© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi)
1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2Department of Regulation-Biology, Saitama University, Saitama 338-8570, Japan
To whom correspondence should be addressed. E-mail: sfuji{at}for.agr.hokudai.ac.jp
Received 21 May 2007; Revised 20 August 2007 Accepted 22 August 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Xylem parenchyma cells (XPCs) in larch adapt to subfreezing temperatures by deep supercooling, while cortical parenchyma cells (CPCs) undergo extracellular freezing. The temperature limits of supercooling in XPCs changed seasonally from –30 °C during summer to –60 °C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from –60 °C to –30 °C. As an approach to clarify the mechanisms underlying the change in supercooling capability of larch XPCs, genes expressed in association with increased supercooling capability were examined. By differential screening and differential display analysis, 30 genes were found to be expressed in association with increased supercooling capability in XPCs. These 30 genes were categorized into several groups according to their functions: signal transduction factors, metabolic enzymes, late embryogenesis abundant proteins, heat shock proteins, protein synthesis and chromatin constructed proteins, defence response proteins, membrane transporters, metal-binding proteins, and functionally unknown proteins. All of these genes were expressed most abundantly during winter, and their expression was reduced or disappeared during summer. The expression of all of the genes was significantly reduced or disappeared with deacclimation of winter twigs. Interestingly, all but one of the genes were expressed more abundantly in the xylem than in the cortex. Eleven of the 30 genes were thought to be novel cold-induced genes. The results suggest that change in the supercooling capability of XPCs is associated with expression of genes, including genes whose functions have not been identified, and also indicate that gene products that have been thought to play a role in dehydration tolerance by extracellular freezing also have a function by deep supercooling.
Key words: Cold acclimation, deep supercooling, gene expression, larch (Larix kaempferi), xylem parenchyma cells
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Trees in cold regions acquire the highest freezing resistance within the plant kingdom (Sakai and Larcher, 1987). Among the tissues in trees, however, the mechanisms of adaptation to subfreezing temperatures are different in xylem and cortical tissue cells. Cortical parenchyma cells (CPCs) in trees adapt to subfreezing temperatures by extracellular freezing, whereas xylem parenchyma cells (XPCs) adapt to subfreezing temperatures by deep supercooling (Quamme et al., 1982; Ashworth et al., 1988; Malone and Ashworth, 1991). While extracellular freezing is the most common mechanism of adaptation to freezing for many plant cells, adaptation by deep supercooling is unique to XPCs in trees (Sakai and Larcher, 1987).
In the process of adaptation by extracellular freezing, when extracellular water is frozen at relatively warm subzero temperatures, the difference between vapour pressures of extracellular ice and intracellular water causes living cells to dehydrate effectively in direct parallel to temperature reductions (Steponkus, 1984). By this equilibrium dehydration, cells are able to avoid the occurrence of lethal intracellular freezing. The survival of cells undergoing the process of adaptation by extracellular freezing depends mainly on the dehydration tolerance, and the range of temperatures for survival varies widely from just below zero to the temperature of liquid nitrogen (Sakai, 1960; Sakai and Larcher, 1987). As a result of seasonal cold acclimation, extracellular freezing CPCs in boreal trees, including larch, obtain extremely high freezing tolerance and can tolerate liquid nitrogen temperature during winter (Sakai and Larcher, 1987).
XPCs in boreal trees, including larch (Kasuga et al., 2007b), on the other hand, adapt to subfreezing temperatures by metastable deep supercooling (Kuroda et al., 2003). Deep supercooling XPCs are not dehydrated under the condition of freezing of apoplastic water and maintain intracellular water in a liquid state. While XPCs can survive in a supercooling state, the supercooling of intracellular water has a physical limit. When the temperature falls below the limit of supercooling, the capability for maintaining a metastable equilibrium is exceeded and lethal intracellular freezing occurs by homogeneous ice nucleation (Malone and Ashworth, 1991). The limit of temperatures for supercooling in XPCs is generally higher than that for survival of extracellular freezing cells, especially CPCs of boreal trees (Sakai and Larcher, 1987). The freezing resistance of XPCs in boreal trees is therefore weakest among all tissue cells in trees (Quamme et al., 1972, 1982). Therefore, the temperature limits of supercooling capability in XPCs become a critical factor that limits the distribution of trees to cold areas (Burke and Stushnoff, 1979; Sakai and Larcher, 1987).
In order to adapt to environmental temperature changes, XPCs change the limit of temperatures for supercooling. The temperature limits for supercooling in XPCs of trees gradually decrease in parallel with the reduction in latitudinal environmental temperature from tropical toward arctic zones in the range from –10 °C to about –60 °C, possibly as a result of evolutionary cold acclimation (Fujikawa and Kuroda, 2000; Kuroda et al., 2003). The temperature limit of supercooling also changes seasonally. The supercooling capability of XPCs in boreal trees, including larch, is in the range of –20 °C to –30 °C during summer, but is lowered to around –60 °C during winter as a result of seasonal cold acclimation (Kuroda et al., 1999, 2003; Fujikawa and Kuroda, 2000; Kasuga et al., 2007a). The supercooling capability of XPCs is also changed by artificial cold acclimation and deacclimation (Hong and Stuknoff, 1982).
Based on results of in vitro experiments on the supercooling of small isolated water droplets (Fletcher, 1970; MacKenzie, 1977), the mechanism of deep supercooling in XPCs has been explained solely by the physical state of water (Ashworth and Abeles, 1984). It has been suggested that protoplasts of XPCs are isolated from the effects of extracellular (apoplast) ice crystals due to the presence of specific cell walls that allow neither dehydration of protoplasts nor penetration of extracellular ice into protoplasts (George and Burke, 1977; Quamme et al., 1982; George, 1983; Ashworth and Abeles, 1984). Thus, it has been thought that protoplasts of XPCs as an isolated droplet could supercool to the homogeneous ice nucleation temperature of water (–40 °C) and sometimes to lower temperatures by freezing temperature depression that results from the concentration of solutes in protoplasts (Gusta et al., 1983; Kuroda et al., 2003; Kasuga et al., 2007a).
The isolation of protoplasts from the effects of extracellular ice is undoubtedly a prerequisite for supercooling in XPCs. However, it is difficult to explain the cause of the change in supercooling capability of XPCs by cold acclimation and deacclimation only by such a physical state of water as an isolated droplet. On the basis of a previous hypothesis, it has been suggested that the temperature limit of supercooling corresponds to temperatures when cell walls lose their barrier property against penetration of extracellular ice, by which seeding of ice to protoplasts results in breakdown of supercooling (Burke and Stushnoff, 1979; Ashworth and Abeles, 1984). A previous study, however, showed that plant cell walls even in extracellular freezing cells can inhibit penetration of extracellular ice (Yamada et al., 2002). A recent study, furthermore, showed that the supercooling capability of XPCs is changed significantly by release of intracellular contents without a significant change in cell wall properties, suggesting involvement of intracellular substances in the fluctuation of the supercooling capability of XPCs (Kasuga et al., 2006). Therefore, it is thought that the fluctuation of the supercooling capability of XPCs by cold acclimation and deacclimation might be related to intracellular contents and controlled by more complex mechanisms.
Although cold acclimation-induced changes of freezing resistance in association with gene expression have been investigated in many studies, such studies have been limited to plant cells that adapt to subfreezing temperatures by extracellular freezing (Sakai and Larcher, 1987; Guy, 1990; Hughes and Dunn, 1996; Thomashow, 1999). There have been few studies on cold acclimation-induced changes in supercooling capability of XPCs in trees (Kasuga et al., 2007a), and to the best of our knowledge there has been no report on a comprehensive analysis of gene expression in association with changes in supercooling capability of XPCs in trees. In the present study, therefore, the possibility of changes in gene expression in association with a change in supercooling capability of XPCs in larch was examined.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Fresh twigs of
4 years of age were obtained from adult larch (Larix kaempferi) trees growing on the campus of Hokkaido University, Sapporo, Japan.
Measurement of freezing resistance
For evaluation of seasonal changes in freezing resistance of XPCs, xylem tissues were collected on 10 August, 10 September, 15 October, 13 November, and 9 December in 2003, and on 14 January, 9 February, 17 March, 23 April, and 17 July in 2004.
For evaluation of changes in freezing resistance of XPCs during artificial deacclimation, twigs were collected on 22 December 2003. The twigs were cut into lengths of 10 cm and the cross-sectional ends were wrapped with wet paper towels to avoid desiccation, and then they were kept at 23 °C in the dark for 1, 3, and 7 d for artificial deacclimation.
Freezing resistance was determined by electrolyte leakage measurement. Xylem tissues were removed from twigs and cut into small pieces (7x2x1 mm). Several pieces were put in a test tube containing 0.5 ml of distilled water. The test tubes were kept at –3 °C for 30 min in a programmed freezer (MDF-192 freezer, Sanyo Co., Ltd, Tokyo, Japan, equipped with an ES-100P programmable controller, Tajiri Co., Ltd, Sapporo, Japan), frozen by inoculation of ice tips, and further kept for 1 h. The test tubes were then cooled at a rate of 5 °C h–1 to the given temperatures. Soon after reaching the given temperatures, the test tubes were withdrawn from the freezer and thawed overnight at 4 °C in the dark. A 5 ml aliquot of distilled water was added to each test tube and the tubes were incubated at room temperature for 4 h with gentle shaking in the dark. The amounts of electrolytes in the distilled water that were released from the freeze-thawed tissues (Efx) were measured by an electro-conductivity meter (B-173, Horiba Ltd, Kyoto, Japan). After measurement, the tubes containing the samples were boiled for 10 min. The tubes were again gently shaken for 2 h at room temperature in the dark, and the amount of electrolytes in distilled water (Ebx) was measured. The percentage of freezing injury was calculated as (Efx/Ebx–Efc/Ebc)/(1–Efc/Ebc)x100, where Efc was determined as the amount of electrolytes from fresh tissues and Ebc was determined as the amount of electrolytes after boiling of fresh tissues. By plotting the percentage of freezing injury at different freezing temperatures, the temperature at which 50% of the cells were injured (LT50) was determined.
RNA extraction
Total RNAs were isolated from xylem in twigs in different seasons. Total RNAs were also isolated from xylem in winter before and after deacclimation and from cortical tissues in winter.
These tissue samples were fragmented by a mortar and pestle in liquid nitrogen and then homogenized with addition of 10 times the volume of extraction buffer containing 2% (w/v) cetyltrimethylammonium bromide, 0.1 M TRIS-HCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, and 1% (w/v) β-mercaptoethanol. After incubation with the extraction buffer at 65 °C for 10 min followed by chloroform extraction, the upper phase was precipitated by isopropanol. The precipitates were dissolved in diethylpyrocarbonate (DEPC)-treated water, and LiCl was added to make a final concentration of 2.6 M. Precipitates of RNAs were collected by centrifugation at 18 000 g, dissolved in DEPC-treated water, and purified with extraction by phenol and chloroform.
Differential display analysis
For differential display analysis, total RNAs were obtained from xylem tissues of field-grown trees harvested on 13 February 2001. Total RNAs were also obtained from the deacclimated xylem tissues. For deacclimation treatment, twigs harvested on 13 February 2001 were put in a pot with water at 25 °C with a 16 h light/8 h dark cycle for 7 d. By this treatment, leaves began to open. These two samples, xylem tissues before and after deacclimation, were used for differential display analysis to detect genes that change expression, as described by Yoshida et al. (1994).
cDNAs were synthesized by a SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions using 2 µg of total RNAs. The volume of the reaction product was then adjusted to 500 µl with distilled water. PCR was performed using 1 µl of the diluted reaction product with 12-base common primer pairs (Bex, Tokyo, Japan). In total, 300 common primer pairs were used for PCR. The PCR products were analysed by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The PCR bands that decreased by deacclimation treatment were excised from the gel. The DNA fragments in the gel were eluted out in TE buffer [100 mM TRIS-HCl (pH 8.0) and 10 mM EDTA (pH 8.0)] by overnight incubation at 4 °C. The fragments were used as a template for the re-amplification reaction using the same primer pairs and reaction programs. The amplified PCR fragments were subcloned into pGEM-T Easy vector (Promega, Madison, WI, USA) according to the manufacturer's instructions and subjected to dideoxynucleotide sequencing. A homology search of the sequenced clones was carried out in the NCBI database (http://www.ncbi.nlm.nih.gov/) using the BLAST sequence search protocol. To reduce the ‘false-positive’ rate of mRNAs in differential display analysis, each primer pair was used to amplify two different sets of RNAs isolated from different xylem tissues.
Construction of a cDNA library
Total RNAs were extracted from xylem tissues that were harvested on 22 December 2003. The poly(A)+ RNAs were purified from the total RNAs using an oligo(dT)–cellulose column (Sambrook et al., 1989). The cDNA library was constructed in Uni-ZAP vector (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions.
Differential screening using a cDNA filter array
Total RNAs were obtained from xylem tissues that were harvested from field-grown trees on 22 December 2003. Total RNAs were also obtained from the xylem of 4-d-deacclimated twigs harvested on 22 December 2003. Deacclimation conditions were the same as those for samples used for measurement of freezing resistance. These winter samples before and after deacclimation treatments were used for differential screening to detect genes with differences in expression.
To construct cDNA filter arrays, 2400 clones were randomly selected from the cDNA library. These clones were amplified by PCR using the primer pair 5'-CATTAGGCACCCCAGGCTTTACAC-3' and 5'-GTAATACGACTCACTATAGGGC-3'. PCR-amplified DNA fragments were spotted onto Hybond N+ membranes (GE Healthcare, USA) in duplicate. The filters were hybridized with 32P-labelled first-strand cDNAs that were made from the two mRNAs as described by Sambrook et al. (1989). After washing the filters in 0.1x SSC and 0.1% SDS at 60 °C for 1 h, the filters were exposed to X-ray film. For detection of repressed genes by deacclimation, radioactive signals of spots were compared on the arrays, and clones showing a visual decrease in signal intensity of less than one-third were selected. The clones were subjected to dideoxynucleotide sequencing. A homology search of the sequenced clones was carried out in the NCBI (http://www.ncbi.nlm.nih.gov/) database using the BLAST sequence search protocol.
Northern blot analysis
Twigs were harvested on 22 December 2003 and used to investigate changes in gene expression by deacclimation of winter twigs. The deacclimation conditions were the same as those for samples used for measurement of freezing resistance. Xylem tissues in twigs that had been deacclimated for 1, 3, and 7 d were used.
To investigate changes in seasonal gene expression, xylem tissues were collected monthly from July 2002 to June 2003.
For comparison of gene expression in xylem tissues and cortical tissues, both tissues were collected on 15 December 2002.
All samples were frozen in liquid nitrogen and stored at –80 °C until use. Extracted total RNA (5 µg) was electrophoresed on a 1.25% agarose/2.2 mol l–1 formaldehyde gel and transferred onto a Hybond N+ membrane (GE Healthcare) using transfer buffer, 20x SSC. Equal loading of RNAs in each lane was verified by ethidium bromide staining of RNAs in the gel. The membrane was used for hybridization with 32P-labelled DNA probes generated by using a random primer labelling kit (GE Healthcare). The hybridization was carried out at 42 °C for 16 h in ULTRAhyb (Ambion, Austin, TX, USA) with a 32P-labelled DNA probe. After the hybridization, the membranes were washed in 0.2x SSC and 0.1% (w/v) SDS at 55 °C for 1 h and exposed to X-ray film. DNA probes representing the genes identified by differential display analysis were used for EcoRI-digested fragments of subcloned vectors and the genes identified by differential screening were amplified by PCR using the primer pair 5'-CATTAGGCACCCCAGGCTTTACAC-3' and 5'-GTAATACGACTCACTATAGGGC-3'.
Quantitative evaluation of each detected band by northern blot analysis
Imaging X-ray films were scanned with an EPSON scanner (GT-X700, EPSON, Japan) at 300 dpi. The X-ray films were calibrated and analysed to quantify the intensity of each band using NIH Image software (http://rsb.info.nih.gov/nih-image).
For indicating the changes in gene expression during deacclimation, relative expression values during deacclimation were calculated in comparison with the intensity without deacclimation. For indicating the changes in gene expression between cortical tissues and xylem tissues, relative expression values in cortical tissues were calculated in comparison with the intensity of xylem tissues. For indicating seasonal change in gene expression, relative expression values in each month were calculated in comparison with the intensity of maximum expression throughout the entire year.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Changes in freezing resistance of XPCs
The freezing resistance of XPCs in larch grown in the field was significantly different depending on the season. The freezing resistance of XPCs was around –30 °C during summer, increased gradually during autumn, reached a maximum at –60 °C during winter, and then gradually decreased during spring (Fig. 1).
|
Artificial deacclimation of winter twigs also significantly reduced freezing resistance of XPCs (Fig. 2). Freezing resistance of –60 °C before deacclimation was reduced to –36 °C by 3 d of deacclimation and to –31 °C by 7 d of deacclimation.
|
Differential display analysis of genes associated with a change in freezing resistance
In order to find genes that might be associated with a change in freezing resistance of XPCs, gene expression in xylem of winter twigs before deacclimation (with freezing resistance of –60 °C) and after deacclimation (with freezing resistance of –31 °C by 7 d of deacclimation) was compared by differential display analysis.
As a result of differential display analysis, 75 PCR bands that appeared before deacclimation were reduced or disappeared after deacclimation. Among them, nine PCR bands were most distinctly reduced by deacclimation. By using a BLAST search utility, it was found that these genes encoded proteins with sequence similarity to seven functionally known proteins and two functionally unknown proteins (see Supplementary Table S1 at JXB online). Seven functionally known genes encoded nucleotide-binding site (NBS)/leucine-rich repeat (LRR) protein, H+-pyrophosphatase, aminoalcoholphosphotransferase, flavonol 3-o-glucosyltransferase, histone H2A, LRR-containing F-box protein, and Arabidopsis thaliana protein T20L15, 20. Each of these nine genes was designated as Winter-induced gene in Xylem of Larch (WXL). Among these nine genes, five genes were novel cold-induced genes (Table 1).
|
Differential screening of genes associated with a change in freezing resistance
To identify more genes that might be associated with a change in freezing resistance of XPCs, gene expression in xylem of winter twigs before deacclimation (with freezing resistance of –60 °C) and after deacclimation (with freezing resistance of –36 °C by 3 d of deacclimation) were compared by differential screening using a filter array.
As a result of differential screening of 2400 clones in total, 86 clones of genes that were preferentially expressed when freezing resistance of XPCs was maximum (before deacclimation) were isolated. From the 5’ ends of the genes, a few hundred nucleotides were sequenced in all of these 86 clones. Using the BLAST search utility, it was found that 70 clones had sequence similarity to 18 functionally known proteins, three clones had sequence similarity to three functionally unknown proteins, and 16 clones were not recorded in the NCBI database. The 18 functionally known genes encoded abscisic acid (ABA), stress, and ripening (ASR) protein, water deficit-inducible protein LP3-1 and LP3-3, protein phosphatase 2A regulatory subunit, galactinol synthase, dehydrin or dehydrin-like proteins, small heat shock protein (HSP), peptide chain release factor subunit 1, hypersensitive-induced response protein (HIR), carnitine/acylcarnitine translocase, metallothionein, and Arabidopsis thaliana MtN3-like protein (see Supplementary Table S2 at JXB online). Many of the 70 sequenced clones were found in dehydrin-like proteins (16 clones), ASR protein (15 clones), and galactinol synthase (12 clones) (see Supplementary Table S2 at JXB online). Each of these genes encoding 18 functionally known proteins and three functionally unknown proteins was designated as Supercooling-associated gene in Xylem of Larch (SXL). Among these 21 recorded genes, six genes were novel cold-induced genes (Table 1).
Northern blot analysis of deacclimation-induced changes in gene expression
In order to investigate deacclimation-induced expression changes in WXL and SXL genes (Table 1), which showed higher expression levels before deacclimation in parallel with higher freezing resistance in XPCs, northern blot analysis was carried out using total RNAs extracted from xylem tissues during deacclimation of winter twigs.
Expression levels of the transcripts of all 30 genes were decreased in parallel with prolongation of the deacclimation period (Fig. 3A). Such a decrease occurred more clearly with deacclimation periods of >3 d concomitant with periods when freezing resistance of XPCs was dramatically decreased (Fig. 2). The expression of 27 of the 30 genes (WXL1, 4, 5, 6, 7, 10, and 11, and SXL1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 20, 22, 23, 24, and 25) was rapidly down-regulated by 3 d of deacclimation, whereas the expression levels of the other three genes (WXL3, WXL9, and SXL21) gradually decreased during 7 d of deacclimation (Fig. 3B).
|
Northern blot analysis of seasonal changes in gene expression
In order to investigate seasonal changes in expression of WXL and SXL genes (Table 1), northern blot analysis was carried out using total RNAs extracted from xylem tissues in field-grown twigs collected at monthly intervals. Expression levels of almost all of these genes increased during autumn, with the maximum expression level being maintained during winter, when freezing resistance of XPCs was maximal, and then the expression levels decreased during spring and became minimal during summer, when freezing resistance was minimum (Figs 1, 4A).
|
The patterns of induction and repression of these genes were slightly different (Fig. 4B–E). Twenty-one genes (WXL1, 3, 5, 7, 9, and 10, and SXL1, 2, 3, 4, 5, 6, 8, 9, 12, 13, 14, 20, 23, 24, and 25) were abundantly induced in November and retained distinct expression until March or April (Fig. 4B). Five genes (WXL4 and 11, and SXL7, 10, and 22) were abundantly induced from October and retained distinct expression until March or April (Fig. 4C). Three genes (WXL6, and SXL15 and 17) were gradually induced from November and their expression levels peaked in mid-winter and gradually decreased to April (Fig. 4D). Among these three genes, the gene encoding small HSP (SXL15) was also expressed in July. Only one gene (SXL21) was induced from November, and its expression level gradually increased with a peak around April, and then gradually decreased and disappeared in July (Fig. 4E).
Northern blot analysis of tissue-specific changes in gene expression
To compare the tissue-specific expression levels of WXL and SXL genes (Table 1), the gene expression levels were investigated in xylem tissues, in which XPCs adapt to subfreezing temperatures by deep supercooling, and in cortical tissues, in which CPCs adapt to subfreezing temperatures by extracellular freezing. Northern blot analysis was carried out using total RNAs isolated from each fresh tissue in twigs collected in winter.
Most of the WXL and SXL genes were more predominantly expressed in xylem tissues than in cortical tissues (Fig. 5A). Based on expression profiles, these genes were tentatively categorized into three groups (Fig. 5B). Twenty-nine genes (WXL1, 4, 5, 6, 7, 9, 10, and 11, and SXL1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 20, 21, 22, 23, 24, and 25) were expressed in xylem tissues at levels >2-fold higher than those in cortical tissues. Among these 29 genes, furthermore, there was a notable difference in expression patterns of 17 genes (WXL1 and 6, and SXL1, 3, 6, 7, 8, 9, 10, 12, 13, 15, 17, 20, 21, 23, and 24), which were expressed in xylem tissues at levels >5-fold higher than those in cortical tissues. Only one gene (WXL3) was expressed similarly in xylem tissues and cortical tissues. No genes with higher expression in cortical tissues than in xylem tissues were detected.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Deep supercooling adaptation in larch XPCs during winter has been clearly confirmed by a recent study using a cryo-scanning electron microscope (Kasuga et al., 2007b). During supercooling, XPCs can survive, but below the temperature limit of supercooling lethal intracellular freezing occurs (Kuroda et al., 2003). Therefore, many studies have shown correspondence of temperatures between the limit of supercooling and occurrence of freezing injury in XPCs (Sakai and Larcher, 1987). In the case of larch XPCs, the temperature limit of supercooling (Kasuga et al., 2007b) also corresponds to the temperature for survival by the LT50 (Fig. 1).
The supercooling capability of larch XPCs as revealed by their LT50 was significantly changed by seasonal temperature fluctuation in field-grown larch (Fig. 1). The supercooling capability of XPCs in larch becomes maximal during winter (Kasuga et al., 2007b) and decreases during summer (Fujikawa et al., 1999). Similar seasonal changes in the supercooling capability of XPCs have also been shown by differential thermal analysis (DTA) in many trees (George and Burke, 1977; Kaku and Iwaya, 1978; Wisniewski and Ashworth, 1986; Wiesniewski and Davis, 1989; Kuroda et al., 2003). Although there have been few studies on the effects of artificial cold acclimation and deacclimation on supercooling capability of XPCs, reduction of supercooling capability by artificial deacclimation of apple twigs during winter has been shown by a DTA study (Hong and Stukoff, 1982). The present study also showed that artificial deacclimation of winter twigs of larch significantly reduced the supercooling capability of XPCs as revealed by the LT50 (Fig. 2).
The purpose of the present study was to determine whether or not such a change in supercooling capability of XPCs is associated with changes in gene expression. For this purpose, two different gene-screening techniques were used, differential display with 300 common primer pairs and a cDNA filter array representing 2400 clones, and changes in gene expression in xylem of winter twigs were compared before deacclimation (with high supercooling capability in XPCs) and after deacclimation (with reduced supercooling capability in XPCs). As a result of these screenings, 161 clones (75 by differential display and 86 by the filter array) with repressed expression after deacclimation were isolated. These clones corresponded to 30 genes [nine genes by differential display (see Supplementary Table S1 at JXB online) and 21 genes by the filter array (see Supplementary Table S2 at JXB online)]. It is thought that these 30 genes (Table 1), the expression levels of which were reduced by deacclimation in parallel with reduced supercooling capability in XPCs, were associated with high supercooling capability in XPCs before deacclimation.
Each screening technique provided different genes without correspondence between the two techniques. It is thought that the discrepancy in genes detected by different screening techniques is due to a difference in cold acclimation duration (7 d of deacclimation for differential display and 4 d of deacclimation for the filter array) and also due to an inherent difference in the properties of the two techniques to detect genes. Since differential display analysis is more sensitive than a filter array (Kuhn, 2001), differential display might preferentially detect genes with a greater difference in the expression rate of mRNA, even though the quantity of expressed mRNAs is less. On the other hand, in a filter array, it is possible to examine a large number of clones by analysing a large number of cDNA clones.
Despite the difference in genes detected by the two techniques, northern blot analysis confirmed that all of the 30 genes detected by both techniques were associated with increased supercooling capability in XPCs. Expression of these genes as revealed by northern blot analysis was down-regulated by artificial deacclimation of winter twigs (Fig. 3) in parallel with the decrease in supercooling capability of XPCs by deacclimation (Fig. 2). Expression of these genes as revealed by northern blot analysis was closely associated with seasonality, showing a higher expression level during winter (Fig. 4), when the supercooling capability of XPCs reached the maximum (Fig. 1), and, conversely, showing a lower expression level or disappearance during summer (Fig. 4) when the supercooling capability of XPCs reached the minimum (Fig. 1). Furthermore, expression levels of almost all of these genes as revealed by northern blot analysis were much higher in xylem tissues containing deep supercooling XPCs than in cortical tissues containing extracellular freezing CPCs, except for one gene (WXL3) encoding H+-pyrophosphatase as one of the membrane transporters (Fig. 5).
About two-thirds of the 30 genes that are expressed in association with high supercooling capability in XPCs are already known as cold-induced genes (Table 1). These known cold-induced genes have been shown to be related to low temperature signal transduction by ASR and PP2A proteins (SXL2, 8, 9, and 10) (Schneider et al., 1997; Monroy, 1998; Maskin et al., 2001; Kim et al., 2002; Yang et al., 2005), change of metabolism under low temperatures, including biosynthesis of raffinose family oligosaccharides (RFOs) by galactinol synthase (SXL3) (Taji et al., 2002; Pennycooke et al., 2003), phospholipid synthesis by aminoalcoholphosphotransferase (WXL4) (Qi et al., 2003), and glucosylation of flavonoids by flavonol-3-o-glucosyltransferase (WXL5) (Christie et al., 1994; Chalker-Scott, 1999; Winkel-Shirley, 2002; Lo Piero et al., 2005), protection of macromolecules from freezing-induced dehydration by LEA proteins (SXL1, 5, 4, 7, 12, 13, and 14) (Houde et al., 1995; Close, 1997; Danyluk et al., 1998; Wisniewski et al., 1999; Fujikawa et al., 2006), membrane stabilizer, molecular chaperone, and cryoprotectant under freezing by HSP (SXL15) (van Berkel et al., 1994; Sabehat et al., 1998; Ukaji et al., 1999; Török et al., 2001; van Montfort et al., 2001; Fujikawa et al., 2006), regulation of protein synthesis and chromatin construction under low temperature by eRF1 and histone H2A proteins (SXL17 and WXL6) (Seki et al., 2002), and maintenance of the pH gradient between the cytoplasm and vacuole under low temperature by H+-pyrophosphatase (WXL3) (Carystinos et al., 1995). Additionally, metallothionein genes, encoding metal-binding protein (SXL22), are induced by cold (Reid and Ross, 1997; Cho et al., 2006), although the functional roles under cold stress are unknown.
It is thought that because many of these genes might be mainly involved in adaptation to low temperatures rather than adaptation to dehydration, most of these already known cold-induced genes identified in extracellular freezing cells might also appear in supercooling XPCs by cold acclimation. Among them, on the other hand, accumulation of dehydrins and soluble sugars, especially RFOs, plays an important role in adaptation to dehydration under extracellular freezing. While there are still questions about the higher expression levels of almost all of these genes in xylem tissues than in cortical tissues, the main issue of interest is the greater accumulation of genes involved in dehydration tolerance, such as dehydrins and soluble sugars, in xylem tissues with deep supercooling XPCs than in cortical tissues with extracellular freezing CPCs (Fig. 5).
LEA proteins, especially dehydrins, accumulate by cold acclimation and confer dehydration tolerance in many plant cells adapting to extracellular freezing (Close, 1997; Danyluk et al., 1998). Thus, the greater accumulation of these dehydrin family proteins in essentially non-dehydrated deep supercooling XPCs than in extracellular freezing CPCs with heavy dehydration seems to be unreasonable. However, one possibility is that some of the dehydrin family proteins in deep supercooling larch XPCs might function as antifreeze proteins that may enhance the supercooling capability. Facilitation of supercooling of water droplets by addition of crude extracts containing antifreeze proteins from insects has been reported (Patterson and Duman, 1978). It has been reported that peach dehydrin, PCA60, exhibits not only cryoprotective activity toward freezing-susceptible l-lactate dehydrogenase but also antifreeze activity as evidenced by inhibition of ice crystal growth and development of thermal hysteresis (Wisniewski et al., 1999). The function of dehydrin family proteins from larch xylem with deep supercooling XPCs as antifreeze proteins is under examination in our laboratory. Another possible reason for the greater accumulation of genes encoding LEA proteins is a higher intracellular osmotic concentration in XPCs than in CPCs. A previous study showed that deep supercooling XPCs in birch have a higher intracellular osmotic concentration than that in extracellular freezing CPCs during winter (Kasuga et al., 2007a). For adaptation to a higher osmotic concentration during winter, XPCs might produce more dehydrins than those produced in CPCs (Fig. 5). It has been shown that dehydrins were accumulated in woody plant cells at the onset of dormancy by short daylength due to seasonal cold acclimation in parallel with an increased intracellular osmotic concentration (Welling et al., 2002). It has also been shown that the higher intracellular osmotic concentration in XPCs of birch during winter is produced by a seven times higher accumulation of soluble sugars, including RFOs, than in CPCs (Kasuga et al., 2007a). While it has been generally suggested that soluble sugars, including RFOs, stabilize membrane and macromolecular structures against freezing-induced dehydration under extracellular freezing (Pennycooke and Towill, 2001), it has also been suggested that greater accumulation of soluble sugars in XPCs may also have a role in enhancing supercooling of XPCs by melting point depression and consequent reduction of the nucleation temperature to promote supercooling capability (Rasmussen and MacKenzie, 1972; Charoenrein and Reid, 1989). Higher expression levels of galactinol synthase genes in xylem than in cortical tissues (Fig. 5) may be related to greater accumulation of soluble sugars, including RFOs, in larch XPCs.
Among the 30 genes that were expressed in association with high supercooling capability in XPCs, about one-third were novel cold-induced genes (Table 1). All of these novel cold-induced genes also showed higher expression levels in xylem rather than in cortical tissues (Fig. 5). The majority of the novel cold-induced genes are functionally unknown genes (WXL9, 10, 11, SXL6, 23, 24, and 25), and the other genes are involved in signal transduction (WXL1 and 7), defence response (SXL20), and membrane transport (SXL21). As signal transduction factors, two novel cold-induced genes (WXL1 and 7) encoded NBS/LRR proteins and LRR-containing F-box protein. NBS/LRR proteins, which develop the largest class of disease-resistance proteins, are involved in signal transduction by pathogen infection, and confer resistance against a wide variety of phytopathogens (Meyers et al., 2002), but also confer drought tolerance (Grant et al., 2003; Chini et al., 2004). LRR-containing F-box protein is responsible for defence against pathogens (Xie et al., 1998; Xu et al., 2002). Although these signal transduction-related genes are novel as cold-induced genes, it should be noted that pathogen, drought, and cold signalling pathways are often involved in cross-talk (Chinnusamy et al., 2004). One novel cold-induced gene (SXL20) encoded a protein with homology to hypersensitive-induced response protein (HIR). SXL20 shared the Band 7 domain (unpublished results), which is conserved in the family of stomatin, prohibitin, flotillin, and HIR in Arabidopsis (Borner et al., 2005). Many of these proteins are associated with a detergent-resistant sphingolipid-rich and sterol-rich membrane domain, and may be involved in signalling and programmed cell death through control of ion channel activity (Nadimpalli et al., 2000). However, roles for HIR in abiotic stresses have not been reported. One novel cold-induced gene (SXL21) encoded a protein with homology to carnitine/acylcarnitine translocase. Arabidopsis carnitine/acylcarnitine translocase, BOU, is distributed in mitochondria and plays a role in seedling development by degradation of storage lipids in the cotyledon (Lawand et al., 2002). Expression of carnitine/acylcarnitine translocase under abiotic stresses has not been reported.
Although the specific roles of these 30 genes in association with increased supercooling capability in larch XPCs is not yet clear, the present results provide the first evidence that changes in supercooling capability of XPCs may be associated with gene expression. This finding suggests that the change in supercooling capability of XPCs as a result of cold acclimation and deacclimation may be controlled by more complex mechanisms rather than simply by the physical state of protoplasts as an isolated water droplet (Burke and Stushnoff, 1979; Ashworth and Abeles, 1984). Furthermore, it is thought that adaptation to subfreezing temperatures by supercooling or extracellular freezing has both similarities and differences, because of the expression of similar and different genes in both tissues with contrasting freezing adaptation mechanisms. Currently, the functions of these genes are being examined in detail in relation to the change in deep supercooling capability of XPCs.
Supplementary material
Supplementary material in the form of two tables can be found at JXB online.
Table S1. Deep supercooling-associated genes isolated by differential display analysis.
Table S2. Deep supercooling-associated genes isolated by differential screening using a cDNA filter array.
|
Acknowledgements |
|---|
We thank Dr Anzu Minami for her technical advice and support. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Sports, Culture, Science and Technology of Japan (17380101 to SF) and a grant from the Japan Society for the Promotion of Science (17·9014 to JK).
|
Footnotes |
|---|
* Present address: United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
|
Abbreviations |
|---|
ABA, abscisic acid; ASR, ABA, stress, and ripening; CPC, cortical parenchyma cell; DTA, differential thermal analysis; HIR, hypersensitive-induced response protein; HSP, heat shock protein; LEA, late embryogenesis abundant; LRR, leucine-rich repeat; NBS, nucleotide-binding site; RFO, raffinose family oligosaccharide; PP2A, protein phosphatase 2A; SXL, supercooling-associated gene in xylem of larch; WXL, winter-induced gene in xylem of larch; XPC, xylem parenchyma cell.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ashworth EN, Abeles FB. Freezing behavior of water in small pores and the possible role in the freezing of plant tissues. Plant Physiology (1984) 76:201–204.
Ashworth EN, Echlin P, Pearce RS, Hayes TL. Ice formation and tissue response in apple twigs. Plant, Cell and Environment (1988) 11:703–710.[CrossRef]
Borner GH, Sherrier DJ, Weimar T, Michaelson LV, Hawkins ND, Macaskill A, Napier JA, Beale MH, Lilley KS, Dupree P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiology (2005) 137:104–116.
Burke MJ, Stushnoff C. Frost hardiness: a discussion of possible molecular causes of injury with particular reference to deep supercooling of water. In: Stress physiology in crop plants—Mussell H, Staples RC, eds. (1979) New York: Wiley. 197–225.
Carystinos GD, MacDonald HR, Monroy AF, Dhindsa RS, Poole RJ. Vacuolar H+-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiology (1995) 108:641–649.[Abstract]
Chalker-Scott L. Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology (1999) 70:1–9.[CrossRef][ISI]
Charoenrein S, Reid DS. The use of DSC to study the kinetics of heterogeneous and homogeneous nucleation of ice in aqueous systems. Thermochimica Acta (1989) 156:373–381.[CrossRef][ISI]
Chini A, Grant JJ, Seki M, Shinozaki K, Loake GJ. Drought tolerance established by enhanced expression of the CC-NBS-LRR gene, ADR1, requires salicylic acid, EDS1 and ABI1. The Plant Journal (2004) 38:810–822.[CrossRef][ISI][Medline]
Chinnusamy V, Schumaker K, Zhu JK. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany (2004) 55:225–236.
Cho SH, Hoang QT, Kim YY, Shin HY, Ok SH, Bae JM, Shin JS. Proteome analysis of gametophores identified a metallothionein involved in various abiotic stress responses in Physcomitrella patens. Plant Cell Reports (2006) 25:475–488.[CrossRef][ISI][Medline]
Christie PJ, Alfenito MR, Walbot V. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta (1994) 194:541–549.[CrossRef][ISI]
Close TJ. Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiologia Plantarum (1997) 100:291–296.[CrossRef]
Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, Sarhan F. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. The Plant Cell (1998) 10:623–638.
Fletcher NH. The chemical physics of ice (1970) London: Cambridge University Press. 73–103.
Fujikawa S, Kuroda K. Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species. Micron (2000) 31:669–686.[CrossRef][ISI][Medline]
Fujikawa S, Kuroda K, Jitsuyama Y, Sano Y, Otani J. Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell wall. Protoplasma (1999) 206:31–40.[CrossRef][ISI]
Fujikawa S, Ukaji N, Yamane K, Nagao M, Takezawa D, Arakawa K. Functional role of winter-accumulating proteins from mulberry tree in adaptation to winter-induced stresses. In: Cold hardiness in plants: molecular genetics, cell biology and physiology—Chen T, Uemura M, Fujikawa S, eds. (2006) Wallingford, UK: CABI Publishers. 181–202.
George MF. Freezing avoidance by deep supercooling in woody plant xylem: preliminary data on the importance of cell wall porosity. In: Current topics in plant biochemistry and physiology—Randall DD, Blevins DG, Larson RL, Rapp BJ, eds. (1983) Columbia, MO: University of Missouri Press. 84–95.
George MF, Burke MJ. Cold hardiness and deep supercooling in xylem of shagbark hickory. Plant Physiology (1977) 59:319–325.
Grant JJ, Chini A, Basu D, Loake GJ. Targeted activation tagging of the Arabidopsis NBS-LRR gene, ADR1, conveys resistance to virulent pathogens. Molecular Plant–Microbe Interactions (2003) 16:669–680.[CrossRef]
Gusta LV, Tyler NJ, Chen THH. Deep undercooling in woody taxa growing north of the –40 °C isotherm. Plant Physiology (1983) 72:122–128.
Guy CL. Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual Review of Plant Physiology and Plant Molecular Biology (1990) 41:187–223.[ISI]
Hong SG, Stukoff E. Rapid increase in deep supercooling of xylem parenchyma. Plant Physiology (1982) 69:697–700.
Houde M, Daniel C, Lachapelle M, Allard F, Laliberte S, Sarhan F. Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. The Plant Journal (1995) 8:583–593.[CrossRef][ISI][Medline]
Hughes MA, Dunn MA. The molecular biology of plant acclimation to low temperature. Journal of Experimental Botany (1996) 47:291–305.
Kaku S, Iwaya M. Low temperature exotherms in xylems of evergreen and deciduous broad-leaved trees in Japan with reference to freezing resistance and distribution range. In: Plant cold hardiness and freezing stress—Sakai A, Li PH, eds. (1978) New York: Academic Press. 227–239.
Kasuga J, Arakawa K, Fujikawa S. High accumulation of soluble sugars in deep supercooling Japanese white birch xylem parenchyma cells. New Phytologist (2007a) 174:569–579.[CrossRef][ISI][Medline]
Kasuga J, Takata N, Yamane K, Kuroda K, Arakawa K, Fujikawa S. Larch (Larix kaempferi) xylem parenchyma cells respond to subfreezing temperature by deep supercooling. Cryoletters (2007b) 28:77–81.[Medline]
Kasuga J, Mizuno K, Miyaji N, Arakawa K, Fujikawa S. Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells. CryoLetters (2006) 27:305–310.[Medline]
Kim HJ, Kim YK, Park JY, Kim J. Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. The Plant Journal (2002) 29:693–704.[CrossRef][ISI][Medline]
Kuhn E. From library screening to microarray technology: strategies to determine gene expression profiles and to identify differentially regulated genes in plants. Annals of Botany (2001) 87:139–155.
Kuroda K, Kasuga J, Arakawa K, Fujikawa S. Xylem ray parenchyma cells in boreal hardwood species respond to subfreezing temperatures by deep supercooling that is accompanied by incomplete desiccation. Plant Physiology (2003) 131:736–744.
Kuroda K, Ohtani J, Kubota M, Fujikawa S. Seasonal changes in the freezing behavior of xylem ray parenchyma cells in four boreal hardwood species. Cryobiology (1999) 38:81–88.[CrossRef][Medline]
Lawand S, Dorne AJ, Long D, Coupland G, Mache R, Carol P. Arabidopsis A BOUT DE SOUFFLE, which is homologous with mammalian carnitine acyl carrier, is required for postembryonic growth in the light. The Plant Cell (2002) 14:2161–2173.
Lo Piero AR, Puglisi I, Rapisarda P, Petrone G. Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage. Journal of Agricultural and Food Chemistry (2005) 53:9083–9088.[CrossRef][ISI][Medline]
MacKenzie AP. Non-equilibrium freezing behaviour of aqueous systems. Philosophical Transactions of the Royal Society B: Biological Sciences (1977) 278:167–189.[CrossRef]
Maskin L, Gubesblat GE, Moreno JE, Carrari FO, Frankel N, Sambade A, Rossi M, Iusem ND. Differential expression of the members of the Asr gene family in tomato (Lycopersicon esculentum). Plant Science (2001) 161:739–746.
Malone SR, Ashworth EN. Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. Plant Physiology (1991) 95:871–881.
Meyers BC, Morgante M, Michelmore RW. TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsis and other plant genomes. The Plant Journal (2002) 32:77–92.[CrossRef][ISI][Medline]
Monroy AF, Sangwan V, Dhindsa RS. Low temperature signal transduction during cold acclimation: protein phosphatase 2A as an early target for cold-inactivation. The Plant Journal (1998) 13:653–660.[CrossRef][ISI]
Nadimpalli R, Yalpani N, Johal GS, Simmons CR. Prohibitins, stomatins, and plant disease response genes compose a protein superfamily that controls cell proliferation, ion channel regulation, and death. Journal of Biological Chemistry (2000) 275:29579–29586.
Patterson JL, Duman JG. The role of the thermal hysteresis factor in Tenebrio molitor larvae. Journal of Experimental Biology (1978) 74:37–45.
Pennycooke JC, Jones ML, Stushnoff C. Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia. Plant Physiology (2003) 133:901–909.
Pennycooke JC, Towill LE. Medium alterations improve regrowth of sweet potato (Ipomoea batatas Lam.) shoot tips cryopreserved by vitrification and encapsulation-dehydration. CryoLetters (2001) 22:381–389.[Medline]
Qi Q, Huang YF, Cutler AJ, Abrams SR, Taylor DC. Molecular and biochemical characterization of an aminoalcoholphosphotransferase (AAPT1) from Brassica napus: effects of low temperature and abscisic acid treatments on AAPT expression in Arabidopsis plants and effects of over-expression of BnAAPT1 in transgenic Arabidopsis. Planta (2003) 217:547–558.[CrossRef][ISI][Medline]
Quamme HA, Chen PM, Gusta LV. Relationship of deep supercooling and dehydration resistance to freezing injury in dormant stem tissues of ‘Starkrimson Delicious’ apple and ‘Siberian C’ peach. Journal of the American Society for Horticultural Science (1982) 107:299–304.
Quamme HA, Stushnoff C, Weiser CJ. The relationship of exotherms to cold injury in apple stem tissues. Journal of the American Society for Horticultural Science (1972) 97:608–613.
Rasmussen DH, MacKenzie AP. Effect of solute on ice–solution interfacial free energy; calculation from measured homogeneous nucleation temperatures. In: Water structure at the water–polymer interface—Jellinek HHG, ed. (1972) New York: Plenum Press. 126–145.
Reid SJ, Ross GS. Up-regulation of two cDNA clones encoding metallothionein-like proteins in apple fruit during cool storage. Physiologia Plantarum (1997) 100:183–189.[CrossRef]
Sabehat A, Lurie S, Weiss D. Expression of small heat-shock proteins at low temperatures. A possible role in protecting against chilling injuries. Plant Physiology (1998) 117:651–658.
Sakai A. Survival of the twig of woody plants at –196 °C. Nature (1960) 185:393–394.[CrossRef]
Sakai A, Larcher W. Frost survival in plants: responses and adaptations to freezing stress (1987) Berlin: Springer-Verlag.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual (1989) 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Schneider A, Salamini F, Gebhardt C. Expression patterns and promoter activity of the cold-regulated gene ci21A of potato. Plant Physiology (1997) 113:335–345.[Abstract]
Seki M, Narusaka M, Ishida J, et al. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal (2002) 31:279–292.[CrossRef][ISI][Medline]
Steponkus PL. Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology (1984) 35:543–584.[CrossRef][ISI]
Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K. Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. The Plant Journal (2002) 29:417–426.[CrossRef][ISI][Medline]
Thomashow MF. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology (1999) 50:571–599.[CrossRef][ISI][Medline]
Török Z, Goloubinoff P, Horváth I, et al. Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding. Proceedings of the National Academy of Sciences, USA (2001) 98:3098–3103.
Ukaji N, Kuwabara C, Takezawa D, Arakawa K, Yoshida S, Fujikawa S. Accumulation of small heat-shock protein homologs in the endoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation. Plant Physiology (1999) 120:481–490.
van Berkel J, Salamini F, Gebhardt C. Transcripts accumulating during cold storage of potato (Solanum tuberosum L.) tubers are sequence related to stress-responsive genes. Plant Physiology (1994) 104:445–452.[Abstract]
van Montfort R, Slingsby C, Vierling E. Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones. Advances in Protein Chemistry (2001) 59:105–156.[ISI][Medline]
Welling A, Moritz T, Palva ET, Junttila O. Independent activation of cold acclimation by low temperature and short photoperiod in hybrid aspen. Plant Physiology (2002) 129:1633–1641.
Winkel-Shirley B. Biosynthesis of flavonoids and effects of stress. Current Opinion in Plant Biology (2002) 5:218–223.[CrossRef][ISI][Medline]
Wisniewski M, Ashworth ED. A comparison of seasonal ultrastructural changes in stem tissues of peach (Prunus persica) that exhibit contrasting mechanisms of cold hardiness. Botanical Gazette (1986) 147:407–417.
Wisniewski M, Davis G. Evidence for the involvement of a specific cell wall layer in regulation of deep supercooling of xylem parenchyma. Plant Physiology (1989) 91:151–156.
Wisniewski M, Webb R, Balsamo R, Close TJ, Yu XM, Griffith M. Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiologia Plantarum (1999) 105:600–608.[CrossRef]
Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG. COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science (1998) 280:1091–1094.
Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D, Xie D. The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. The Plant Cell (2002) 14:1919–1935.
Yamada T, Kuroda K, Jitsuyama Y, Takezawa D, Arakawa K, Fujikawa S. Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing. Planta (2002) 215:770–778.[CrossRef][ISI][Medline]
Yang CY, Chen YC, Jauh GY, Wang CS. A lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis. Plant Physiology (2005) 139:836–846.
Yoshida KT, Naito S, Takeda G. cDNA cloning of regeneration specific genes in rice by differential screening of randomly amplified cDNAs using RAPD primers. Plant and Cell Physiology (1994) 35:1003–1009.