Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China (X.D., Y.X., Q.M., T.W., K.C.); Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China (W.X., Y.X.); and the National Center for Plant Gene Research, Beijing 100093, China (T.W., Y.X., K.C.)
We used a cDNA microarray approach to monitor the expression profile of rice (Oryza sativa) under cold stress and identified 328 cold-regulated genes. Thirteen such genes encoding MYB, homeodomain, and zinc finger proteins with unknown functions showed a significant change in expression under 72-h cold stress. Among them, OsMYB3R-2 was selected for further study. Unlike most plant R2R3 MYB transcription factors, OsMYB3R-2 has three imperfect repeats in the DNA-binding domain, the same as in animal c-MYB proteins. Expression of OsMYB3R-2 was induced by cold, drought, and salt stress. The Arabidopsis (Arabidopsis thaliana) transgenic plants overexpressing OsMYB3R-2 showed increased tolerance to cold, drought, and salt stress, and the seed germination of transgenic plants was more tolerant to abscisic acid or NaCl than that of wild type. The expression of some clod-related genes, such as dehydration-responsive element-binding protein 2A, COR15a, and RCI2A, was increased to a higher level in OsMYB3R-2-overexpressing plants than in wild type. These results suggest that OsMYB3R-2 acts as a master switch in stress tolerance.
Plants are exposed to environmental conditions that frequently impose constraints on growth and development. Among them, low temperature stress is one of the serious environmental stresses affecting plant growth and agricultural production. On exposure of plants to low temperature, a series of genes are induced, the products of which may either directly protect against stress or further control the expression of other target genes (Yamaguchi-Shinozaki and Shinozaki, 2006  ). In Arabidopsis (Arabidopsis thaliana), a major transcriptional regulatory system that controls abscisic acid (ABA)-independent gene expression in response to low temperatures has been identified (Stockinger et al., 1997; Liu et al., 1998). The system is based on the C-repeat (Baker et al., 1994)/dehydration-responsive element (Yamaguchi-Shinozaki and Shinozaki, 1994) that interacts with C-repeat-binding factors (CBFs). Under cold stress, CBF/dehydration-responsive element-binding protein 1 (DREB1) genes are rapidly and transiently induced and subsequently activate the expression of target genes (Gilmour et al., 1998). Several studies have reported that ectopic overexpression of some CBFs resulted in both activation of target genes and enhanced freezing, salt, or dehydration tolerance of transgenic plants (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Kasuga et al., 1999; Haake et al., 2002).
The CBF pathway is a central component of cold response, but CBF-independent pathways might also be necessary for the cold stress response (Zhu et al., 2004
Rice (Oryza sativa) is a model monocot system and one of the most important food crops in Asia (Khush, 1997
Here, we report on the isolation and functional characterization of a nuclear-localized R1R2R3 MYB TF designated OsMYB3R-2 (O. sativa R1R2R3 MYB-2) in rice. The protein, like animal c-Myb proteins, contains three imperfect repeat sequences in the N-terminal DNA-binding domain (Jin and Martin, 1999
Isolation of Cold-Responsive MYB TFs from Cold-Tolerant Rice
Yuedongdao, a rice variety possessing characteristics of cold tolerance is a crossed progeny of cultivated rice and Dongxiang wild rice (Oryza rufipogon Griff.), which is a population of common wild rice with increased cold stress tolerance from Dongxiang in the Jiangxi province of China. The Dongxiang wild rice rhizome can survive at a freezing temperature to –12.8°C (Liu et al., 2003a
We performed semiquantitative reverse transcription (RT)-PCR to confirm the differentially expressed genes identified by microarray analysis. Twenty-six genes representing different expression profiles were analyzed, of which 25 exhibited expression patterns similar to that from microarray analysis (Fig. 1A ); only one, R0005G01, showed no significant difference in gene expression between the treatment and control with RT-PCR amplification. Moreover, all randomly scattered expressed sequence tags that represent the same gene showed a similar differentially expressed pattern in the microarray analysis (Supplemental Table S1). Thus, our cDNA microarray hybridizations were stable and reliable.
Among the 328 cold-responsive genes, we identified three genes encoding cold-inducible TFs, including two MYB TFs and one homeodomain TF. One, an expressed sequence tag R0481B08 (accession no. BAD81765) encoding a putative R1R2R3 MYB TF was selected for further functional studies. In our microarray hybridization, R0481B08 showed a completely opposite hybridization signal in two dye-exchange replicates (Fig. 1B). The transcript level was increased 2.6-fold when the cold-treated sample was labeled with Cy5 and the untreated sample was labeled with Cy3, whereas in the dye-exchange hybridization, the transcript level was decreased 2.3-fold (Supplemental Table S1). The expression of R0481B08 in the microarray analysis was confirmed by semiquantitative RT-PCR (Fig. 1A). Moreover, real-time PCR used to further examine its expression in microarray hybridizations showed a similar amount of change as the microarray data (Fig. 1D), which strongly supports the validity of the cold-regulated expression pattern from the microarray analysis (Fig. 1, C and D).
To investigate the function of R0481B08, we amplified its full-length cDNA by RT-PCR from rice seedlings treated for 72 h at 2°C. The full-length cDNA contains an open reading frame of 587 amino acids with a calculated molecular mass of 63.9 kD. Homological analysis showed that the gene shared the greatest sequence similarity with the MYB TFs from Arabidopsis, rice, Populus, and tobacco (Nicotiana tabacum) within the MYB domain. The MYB domain is composed of three imperfect repeat sequences (R1, R2, and R3) of 50 to 53 amino acids in the mammalian MYB protein c-MYB and related proteins A-MYB and B-MYB (R1R2R3-MYB; Carr and Mott, 1991
We constructed a phylogenetic tree based on the amino acid sequences of MYB domains of animal and plant Myb proteins (Fig. 2B). The OsMYB3R-2 protein is more similar to AF214117, T48253, MYB3R-1, and NtmybA2 proteins than to plant R2R3-Myb proteins. The OsMYB3R-2 protein showed 66.7% to 74.8% identity with human C-Myb and B-Myb but only 37.6% to 44.1% identity with plant R2R3-Myb proteins. However, OsMYB3R-2 protein did not group with any animal R1R2R3-type Myb proteins (A-Myb, B-Myb, and C-Myb) but, rather, formed a separate branch. OsMYB3R-2 and MYB3R-1 are more closely related to each other than to AJ430051, a putative R1R2R3-type MYB from rice. To examine its subcellular localization, OsMYB3R-2 was fused in frame to the 5' terminus of the green fluorescent protein (GFP) reporter gene under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S). The recombinant constructs of the OsMYB3R-2-GFP fusion gene and GFP alone were introduced into onion (Allium cepa) epidermal cells by particle bombardment. As shown in Figure 2C, the OsMYB3R-2-GFP fusion protein accumulated mainly in the nucleus, whereas GFP alone was present throughout the whole cell. Thus, OsMYB3R-2 is a nuclear-localized protein, which is consistent with its predicted function as a TF (Fig. 2C).
We performed real-time RT-PCR to examine the expression pattern of OsMYB3R-2 under different stress conditions. Under cold stress, the transcripts of OsMYB3R-2 began to increase after 5 h cold treatment and gradually accumulated up to 72 h of treatment (Fig. 3A
), which was consistent with our microarray results. In the case of salt and dehydration stress, transcript levels of OsMYB3R-2 were also induced after 5 h treatment as compared with that of nontreated controls (Fig. 3C). To validate this experiment, we used the OSISAP1, a gene encoding zinc-finger protein from rice, as a positive control. OSISAP1 is induced under cold, salt, and drought stress. Under cold treatment, the transcript level of OSISAP1 was increased to a very high level during a 12-h cold treatment and declined thereafter (Mukhopadhyay et al., 2004
In addition, we examined tissue-specific expression of OsMYB3R-2 in rice using real-time RT-PCR. The OsMYB3R-2 transcripts were detected in all organs tested, but the highest level was in young stems and the lowest in spikes (Fig. 3D). Taken together, these results suggest that OsMYB3R-2 is induced under cold, salt, and drought stimulation, which suggests that it functions during these stresses.
To investigate the function of OsMYB3R-2 in plants, we overexpressed OsMYB3R-2 in transgenic Arabidopsis under control of a CaMV 35S promoter. Transformed lines of Arabidopsis were confirmed by hygromycin selection and Southern blotting. Southern blot was performed by using the DNA digested with HindIII or EcoRI and
To examine the possible phenotypes of transgenic lines, T3 progeny of the OsMYB3R-2-overexpressed lines and the wild-type plants were grown in the greenhouse under identical conditions. Compared with wild-type plants, transgenic plants showed a little retarded growth under normal conditions (Fig. 4C). To investigate the effect of OsMYB3R-2 overexpression on freezing tolerance, T3 transgenic and wild-type seedlings were exposed to –8°C for 10 h. After 6 d recovery at normal conditions, survival was 26.8% for the wild-type and 84.5% for transgenic lines (Fig. 5, A and B ). Phenotypically, most transgenic seedlings were green and could regrow as compared with the wild type, whereas most wild-type seedlings became white and did not regrow after removed to normal conditions. The survival percentage under different low temperatures also showed dramatic difference between the transgenic plants and wild-type plants (Fig. 5, C and D). At –10°C, the proportion of survived wild-type plants decreased to 5.6%, whereas more than 42.8% of transgenic plants survived. Thus, OsMYB3R-2-overexpression plants show high tolerance to freezing stress.
To determine the effect of OsMYB3R-2 overexpression on drought tolerance, 14-d-old plants grown on soil were not watered for 2 weeks and then watered and grown under normal conditions for 7 d (Fig. 6A ). After watering was restarted, transgenic plants showed a stronger growth recovery phenotype than wild-type plants. Only 26.7% of the wild-type plants survived this treatment. In contrast, more than 85% of OsMYB3R-2-overexpressed plants survived (Table II ), which suggests that the overexpression of OsMYB3R-2 in transgenic Arabidopsis results in greater tolerance to drought stress than in the wild type. The drought-tolerance phenotype of transgenic plants overexpressing OsMYB3R-2 was consistent with slower water loss in detached rosette leaves as compared with the wild type (Fig. 6B).
To test the effect of OsMYB3R-2 overexpression on salt tolerance, transgenic and wild-type seedlings were grown as described in "Materials and Methods." Seedlings of both genetic backgrounds grew normally in NaCl up to 150 mM, but the transgenic seedlings formed longer roots than the wild type when grown vertically under NaCl treatment (Fig. 6, C and D). When NaCl concentration was increased to 200 mM, the growth of the wild type was completely inhibited and the seedlings showed absence of greening, whereas transgenic seedlings were still green and continued to grow (Fig. 6C).
We tested the effect of ABA and NaCl on germination of OsMYB3R-2-overexpressed seeds. There was no difference in seed germination between the wild-type and transgenic plants under normal conditions (Fig. 7A ). In the presence of exogenous ABA, the germination of both wild-type and OsMYB3R-2-overexpressed seeds was inhibited significantly, but transgenic seeds inhibited to a lesser extent (Fig. 7B). For example, at 0.5 µM ABA, approximately 80% of OsMYB3R-2-overexpressed seeds germinated comparing with only 30% seeds of the wild type. Under 1.0 µM ABA treatment, most seeds of the wild type did not germinate. In contrast, about one-half the seeds of the transgenic plants germinated and developed green cotyledons and true leaves (Fig. 7, C and D). At ABA levels higher than 2.0 µM, the germination of both wild-type and transgenic seeds was inhibited completely.
We also observed that the germination of transgenic seeds was more tolerant to NaCl than that of wild type under different NaCl treatments (Fig. 7, E and F). At 50 mM NaCl, nearly 79% to 90% of transgenic seeds germinated at day 2 compared with only 35% seed germination for the wild type (Fig. 7E). At 75 mM NaCl, germination of both wild-type and OsMYB3R-2-overexpressed seeds was completely inhibited at day 2 after imbibition. At day 3, although seeds of both plants began to germinate, the germination in the transgenic plants was significantly higher than that of the wild type (Fig. 7F). Nevertheless, both wild-type and transgenic seeds were not observed to germinate at day 5 after imbibition when NaCl concentrations were at 100 mM (data not shown). Thus, overexpression of OsMYB3R-2 in Arabidopsis increased tolerance to NaCl and ABA during seed germination.
To elucidate the molecular mechanism of OsMYB3R-2 in the cold response, we monitored the expression of cold-responsive genes identified in the regulated pathways by real-time PCR analysis. Under 4°C cold treatment for 6 h, the tested marker genes, including RD29A, CBF1, CBF2, CBF3, KIN1, and COR47, showed slight induction in both wild-type and transgenic plants under cold-stress conditions, consistent with previous studies (Kurkela and Franck, 1990
OsMYB3R-2 Encodes a Cold-Responsive R1R2R3 MYB TF
In plants, the transcripts of genes encoding several families of TFs, such as AP2/EREBP, bZIP/HD-ZIP, and several classes of zinc finger domains, are induced after exposure to various abiotic stresses (Shinozaki and Yamaguchi-Shinozaki, 2000
R1R2R3-MYB genes seem to constitute a small gene family in plants. In Arabidopsis, five R1R2R3-type Myb genes have been described (Braun and Grotewold, 1999
Usually transcriptional factors are induced rapidly during the early phase of the response to cold, drought, and salt stress, reach maximal induction at several hours, and then decrease in expression level (Thomashow, 2001
Certain stress-induced proteins have been shown to impart stress tolerance. Overexpression of genes such as CBF/DREB1, OSISAP1, and HVA1 could confer stress tolerance in transgenic plants (Browse and Xin, 2001
Several lines of evidence indicate that other signal pathways in addition to those mediated by CBF TFs are involved in cold stress (Fowler and Thomashow, 2002
Usually, enhanced drought tolerance accompanies hypersensitivity to ABA treatments during seed germination and early seedling development (Hu et al., 2006 OsMYB3R-2 differs from HOS10 and Osmyb4. Importantly, the activated genes in 35S::OsMYB3R-2 Arabidopsis also differ from those in Osmyb4 transgenic Arabidopsis. These results suggest that OsMYB3R-2 is a novel member of the R1R2R3-type MYB family in rice and is involved in stress response. This study has characterized an R1R2R3-type MYB protein localized at the nucleus in rice and induced by cold, drought, and salt stress. The enhanced stress tolerance of 35S::OsMYB3R-2 Arabidopsis plants reveals that OsMYB3R-2 could mediate signal transduction, regulating some stress-responsive genes involved in CBF-dependent or -independent pathways. Although the detailed mechanism of OsMYB3R-2 involvement in stress is not yet clear, the characterization of OsMYB3R-2 function will provide new insights into stress pathways. This report provides beneficial information for molecular breeding leading to improved stress tolerance of agricultural crops.
Plant Materials We used rice (Oryza sativa) L. cv Yuedongdao, which is insensitive to cold stress. Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used in gene transformation. Rice seeds were surface sterilized for 5 min with ethanol (75% v/v) and 10 min with commercially diluted (1:3 v/v) NaOCl, followed by several rinses with sterile water. Germination was carried out for 72 h on sterile Murashige and Skoog (MS) medium in the dark at 28°C, then grown under 28°C-day/25°C-night temperatures, 12-h-light/12-h-dark cycle, and 50% humidity. After 2 weeks of germination, seedlings underwent several treatments: cold, 2°C for 0.5, 1, 2, 5, 10, 24, 36, 48, 64, and 72 h; drought, transferred to Whatman 3MM paper in a sterile petri dish for 5 h; and high salinity, 250 mM NaCl for 5 h. After all the treatments, seedlings were harvested, frozen in liquid nitrogen, and stored at –70°C for further analysis. Control plants were harvested at the same time as the treated plants.
Rice seedlings were exposed to cold for 72 h; total RNAs from treated plants and nontreated plants were used for preparation of Cy5- and Cy3-labeled cDNA probes. P100S cDNA microarray (Biostar Genechip) were hybridized with Cy5- and Cy3-labeled probe pairs of cold-treated and nontreated plants. Labeling, hybridization, and washing were performed as described for the CyScribe Post-Labeling kit. Hybridized slides were scanned with use of a GenePix 4000B scanner (Axon Instruments) at 532 and 635 nm to capture the emission of Cy3 and Cy5, respectively. The intensity of each spot at the two wavelengths was transformed into a ratio value with use of the GenePix 4.0 software. Overall intensity of the hybridized slide was normalized by use of GenePix 4.0 software. With the removal of the spots automatically flagged Bad or Not Found by the software, the spots whose ([S – B]/B) < 3, referring to ([media of signal – media of background]/media of background) < 3, were also deleted. Thus, only the spots whose signal intensity was at least 4-fold that of its background were further analyzed, whereas those less than 4-fold that of the background were removed. In addition, we ruled out spots whose regulation pattern was contradictory in two dye-exchange replicates. In this article, only data with |Log2 ratio| To further annotate the genes differentially expressed during cold stress, similarity analysis for each sequence involved use of databases from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and The Institute for Genomic Research rice genome project (http://www.tigr.org) with both BLASTn and BLASTp. Functional classification involved gene ontology searches (http://www.geneontology.org).
Total RNA was extracted from Arabidopsis or rice seedlings with use of Trizol reagent (Invitrogen) and treated with RNase free DNase (Progma). To confirm the reliability of microarray hybridization, semiquantitative RT-PCR involved use of the One Step RNA PCR kit (AMV; TaKaRa) with gene-specific primers (Supplemental Table S2). Total RNA was isolated from materials collected for microarray hybridization. One microgram of total RNA was used as template in one reaction. The same amplification reaction was conducted with a rice Tubulin gene used as template RNA loading control. RT-PCR reactions were repeated five times. For real-time PCR, 2 µg total RNA was used for RT with SuperScripts II reverse transcriptase (Invitrogen). The cDNA samples were diluted to 2 and 8 ng/µL. Triplicate quantitative assays were performed on 1 µL of each cDNA dilution with the SYBR GreenMaster mix and an ABI 7900 sequence detection system according to the manufacturer's protocol (Applied Biosystems). The relative quantification method (Delta-Delta CT) was used to evaluate quantitative variation between the replicates examined. The amplification of Actin was used as an internal control to normalize all data. Gene-specific primers for OsMYB3R-2 were 5'-CAG GGT TTC TAT CTC GTT CC-3' and 5'-ATT TCC AAG CCC TTA CCA C-3'; for OSISAP1, 5'-GAT CAG GAG CCG ACG GAG CT-3' and 5'-GAC AAA GAA GAC GGC GAC GAG-3'; for DREB2A, 5'-AAG GTA AAG GAG GAC CAG AG-3' and 5'-ACA CAA CCA GGA GTC TCA AC-3'; for COR15a, 5'-CTC AGT TCG TCG TCG TTT C-3' and 5'-CAT CTG CTA ATG CCT CTT T-3'; for RCI2A, 5'-ATC GCC ATC CTC TTG CCT CC-3' and 5'-TAG GAG AAC ACG ACG GAA C-3'; for CBF1, 5'-CTT CGC TGA CTC GGC TTG G-3' and 5'-ACG CAC CTT CAC TCT GTT CC-3'; for CBF2, 5'-AAC CAG CGG GAA GGA AGA AGT-3' and 5'-TTT CCT TGG CAC AGG TTG ATT-3'; for CBF3, 5'-GAT CAG CCT GTC TCA ATT TC-3' and 5'-CTT CTG CCA TAT TAG CCA AC-3'; for COR 47, 5'-TAT CAT GCC AAG ACC ACT GAA-3' and 5'-CAA CGA AAG CCA CAA TAA CAA-3'; for RD29A, 5'-ATC ACT TGG CTC CAC TGT TGT TC-3' and 5'-ACA AAA CAC ACA TAA ACA TCC AAA GT-3'; for KIN1, 5'-ACC AAC AAG AAT GCC TTC CA-3' and 5'-CCG CAT CCG ATA CAC TCT TT-3'; for Actin in Arabidopsis, 5'-GGT AAC ATT GTG CTC AGT GGT GG-3' and 5'-AAC GAC CTT AAT CTT CAT GCT GC-3'; and for Actin in rice, 5'-GAA CTG GTA TGG TCA AGG CTG-3' and 5'-ACA CGG AGC TCG TTG TAG AAG-3'.
The localization assay was performed as described by Wang et al. (2004)
The digestion product OsMYB3R-2 from pT-OsMYB3R-2 was directionally cloned into the KpnI-BamHI sites of an SN1301 vector to create SN1301-OsMYB3R-2, which carried a GUS marker. OsMYB3R-2 was driven by a CaMV 35S promoter in the construct. The construct was electroporated into the Agrobacterium tumefaciens C58. Arabidopsis plants were transformed by the floral dip method (Clough and Bent, 1998
DNA gel-blot analysis was performed as described by Wang et al. (2004)
Total RNA was extracted from 3-week-old seedlings of Arabidopsis with use of Trizol reagent (Invitrogen). Total RNA of 20 µg was electrophoresed on 1.2% agarose gel containing 0.4 M formaldehyde and transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech). Probes labeled with
Arabidopsis was grown in 10-cm pots filled with a 1:1 mixture of perlite and vermiculite under a long-day photoperiod (16-h light/8-h dark) at 22°C. Freezing stress involved transferring the 4-week-old or 3-week-old plants into a chamber, decreasing the temperature to –2°C, –4°C, –6°C, –8°C, and –10°C for 10 h respectively, then returning the temperature to 22°C. Plants were analyzed after recovery for 6 d in normal growth conditions. For the drought stress evaluation, seedlings were grown in pots (10-cm diameter) filled with vermiculite for 2 weeks with constant watering before water was withheld. After 2 weeks without water, all the pots were rewatered simultaneously, and the plant regrowth was scored 7 d later. Plants were considered dead if all the leaves were brown and there was no regrowth 7 d after rewatering. For water-loss analysis, 10 fully expanded leaves from wild-type and 35S::OsMYB3R-2 plants that had developed approximately 14 leaves were detached and weighed at different times to determine the rate of water loss. Each experiment was carried out at least three times. For the salt tolerance assay, transgenic and wild-type seeds were planted on MS agar plates for germination. Two days after germination, seedlings from each line were carefully transferred to a new MS agar plate supplemented with different concentrations of NaCl. After 7-d growth in treatment media, plants with absent green or dead cotyledons were scored. The root length of the seedlings was measured. We repeated freezing, drought, and salt tolerance experiments three times.
The sensitivity of seed germination to ABA and NaCl was assayed on MS agar plates saturated with ABA and NaCl solution (Xiong et al., 2001 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number BAD81765.
The following materials are available in the online version of this article.
We are grateful to Joshua Gendron and Dr. Zhiyong Wang (Stanford University) for their critical reading, Prof. Dazhou Chen (Rice Research Institute, Jiangxi Academy of Agricultural Sciences) for supplying the rice seeds of Yuedongdao, and Mr. Cheng Yuan (Institute of Botany, Chinese Academy of Sciences) for assisting with microscope techniques. Received December 11, 2006; accepted February 3, 2007; published February 9, 2007.
1 This work was supported by the Major State Basic Research Program of the People's Republic of China (grant no. 2005CB120806), the National Natural Science Foundation of China (grant nos. 30525026 and 30470866), the State Project of Transgenic Plants (grant no. JA03–A–09), and by the State High-Tech Project (grant no. 2006AA10Z169).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kang Chong (chongk{at}ibcas.ac.cn).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.094532 * Corresponding author; e-mail chongk{at}ibcas.ac.cn; fax 86–010–82594821.
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TOP
ABSTRACT
RESULTS
1) as cold-inducible or cold-repressive genes. A total of 328 genes showing reproducible 2-fold up- or down-regulation were selected. Among them, 157 genes were cold inducible and 171 cold repressive (
-glucuronidase (GUS) gene as a probe. Three transgenic lines were randomly selected and showed different hybridized patterns to the GUS probe (
-32P-dCTP and 
