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Plant Physiol. Jun 2005; 138(2): 837–846.
PMCID: PMC1150401

Starch-Related α-Glucan/Water Dikinase Is Involved in the Cold-Induced Development of Freezing Tolerance in Arabidopsis1

Abstract

Cold-induced soluble sugar accumulation enhances the degree of freezing tolerance in various cold-hardy plants including Arabidopsis (Arabidopsis thaliana), where soluble sugars accumulate in only a few hours at 2°C. Hence, along with photosynthesis, starch degradation might play a significant role in cold-induced sugar accumulation and enhanced freezing tolerance. Starch-related α-glucan/water dikinase (EC 2.7.9.4), encoded by Arabidopsis STARCH EXCESS 1 (SEX1), is hypothesized to regulate starch degradation in plastids by phosphorylating starch, thereby ensuring better accessibility by starch-degrading enzymes. Here, we show that Arabidopsis sex1 mutants, when incubated at 2°C for 1 d, were unable to accumulate maltooligosaccharides or normal glucose and fructose levels. In addition, they displayed impaired freezing tolerance. After 7 d at 2°C, sex1 mutants did not show any of the above abnormal phenotypes but displayed slightly higher leaf starch contents. The impaired freezing tolerance of sex1 mutants was restored by overexpression of wild-type SEX1 cDNA using the cauliflower mosaic virus 35S promoter. The results demonstrate a genetic link between the SEX1 locus and plant freezing tolerance, and show that starch degradation is important for enhanced freezing tolerance during an early phase of cold acclimation. However, induction of starch degradation was not accompanied by significant changes in α-glucan/water dikinase activity in leaf extracts and preceded cold-induced augmentation of SEX1 transcripts. Therefore, we conclude that augmentation of SEX1 transcripts might be a homeostatic response to low temperature, and that starch degradation during an early phase of cold acclimation could be regulated by a component(s) of a starch degradation pathway(s) downstream of SEX1.

Cold-hardy plants, including Arabidopsis (Arabidopsis thaliana), increase their degree of freezing tolerance in response to low, nonfreezing temperatures. This is a phenomenon known as cold acclimation (Thomashow, 1999). Cold acclimation is accompanied by numerous changes in plant cells (Levitt, 1980), such as changes in sugar and Pro content (Sakai, 1962; Siminovitch, 1981; Rudolph and Crowe, 1985; Guy et al., 1992; Koster and Lynch, 1992; Wanner and Junttila, 1999; Takagi et al., 2003), membrane lipid composition (Steponkus, 1984; Uemura and Steponkus, 1994; Uemura et al., 1995), soluble and membrane protein compositions (Yoshida, 1984; Guy, 1990; Kawamura and Uemura, 2003), leaf ultrastructure (Ristic and Ashworth, 1993), and expression profiles of genes, including various stress-responsive genes (Guy et al., 1985; Fowler and Thomashow, 2002; Seki et al., 2002). The cold-inducible transcription factor family CBF/DREB1 (for C-repeat binding factor/dehydration-responsive element binding protein 1) and other related components have been identified as the chief conductors of genome-wide responses to low temperature (Xiong et al., 2002; Chinnusamy et al., 2003; Shinozaki et al., 2003; Zhu et al., 2004). However, the significance of individual cold-induced changes is currently under investigation, most effectively using Arabidopsis as a model system.

Several lines of evidence suggest that cold-induced sugar accumulation enhances the degree of plant freezing tolerance. In Arabidopsis rosettes, a large increase in the degree of freezing tolerance that occurs within 1 d at 2°C is positively correlated with soluble sugar content (Wanner and Junttila, 1999; Takagi et al., 2003), and the heterosis of leaf freezing tolerance generated by crossing between different ecotypes is positively correlated with leaf sugar content (Rohde et al., 2004). Increased Suc levels in transgenic Arabidopsis plants overexpressing a gene for Suc phosphate synthase paralleled the freezing tolerance (Strand et al., 2003). Conversely, sensitive to freezing 4 mutants exhibited an impaired cold acclimation capacity due to a reduced accumulation of Glc and Suc at low temperature relative to the wild type (McKown et al., 1996). The impaired cold acclimation capacity of sensitive to freezing 4 protoplasts was restored by providing sugars before protoplast isolation. This decreased the incidence of freeze-induced protoplast membrane lesions (Uemura et al., 2003). However, the exact molecular mechanism for cold-induced sugar accumulation, especially during an early phase of cold acclimation (within 24 h), is unclear, although increased sugar levels in transgenic Arabidopsis plants overexpressing CBF3/DREB1A suggested a possible link between the CBF/DREB1 pathway and cold-induced sugar accumulation (Gilmour et al., 2000; Cook et al., 2004). Because of the rapid increases in sugar levels, we herein address a question of whether, along with photosynthesis, starch degradation could play a significant role during an early phase of cold acclimation in Arabidopsis.

Recent studies with potato (Solanum tuberosum) and Arabidopsis revealed that starch-related α-glucan/water dikinase (GWD; EC 2.7.9.4) is a global regulator of starch catabolism. GWD, a starch-associated protein initially termed R1 in the potato, phosphorylates the C-3 and C-6 positions of α-glucans with the β-phosphate of ATP (Ritte et al., 2000, 2002). Antisense repression of potato GWD not only inhibits the so-called cold sweetening of cold-stored tubers, but also leads to reduced starch phosphorylation levels and the inhibition of starch degradation in leaves at ambient temperature (Lorberth et al., 1998). In Arabidopsis, six allelic mutants showing impaired starch degradation in dark-adapted leaves have been isolated. This mutation is termed sex1 (for starch excess 1; Caspar et al., 1991; Yu et al., 2001). Map-based cloning revealed that SEX1 encodes a protein whose C-terminal domain is similar to the N-terminal domain of bacterial and plant pyruvate/orthophosphate dikinase (EC 2.7.9.1) or bacterial pyruvate/water dikinase (EC 2.7.9.2; Yu et al., 2001). It has been demonstrated that sex1 leaves exhibit reduced starch phosphorylation levels (Yu et al., 2001) and severely reduced GWD activity (Ritte et al., 2003). Conversely, increased starch phosphorylation is associated with starch degradation in potato leaves and green algae (Ritte et al., 2004). Although it is not exactly understood how GWD/SEX1-dependent starch phosphorylation promotes starch degradation, starch phosphorylation is hypothesized to increase the hydrophilicity of water-insoluble glucans, thereby ensuring better accessibility by starch-degrading enzymes (Yu et al., 2001; Ritte et al., 2004).

Although the aforementioned studies clearly demonstrate the involvement of GWD/SEX1 in some aspects of starch degradation in Arabidopsis and other plants, it remains unclear whether SEX1-dependent starch degradation is involved in cold-induced sugar accumulation in leaves. Furthermore, to the best of our knowledge, neither the expression profile of the SEX1 gene at low temperature nor the cryobehavior of sex1 mutants has ever been evaluated. A minor increase of microarray signals for the SEX1 transcripts in CBF-overproducing Arabidopsis plants was not interpreted as due to typical cold-responsible genes (Fowler and Thomashow, 2002). Here, we show that Arabidopsis sex1 mutants during an early phase of cold acclimation are defective in accumulating maltooligosaccharides (MOS), as well as normal levels of Glc and Fru, and display an impaired freezing tolerance, and that the introduction of a wild-type SEX1 cDNA under the control of a constitutive cauliflower mosaic virus 35S promoter restores the impaired freezing tolerance of the mutant. However, sex1 mutants did not show any of the above abnormal phenotypes in a fully cold-acclimated state. This suggests that SEX1-dependent starch degradation may contribute to soluble sugar accumulation as well as enhanced freezing tolerance in Arabidopsis rosettes during an early phase of cold acclimation. We also show that the induction of starch degradation is not accompanied by significant changes of GWD activity and also precedes cold-induced augmentation of SEX1 transcripts. Therefore, we conclude that the augmentation of SEX1 transcripts at low temperature might be a homeostatic response required for prolonged cold acclimation and that other component(s) of starch degradation pathways downstream of SEX1 could play a regulatory role in starch degradation during an early phase of cold acclimation.

RESULTS

sex1 Mutant Characterization

To investigate the role of starch degradation in the cold acclimation of Arabidopsis rosettes, two allelic sex1 mutants were obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, Columbus, OH; Fig. 1A). The sex1-1 mutant contains a single nucleotide substitution that causes a G1268E substitution in the C-terminal region of the SEX1 protein (Caspar et al., 1991; Yu et al., 2001). A knockout sex1 mutant, termed sex1-7, was also purified from the T-DNA-tagging line SALK_062752 by backcrossing. The sex1-7 mutant was determined to carry a T-DNA insertion within the 20th exon of the SEX1 gene (Fig. 1A). An RNA gel-blot analysis showed that the wild-type and homozygous sex1-1 plants accumulated SEX1 transcripts of similar size, whereas homozygous sex1-7 plants accumulated SEX1 transcripts of smaller size. They were also less extensive when compared with the wild type (Fig. 1B). Each of the sex1-1 and sex1-7 mutants grew indistinguishably from the wild type when grown at 23°C under continuous illumination. However, the leaf starch content was significantly higher in both sex1 mutants than in the wild type (Fig. 2A). These results were consistent with reported phenotypes of sex1 mutants (Caspar et al., 1991; Yu et al., 2001). Crosses between homozygous sex1-1 and homozygous sex1-7 plants produced F1 progenies with the mutant phenotype exclusively, thereby verifying allelism between these mutations.

Figure 1.
Structural features of the Arabidopsis sex1 loci and their effects on the level of SEX1 transcripts. A, A schematic representation of the structures of the wild type (At1g10760) and mutant SEX1 genes used in this study. Exons are indicated by black boxes. ...
Figure 2.
Cold-induced changes of starch and MOS content in the wild-type and sex1 mutants of Arabidopsis. A, Changes of starch (top) and MOS content (bottom) in the third to fifth leaves of 18-d-old wild-type ([open diamond]), sex1-1 ([filled square]), and sex1-7 plants ...

Cold-Induced Starch Degradation in Arabidopsis Rosettes

When wild-type plants grown at 23°C for 18 d under continuous illumination at a photon flux density of 65 μmol m−2 s−1 were shifted to 2°C for 24 h under continuous illumination at a photon flux density of 35 μmol m−2 s−1, decreases in leaf starch content, as determined for the third to fifth leaves of the rosettes, were noticeable. The amount of starch was reduced by the equivalent of 5.0 and 7.7 μmol Glc g−1 fresh weight after 3 and 6 h at 2°C, respectively, and starch levels did not begin to recover until after 24 h at 2°C (Fig. 2A). However, the starch content became more than 4-fold greater after 7 d at 2°C when compared with that in nonacclimated plants. This suggested that starch accumulation was restored at 2°C after prolonged incubation (Fig. 2B).

To obtain another line of evidence for starch degradation, we examined the levels of MOS recovered in soluble sugar extracts from the leaves. The MOS content was defined as the amount of Glc that was released from the soluble sugar extracts by amyloglucosidase digestion. According to literature (Critchley et al., 2001), the MOS content thus determined should reflect the amount of soluble intermediates of starch degradation, such as maltose, maltotriose, and higher oligoglucosides. Before the cold treatment, wild-type plants contained a negligible amount of MOS that was equivalent to 0.04 μmol Glc g−1 fresh weight. In accordance with an early reduction of leaf starch content in wild-type plants subjected to the 2°C treatment (Fig. 2A), the MOS content increased as early as 3 h at 2°C. The MOS levels after 3, 6, and 24 h at 2°C were equivalent to 4.6, 5.9, and 3.4 μmol Glc g−1 fresh weight, respectively. The increased MOS levels were largely sustainable at 2°C for 7 d (Fig. 2B). These results were consistent with a view that starch degradation is accelerated in wild-type plants at 2°C under our cold acclimation conditions.

Starch and MOS contents were then measured in the sex1-1 and sex1-7 plants that were incubated at 2°C under the same conditions as described above for the wild-type plants. The leaf starch content in both mutants increased within 3 h at 2°C, then gradually increased in the sex1-1 mutants until 24 h or increased in the sex1-7 mutants until 6 h to reach a transient plateau. In the latter mutant, the starch content was also measured after 7 d at 2°C. It displayed a 4-fold increase when compared with the level before the 2°C treatment and was slightly higher than that in the wild type after 7 d at 2°C (Fig. 2B). In both sex1 mutants, the MOS content increased very slowly for 24 h at 2°C up to less than 1 μmol Glc g−1 fresh weight. This level was almost 25% of that found in wild-type leaves subjected to the same treatment (Fig. 2A). However, after 7 d at 2°C, the MOS content in the sex1-7 mutants increased to 2.2 μmol Glc g−1 fresh weight, a level comparable to that in wild-type plants subjected to the same treatment (Fig. 2B). These results suggested that the sex1 mutants used in this work are likely to be impaired in starch degradation, at least during 24 h under our cold acclimation conditions. Although previous studies have revealed the role of GWD in the regulation of starch degradation in dark-adapted leaves or tubers after a long-term cold treatment, our results suggested that GWD also plays an important role in leaf starch degradation in Arabidopsis after a short-term cold treatment.

Cold-Induced Sugar Accumulation in sex1 Leaves

To investigate whether the sex1 mutation affects soluble sugar levels in leaves of Arabidopsis at low temperature, we determined the content of Glc, Fru, and Suc in the third to fifth leaves of wild-type and sex1 plants during incubation at 2°C for 0, 3, 6, and 24 h. Before the cold treatment (0 h), there was no significant difference in soluble sugar levels between the wild-type, sex1-1, and sex1-7 leaves (Fig. 3A). However, after 3 h at 2°C, increases in the content of Glc, Fru, and Suc were noticeable in both wild-type and mutant leaves. However, sex1-1 and sex1-7 leaves tended to accumulate smaller amounts of Glc and Fru than wild-type leaves, at least during the first 24 h at 2°C (Fig. 3A). By contrast, there was no significant difference in the content of Suc between wild-type, sex1-1, and sex1-7 leaves for up to 24 h at 2°C (Fig. 3A). These results demonstrated that the sex1 mutation affects Glc and Fru levels but not Suc levels at 2°C for at least 24 h. However, after 7 d at 2°C, the content of Glc, Fru, and Suc did not differ significantly between wild-type and sex1-7 leaves (Fig. 3B). Therefore, SEX1-dependent starch degradation appeared to affect Glc and Fru levels in Arabidopsis leaves during an early phase of cold acclimation.

Figure 3.
Soluble sugar content in the wild-type, sex1-1, and sex1-7 plants before and after cold treatment. A and B, The content of Glc (top), Fru (middle), and Suc (bottom) in the third to fifth leaves of 18-d-old wild-type ([open diamond]), sex1-1 ([filled square]), and ...

Impaired Freezing Tolerance in sex1 Mutants after 1 d at 2°C

To evaluate the effect of the sex1 mutation on the cold acclimation capacity of Arabidopsis rosettes, we determined the degree of electrolyte leakage from leaves subjected to a freeze/thawing treatment (electrolyte leakage tests). For this purpose, the third to fifth leaves from wild-type or sex1 rosettes before and after incubation at 2°C for 1 d were used. Before the cold treatment, there was no significant difference between the wild type and sex1 mutants in the degree of freeze/thaw-induced electrolyte leakage from the leaves (Fig. 4A). The freezing temperature that caused a 50% electrolyte leakage (TEL50 value) was −4.0°C ± 0.8°C, −3.7°C ± 0.9°C, and −3.6°C ± 0.6°C for wild-type, sex1-1, and sex1-7 leaves, respectively. After 1 d at 2°C, TEL50 values for wild-type, sex1-1, and sex1-7 leaves were reduced to −8.5°C ± 0.8°C, −6.6°C ± 1.1°C, and −5.4°C ± 0.8°C, respectively. This suggested that the freezing tolerance of sex1-1 and sex1-7 leaves, especially of the latter, did not greatly increase at low temperature when compared with wild-type leaves (Fig. 4B). These results were also confirmed by whole-plant freezing tests. When soil-grown plants treated at 2°C for 1 d were frozen at −9.0°C for 10 h and then thawed at 4°C, both sex1-1 and sex1-7 suffered more severe injury than the wild-type plants (Fig. 4C). Furthermore, an impaired freezing tolerance of sex1-7 was restored when a wild-type SEX1 cDNA under the control of cauliflower mosaic virus 35S promoter was introduced into the mutant (Fig. 4D). These results clearly demonstrated a genetic link between the SEX1 locus and the development of freezing tolerance during an early phase of cold acclimation.

Figure 4.
Impaired freezing tolerance of sex1 mutants after 1 d at 2°C. Freezing tolerance of the wild-type, sex1-1, and sex1-7 plants was evaluated using electrolyte leakage tests (A, B, and E) or whole-plant freezing tests (C and D). A, B, and E, Electrolyte ...

We also compared the degree of freezing tolerance between wild-type and sex1-7 leaves from fully cold-acclimated plants (after 7 d at 2°C) using electrolyte leakage tests. TEL50 values for wild-type and sex1-7 leaves were −12.2°C ± 0.4°C and −11.7°C ± 0.5°C, respectively, and there was no significant difference in the degree of freezing tolerance between their leaves (Fig. 4E). It seems likely that the maximum degree of freezing tolerance is not affected by the sex1 mutation.

Transcript Levels of Stress-Induced Genes in the sex1 Mutants

Although it seems very unlikely, the above results did not exclude the possibility that the impaired freezing tolerance of sex1 mutants was caused by an inhibition of stress-inducible signaling pathways such as CBF/DREB1- or abscisic acid (ABA)-dependent pathways. To eliminate this possibility, we performed RNA gel-blot analyses for two representative stress-inducible genes. The result showed that the transcript levels of the cold-responsive gene COR78/RD29A (cold regulated/responsive to dehydration) and the ABA-responsive gene dehydrin/RAB18 were not significantly different between the wild type and sex1-1 mutants (Fig. 5). This suggested that the sex1 mutation does not affect the transcript levels of stress-responsive genes and that the impaired freezing tolerance in sex1 mutants is not related to any defect in the CBF/DREB1- or ABA-signaling pathways.

Figure 5.
Effects of the sex1 mutation on the expression of stress-inducible genes. RNA gel-blot analysis for COR78/RD29A or dehydrin/RAB18 transcripts in the wild-type and sex1-1 plants is shown. Total RNA was isolated from plants grown for 18 d at 23°C ...

The SEX1 Gene Is a Cold-Responsive Gene

To evaluate the effect of low temperature on the levels of SEX1 transcripts, RNA gel-blot analyses were performed with RNA extracted from whole rosettes of wild-type plants subjected to cold acclimation conditions at 2°C or to subsequent deacclimation conditions at 23°C (Fig. 6A). Although SEX1 transcripts were constitutively detected in Arabidopsis rosettes, the level of SEX1 transcripts significantly increased within 6 h at 2°C and continued to increase until 48 h at 2°C. When cold-treated plants were subsequently incubated at 23°C, the level of SEX1 transcripts began to decrease within 2 h to return after 12 to 24 h to a level found before the cold treatment. These results provide firm evidence that the SEX1 gene is a cold-responsive gene. However, as described above, starch degradation that appeared to be activated as early as 3 h at 2°C (Fig. 2A) preceded the maximum accumulation of SEX1 transcripts that occurred after 24 to 48 h at 2°C in Arabidopsis rosettes (Fig. 6A). Therefore, the cold-induced augmentation of SEX1 transcripts is likely to be unrelated to the activation of starch degradation during an early phase of cold acclimation.

Figure 6.
Effects of temperature on the levels of SEX1 transcripts and GWD activity. A, RNA gel-blot analysis for SEX1 transcripts in wild-type plants. Total RNA was isolated from 21-d-old wild-type plants subjected to cold treatment for up to 48 h at 2°C, ...

GWD Activity Did Not Change, at Least for 12 h at 2°C

Because the induction of starch degradation at 2°C appeared to precede the cold-induced augmentation of SEX1 transcripts in wild-type plants (Figs. 2A and and6A),6A), changes in GWD activity were studied in wild-type plants subjected to the 2°C treatment. For this purpose, the GWD activity recovered in the leaf soluble extracts was measured using a randomized mixture of [β-32P]ATP and [γ-32P]ATP, and using starch granules as substrates (Ritte et al., 2002, 2003). Under our assay conditions, GWD activity was roughly proportional to the volumes of leaf extracts (up to approximately 20 μg of protein) added to the reaction mixtures (see WT and WT1/2 in Fig. 6B). The sex1-7 leaf extracts exhibited about 20% of GWD activity when compared with wild-type leaf extracts, although the origin of this residual GWD activity in sex1-7 leaf extracts remained unclear. However, GWD activity in wild-type leaf extracts did not change significantly for up to 12 h at 2°C. This demonstrated that cold-induced starch degradation in Arabidopsis rosettes during an early phase of cold acclimation is not accompanied by the activation of GWD activity.

DISCUSSION

Cold-induced starch degradation in Arabidopsis rosette leaves is supported by data presented in this work as well as those from other groups. Ristic and Ashworth (1993) first reported that the leaf starch content in Arabidopsis increased in association with a temperature shift from 22°C to 23°C to 4°C under continuous illumination at a photon flux density of 120 μmol m−2 s−1. However, these authors noted a transient decrease in the average area of starch grain sections on electron micrographs after 12 h at 4°C. This suggested an active metabolic status of starch grains during an early phase of cold acclimation. Recently, Kaplan and Guy (2004) presented more direct evidence for cold-induced starch degradation in Arabidopsis. They found that an Arabidopsis chloroplast-targeted β-amylase isogene encoded by BMY8 positively responds to low temperature, and the cold-induced enhancement of BMY8 transcripts is correlated with maltose accumulation.

In this work, we demonstrated that starch content reduction and MOS accumulation, both of which are indicative of starch degradation, occur in Arabidopsis rosette leaves within 24 h under our cold acclimation conditions. We also found that there is a significant difference in the time courses of MOS accumulation between the wild type and the sex1 mutants after 2°C exposure (Fig. 2A). In the wild type, the MOS content increases rapidly to reach the maximum around 6 h and then decreases gradually to a substantial level until after 24 h at 2°C. By contrast, the MOS content increases very slowly in the sex1 mutants until after 24 h at 2°C. This difference probably reflects the fact that the GWD activity in the sex1-7 leaf extracts is only 20% of that detected in the wild-type leaf extracts (Fig. 6B). Therefore, GWD/SEX1 appears to be a limiting factor for starch degradation at low temperature, although a direct proof for this view requires a measurement of the starch phosphorylation status. Our results complemented well with the aforementioned previous studies (Ristic and Ashworth, 1993; Kaplan and Guy, 2004), adding a finding that SEX1 is required for the rapid degradation of starch in Arabidopsis rosettes during an early phase of cold acclimation.

Because the leaf GWD activity remains almost constant in the wild type for the first 12 h after 2°C exposure (Fig. 6B), the rapid rise of MOS content in the wild type within 3 h at 2°C (Fig. 2A) appears to be not due to GWD activation. Therefore, we conclude that the cold-induced starch degradation is regulated by a component(s) of the starch degradation pathway(s) downstream of GWD/SEX1. Kaplan and Guy (2004) deductively concluded that the cold-induced augmentation of BMY8 transcripts is responsible for the cold-induced accumulation of maltose. However, it seems difficult to demonstrate specific changes in β-amylase activity associated with the changes of BMY8 transcript levels, and the genetic dissection of this phenomenon is awaited. Elucidation of pathways and regulatory steps for cold-induced starch degradation remains a topic for future research.

The sex1 mutation was found to affect cold-induced sugar accumulation during an early phase of cold acclimation. In Arabidopsis, maltose produced in the stroma by starch degradation is transported to the cytosol by a recently identified maltose-specific transporter, MALTOSE EXCESS 1, and then broken down to Glc by enzymes such as putative α-glucosidase (Smith et al., 2003; Niittylä et al., 2004) or cytoplasmic disproportionating enzymes such as DPE2 (Chia et al., 2004). According to the data presented in Figure 2A, the apparent starch degradation that occurred in wild-type leaves during 1 d at 2°C is calculated to produce approximately 13 μmol Glc g−1 fresh weight, which accounts for approximately 40% of the sum of Glc and Fru that accumulated during this period (33 μmol g fresh weight−1). However, the remainder of Glc and Fru must be coming from nonstarch degradation pathways, most probably from continued photosynthesis. After 1 d at 2°C, the total sugar levels in sex1-1 and sex1-7 leaves were 30% and 50% lower than the amount accumulated in wild-type leaves, respectively. These decreases were largely ascribed to a reduction in Glc and Fru levels (Fig. 3B). Therefore, lower levels of Glc and Fru in sex1 mutants subjected to low temperature may reflect a decreased rate of starch degradation in sex1 mutants when compared with the wild type. However, sex1 mutation did not block accumulation of Glc and Fru, and, therefore, there are starch degradation-independent pathways leading to Glc and Fru also in the sex1 mutants. By contrast, there was no significant change in the level of Suc between the wild type and sex1-1 or sex1-7 mutant. This result suggests that Suc accumulation is independent of starch degradation, at least with respect to GWD.

We showed that the sex1 mutation inhibits the development of freezing tolerance during the initial day of cold acclimation (Fig. 4) without affecting the transcript levels of the COR78/RD29A and dehydrin/RAB18 genes, which are regulated by CBF/DREB1 transcription factors and ABA, respectively (Fig. 5). We also showed that the impaired freezing tolerance of sex1 is restored by introducing a wild-type SEX1 cDNA under the control of CaMV 35S promoter into sex1-7 plants (Fig. 4D). Because sugar levels in the wild-type, sex1-1, and sex1-7 plants are positively correlated with the degree of freezing tolerance (Fig. 7), it is very likely that altered sugar levels in the sex1 mutants are the direct cause of the impaired freezing tolerance in these mutants.

Figure 7.
Correlation between the content of soluble sugars and the degree of freezing tolerance in plants before and after cold treatment for 1 d at 2°C. Plotted data and error bars (sd) for soluble sugar content were taken from Figure 3A. Total sugar ...

We also demonstrated that SEX1 is a cold-responsive gene in Arabidopsis, although it is not required for the activation of starch degradation at low temperature. However, as shown in Figure 2B, MOS levels in the wild-type and sex1-7 plants after 7 d at 2°C are substantially higher than those before the cold treatment. This suggests that cold-induced starch degradation sustains, at least as long as 7 d at 2°C. Because GWD activity is a limiting factor for starch degradation at low temperature as described above, the cold-induced augmentation of SEX1 transcripts might be related to a homeostatic response to low temperature. In this context, it should be noted that the 5′ and 3′ flanking sequences of the SEX1 gene do not contain canonical CRT/DRE motifs. Strand et al. (1999) reported that starch remobilization is apparently arrested in Arabidopsis leaves incubated for 10 d at 5°C under a day/night photoregime but is restored in new leaves that fully developed after 40 d under the same cold acclimation conditions. This suggests the importance of starch phosphorylation in leaves also after a long-term cold treatment. Obviously, further studies are required for clarifying the reason and the mechanism for the cold-induced augmentation of SEX1 transcripts.

CONCLUSION

We demonstrated that starch degradation is involved in the freezing tolerance enhancement of Arabidopsis rosettes during an early phase of cold acclimation. SEX1 plays an essential role in the cold-induced starch degradation, sugar accumulation, and freezing tolerance enhancement during an early phase of cold acclimation. However, it seems likely that starch degradation in Arabidopsis rosettes during an early phase of cold acclimation is regulated by a component of a starch degradation pathway(s) downstream of SEX1. Future studies with knockout mutants for chloroplast-targeted β-amylases or other starch-related enzymes will uncover the precise molecular mechanisms of cold-induced starch to sugar conversion and the development of freezing tolerance in leaves. Investigation of the mechanism of SEX1 transcription at low temperature may provide an insight into the transcriptional regulation of genes related to homeostatic responses to low temperature.

MATERIALS AND METHODS

Plant Materials

Seeds of the wild-type Arabidopsis (Arabidopsis thaliana; ecotype Columbia) were purchased from Lehle Seeds (Round Rock, TX), and seeds of the sex1-1 mutants (Columbia background) were obtained from the ABRC. A single nucleotide substitution in the sex1-1 locus was confirmed by StyI cleaving of a SEX1 gene fragment that was amplified by PCR using the primers 5′-TTGCACCCCCAGAACTGG-3′ and 5′-TCACACTTGTGGTCGTGTCTG-3′ (Yu et al., 2001). A sex1-knockout mutant, herein termed sex1-7 according to the previous numbering (Yu et al., 2001), was purified from the T-DNA-tagging line SALK_062752 (ABRC) by crossing with the wild type.

All homozygous sex1-7 mutants that were identified among the F2 progeny from a cross between homozygous sex1-7 plants and the wild type, as well as their self-pollinated offspring (F3), showed the mutant phenotype described in the text. T-DNA insertion in the sex1-7 mutants was confirmed by PCR using the primer for the left border of T-DNA, 5′-CCACCCCAGTACATTAAAAACGTCC-3′, in combination with either of the SEX1-specific primers, 5′-GACCTCTACAGGACTAATAACCTG-3′ or 5′-GACGGTCTTTTGCGTGATCTTG-3′.

Growth Conditions

Seeds were sown on peat sheets (Sakata Seed, Yokohama, Japan) irrigated with water and chilled for vernalization for 48 h at 2°C in darkness. The seedlings were then raised for 18 d or for indicated days at 23°C under continuous illumination at a photon flux density of 65 μmol m−2 s−1. For the cold treatment, 18-d-old seedlings were incubated at 2°C under continuous illumination at a photon flux density of 35 μmol m−2 s−1. For deacclimation, the cold-treated seedlings were incubated at 23°C under the growth conditions described above.

Mutant Restoration by SEX1 cDNA Expression

A 4,200-bp SEX1 cDNA fragment was amplified by PCR using the primers 5′-AAATCTAGAATGAGTAACTCTGTAGTGCATAACTTAC-3′ and 5′-TTTGAGCTCTCACACTTGTGGTCGTGTCTG-3′. The binary vector pPZP211 (Hajdukiewicz et al., 1994) was a generous gift from Dr. Pal Maliga (Rutgers University, Piscataway, NJ). The cDNA fragment was subcloned between the XbaI and SacI sites of the binary vector pPZP211, i.e. between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The resultant binary vector was introduced into the Agrobacterium tumefaciens strain EHA101 by electroporation, and homozygous sex1-7 plants were transformed using a floral dip method (Clough and Bent, 1998). T1 transformants were selected for kanamycin resistance, and T3 progeny were tested for the restoration of mutant phenotypes.

RNA Gel-Blot Analysis

Isolation and gel-blot analyses of RNA were performed as described previously (Takagi et al., 2003). Specific hybridization probes were prepared from DNA fragments that were amplified by PCR using the following primer sets: SEX1, 5′-GACGGTCTTTTGCGTGATCTTG-3′ and 5′-GACCTCTACAGGACTAATAACCTG-3′; COR78/RD29A, 5′-GTATTCGCCGGAATCTGACGG-3′ and 5′-AAGCTCCTTCTGCACCGGAAC-3′; and dehydrin/RAB18, 5′-GTGGTGGCTTGGGAGGAATGCTTCA-3′ and 5′-ATGCGACTGCGTTACAAACCCTCA-3′.

Starch and Sugar Measurements

Frozen samples of the third to fifth leaves from 18-d-old plants were extracted with 75% (v/v) ethanol for 24 h at 4°C with constant shaking and then centrifuged at 10,000g for 20 min. The pellet was washed three times with 75% (v/v) ethanol and subjected to starch analysis, while the supernatant fractions were combined, lyophilized, and dissolved in water to make a total volume of 60 μL. This fraction, referred to as the leaf soluble sugar extracts, was subjected to Glc, Fru, Suc, and MOS determination.

Starch analysis was performed using a starch assay kit (STA-20; Sigma, Tokyo). The pellet from the 75% (v/v) ethanol washes was resuspended in 75% (v/v) ethanol, incubated at 85°C for 5 min, and then centrifuged at 10,000g for 20 min. The resultant pellet was suspended in dimethyl sulfoxide, immersed in boiling water for 5 min, and then subjected to digestion with α-amylase and α-amyloglucosidase according to the manufacturer's protocol. The starch content was represented by the amount of released Glc, which was quantified using a Glc oxidase-mediated method (Glc-Test C2; WAKO, Osaka) using purified potato (Solanum tuberosum) starch (Sigma) as a quantitative standard.

Glc, Fru, and Suc contents were determined by high-performance capillary electrophoresis as described previously (Takagi et al., 2003). The MOS content in the leaf soluble sugar extracts was measured according to Critchley et al. (2001). Briefly, 10 μL of leaf soluble sugar extracts were mixed with 10 μL of either 50 units mL−1 amyloglucosidase solution (S9144; Sigma) or water (control). The resultant mixtures were incubated at 60°C for 15 min and then subsequently at room temperature for 30 min. The amount of Glc released in the presence of amyloglucosidase was determined using a Glc oxidase-mediated method as described above.

Whole-Plant Freezing Tests

Whole-plant freezing tests were performed as described previously (Takagi et al., 2003). Briefly, 18-d-old plants were placed in a copper chamber installed in a programmable freezer (LH-135S; Nihon Ika, Tokyo). The plants were kept for 3 h at −1.5°C before ice nucleation. Ice-nucleated plants were cooled down to a desired freezing temperature at a cooling rate of −1°C h−1, maintained for another 10 h, and then thawed for 24 h at 4°C in darkness. The degree of freezing tolerance was visually assessed after thawed plants were incubated for recovery for 3 d at 23°C in light.

Electrolyte Leakage Tests

Electrolyte leakage tests were performed as described previously (Takagi et al., 2003). Briefly, the third to fifth leaves from 18-d-old plants were placed in capped test tubes placed in a bath-type programmable freezer (NCB-3300; Eyela, Tokyo). Samples were kept for 60 min at −2°C before ice nucleation. Ice-nucleated samples were cooled down to desired sampling temperatures at a cooling rate of −2°C h−1 and held for 30 min at each sampling temperature before sampling. Frozen samples were thawed for more than 18 h at 4°C in darkness and then subjected to electrolyte leakage tests (Takagi et al., 2003). The freezing temperature that causes a 50% electrolyte leakage (TEL50) was calculated from plotted data of relative electrolyte leakage, assuming a sigmoid function.

Assay of GWD Activity

Randomized mixtures of [β-32P]ATP and [γ-32P]ATP were prepared from [γ-32P]ATP (approximately 220 TBq mmol−1; Amersham, Piscataway, NJ) according to Ritte et al. (2002). Extraction and measurements of GWD activity in leaf crude extracts were performed according to Ritte et al. (2002, 2003) with the following modifications: after termination of the GWD reactions, 32P-labeled starch grains were recovered on a size-selection filter (>30 kD) assembled in a Suprec-02 centrifugation cartridge (Takara, Tokyo), washed five times with 2 mm ATP solution, and subjected to radioactivity measurements on a scintillation counter.

Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this paper that would limit their use in noncommercial research purposes.

Acknowledgments

We thank Atsuhiko Aoyama for his technical assistance and Prof. Yoshibumi Komeda for discussions.

Notes

1This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.056374.

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