Skip to main content

Main menu

  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
  • About
    • Editorial Board and Staff
    • About the Journal
    • Terms & Privacy
  • More
    • Alerts
    • Contact Us
  • Submit a Manuscript
    • Instructions for Authors
    • Submit a Manuscript
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Teaching Tools in Plant Biology
    • ASPB
    • Plantae

User menu

  • My alerts
  • Log in

Search

  • Advanced search
Plant Cell
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Teaching Tools in Plant Biology
    • ASPB
    • Plantae
  • My alerts
  • Log in
Plant Cell

Advanced Search

  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
  • About
    • Editorial Board and Staff
    • About the Journal
    • Terms & Privacy
  • More
    • Alerts
    • Contact Us
  • Submit a Manuscript
    • Instructions for Authors
    • Submit a Manuscript
  • Follow PlantCell on Twitter
  • Visit PlantCell on Facebook
  • Visit Plantae
Research ArticleResearch Article
You have accessRestricted Access

HOS1, a Genetic Locus Involved in Cold-Responsive Gene Expression in Arabidopsis

Manabu Ishitani, Liming Xiong, Hojoung Lee, Becky Stevenson, Jian-Kang Zhu
Manabu Ishitani
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Liming Xiong
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hojoung Lee
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Becky Stevenson
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jian-Kang Zhu
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: jkzhu@ag.arizona.edu

Published July 1998. DOI: https://doi.org/10.1105/tpc.10.7.1151

  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading
  • © 1998 American Society of Plant Physiologists

Abstract

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19°C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10°C or higher. Compared with the wild type, hos1-1 plants are less cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.

INTRODUCTION

Low temperature is one of the most important environmental factors limiting the geographic distribution of land plants (Levitt, 1980). Plants from temperate regions can increase in freezing tolerance by being exposed to low nonfreezing temperatures. This process is known as cold acclimation (Guy, 1990). Changes in gene expression (Thomashow, 1994), membrane composition and cryobehavior (Lynch and Steponkus, 1987), enzyme activities, and accumulation of cryoprotectants, such as sugars and polyamines, have been associated with cold acclimation (Levitt, 1980). However, the underlying mechanisms by which plant cells perceive and transduce the cold signal to activate these cellular responses are unclear.

Transient increases in cytosolic calcium appear to be an early step in cold signaling (M.R. Knight et al., 1991; Monroy and Dhindsa, 1995; H. Knight et al., 1996). The phytohormone abscisic acid (ABA) is known to play an important role in cold acclimation. Treatment with ABA at normal growth temperatures increases the freezing tolerance of a wide range of plants (Chen et al., 1983; Mohapatra et al., 1988). Furthermore, exposure to low temperature transiently increases ABA levels in a number of plant species, including Arabidopsis (Chen et al., 1983; Guy and Haskell, 1988; Lång, et al., 1994). Recently, the activity of a MAP kinase has been shown to increase in alfalfa when cold shocked (Jonak et al., 1996). However, this increase is not specific to cold because drought stress can also activate the kinase (Jonak et al., 1996).

The expression of a number of genes in plants is regulated by low temperature (Dhindsa and Monroy, 1994; Guy et al., 1994; Palva et al., 1994; Thomashow, 1994). Most of these genes maintain high levels of expression throughout cold treatment, but their expression decreases rapidly upon return from cold to normal growth temperatures (Thomashow, 1994). The cold-responsive genes encode a diverse array of proteins, such as enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids and antioxidants, molecular chaperones, antifreezing proteins, and many others without a known function (Guy et al., 1994).

Arabidopsis cold-regulated genes identified to date can also be induced by ABA or osmotic stress treatment (Hajela et al., 1990; Kurkela and Borg-Franck, 1992; Nordin et al., 1993). However, the cold induction of these genes may not necessarily depend on endogenous ABA. For example, although the RD29A gene (also known as COR78 or LTI78) can be induced by either cold or ABA treatment, its cold regulation is ABA independent (Gilmour and Thomashow, 1991; Nordin et al., 1993; Yamaguchi-Shinozaki and Shinozaki, 1994). Cold induction of RD29A is not impaired by the ABA-deficient aba1 mutation or the ABA-insensitive abi1 mutation (Gilmour and Thomashow, 1991; Nordin et al., 1993). Yamaguchi-Shinozaki and Shinozaki (1994) and Stockinger et al. (1997) have identified a DRE/C repeat element from the promoters of RD29A and COR15A genes. The DRE/C repeat mediates ABA-independent regulation of gene expression by cold or osmotic stress. A transcriptional factor that binds to the DRE/C repeat has been cloned from Arabidopsis (Stockinger et al., 1997). Other components in the ABA-independent cold signal transduction pathway remain to be identified.

We are interested in the mechanism of ABA-independent regulation of gene expression by cold. Our group has taken a genetic approach by identifying Arabidopsis mutants with altered regulation of gene expression in response to low temperature (Ishitani et al., 1997). We present here a detailed characterization of a plant mutant, hos1-1, that shows enhanced gene expression in response to low-temperature treatments. This mutant defines a genetic locus important not only for cold-regulated gene transcription but also for freezing tolerance and the control of flowering time.

RESULTS

Identification of the HOS1 Locus

To facilitate genetic analysis of stress responses, we transformed Arabidopsis plants with the chimeric RD29A–LUC gene consisting of the firefly luciferase coding sequence under control of the RD29A promoter (−650 to −1 fragment; Ishitani et al., 1997). The RD29A–LUC plants emit bioluminescence in response to cold, osmotic stress, or ABA treatment. Homozygous plants with a single RD29A–LUC transgene, referred to as the wild type in this study, were mutagenized with ethyl methanesulfonate, and mutants with altered bioluminescence responses to cold, osmotic stress, or ABA were selected as described in Ishitani et al. (1997). Preliminary analysis identified several mutants exhibiting an enhanced luminescence response to cold stress but showing normal responses to ABA and osmotic stress treatments. One of the mutants, designated hos1-1, was chosen for detailed characterization.

Figure 1 shows RD29A–LUC expression in hos1-1 seedlings when no treatment was applied or when treated with low temperature, ABA, or high salt. Similar to the wild type, hos1-1 did not show any luciferase expression in the absence of stress treatments (Figure 1). Significant luciferase expression was induced in the mutant as well as in the wild type when treated with either cold, ABA, or high salt. The hos1-1 mutant, however, showed a much higher level of expression than did the wild type under cold treatment. The increased level of luciferase expression in hos1-1 plants was specific to cold stress because induction by ABA or NaCl in the mutant was not substantially different from that of the wild type. Figure 2 shows the luminescence images of hos1-1 and the wild-type seedlings when treated with cold (Figure 2B), ABA (Figure 2D), or NaCl (Figure 2F). hos1-1 seedlings displayed a substantially brighter luminescence phenotype than did the wild type specifically under cold treatment.

The hos1-1 mutant was backcrossed with the wild type. The resulting F1 plants all exhibited a wild-type phenotype (Table 1). The F2 progeny of the selfed F1 segregated ~3:1 for wild type/mutant (Table 1). These results indicate that the hos1-1 mutant is caused by a recessive mutation in a single nuclear gene. Allelism tests showed that the hos1-1 mutant complemented all other mutants (i.e., mutant lines 1609, 1400, 970, 738, 737, 734, 470, and 56) that show specifically enhanced bioluminescence responses to cold treatment only (L. Xiong and J.-K. Zhu, unpublished data).

Cold-Regulated Gene Expression in hos1-1 Plants

To determine whether the hos1-1 mutation affects endogenous RD29A gene expression, we extracted total RNA from hos1-1 and wild-type seedlings that were treated with ABA, NaCl, or polyethlylene glycol (PEG) or by using different periods of low temperature. Figure 3 shows that the steady state levels of RD29A message were higher in hos1-1 than in the wild type when treated by cold stress. This enhanced RD29A induction can be seen at all of the time points for cold treatment, although the difference is especially great at the 6- and 12-hr time points (Figure 3). Figure 4 shows the quantification of the endogenous RD29A expression level in hos1-1 and wild-type plants under cold treatment. As expected, RD29A message levels in hos1-1 were not different from those of the wild type when treated with ABA or NaCl (Figure 3). However, the hos1-1 mutation appears to reduce the level of RD29A induction by PEG (Figure 3).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

RD29A–LUC Expression (Luminescence Intensity) in hos1-1 and Wild-Type Plants.

Data represent the average of 20 individual seedlings. Error bars are standard deviation. Control, room temperature without treatment; Cold, 0°C for 24 hr; ABA, 100 μM ABA for 3 hr; NaCl, 300 mM NaCl for 5 hr; WT, wild type.

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

Luminescence Images of hos1-1 and Wild-Type Plants.

The color scale at right shows the luminescence intensity from dark blue (lowest) to white (highest).

(A) Wild-type (left) and hos1-1 (right) seedlings.

(B) Luminescence after low-temperature treatment at 0°C for 24 hr.

(C) Wild-type (left) and hos1-1 (right) seedlings.

(D) Luminescence after treatment with 100 μM ABA for 3 hr.

(E) Wild-type (left) and hos1-1 (right) seedlings on filter paper saturated with 300 mM NaCl.

(F) Luminescence after treatment with 300 mM NaCl for 5 hr.

View this table:
  • View inline
  • View popup
Table 1.

Genetic Analysis of hos1-1 Mutants (Wild Type × hos1-1 Cross)a

In addition to RD29A, cold-induced expression of several other genes examined was also higher in hos1-1 than in the wild type (Figure 3). COR47 (Gilmour et al., 1992) induction was higher in the hos1-1 mutant throughout the cold treatment. Like RD29A, COR47 induction in the wild-type plants also peaked at 24 hr of cold treatment. In contrast, hos1-1 plants showed a peak level of COR47 message after only 12 hr of cold exposure (Figure 3). Induction of COR15A (Lin and Thomashow, 1992) transcripts by cold was detectable by 6 hr and peaked at 48 hr in the wild type. In hos1-1 plants, COR15A expression peaked at 24 hr of cold treatment (Figure 3). Cold-induced COR15A expression was higher in the hos1-1 mutant at 6, 12, and 24 hr but not at the 48-hr time point (Figure 3). KIN1 (Kurkela and Franck, 1990) expression was higher in the hos1-1 mutant than in the wild type throughout cold treatment (Figure 3). Cold induction of the alcohol dehydrogenase (ADH; Jarillo et al., 1993) gene in the wild type was detectable after 24 hr of cold treatment (Figure 3). In contrast, the ADH transcript was detected in the hos1-1 mutant after only 6 hr of cold treatment. ADH expression was substantially higher in hos1-1 plants throughout cold treatment (Figure 3).

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

Expression of the Endogenous RD29A Gene and Other Stress-Responsive Genes in hos1-1 and Wild-Type Plants.

Plants were subjected to low temperature (0°C) for the indicated times. ABA, 100 μM ABA for 3 hr; NaCl, 300 mM NaCl for 5 hr; PEG, 30% PEG (average molecular weight, 6000) for 5 hr; WT, wild type.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

Quantification of Steady State RD29A mRNA Levels in hos1-1 and Wild-Type Plants Treated at 0°C for Different Time Periods.

Results are the averages of three independent experiments. Error bars represent standard deviation. WT, wild type.

As shown in Figure 3, the expression of COR47, COR15A, KIN1, and ADH was not higher in the hos1-1 mutant when plants were treated with ABA, NaCl, or PEG. In fact, the induction of COR47, COR15A, and KIN1 by NaCl or PEG was considerably lower in the mutant than in the wild type (Figure 3). ABA induction of COR15A and KIN1 also was inhibited by the hos1-1 mutation (Figure 3).

To further examine the effect of hos1-1 mutation on osmotic stress–regulated genes, we analyzed the expression of RAB18 (Lång and Palva, 1992), RD22 (Yamaguchi-Shinozaki et al., 1992), and RD29B (Yamaguchi-Shinozaki and Shinozaki, 1993) (Figure 3). The expression of these genes was upregulated by osmotic stress and to some extent by ABA as well, but they showed little response to cold stress (Figure 3). Although the expression of RAB18 and RD29B was inhibited in the hos1-1 mutant when plants were treated with NaCl, PEG, or ABA, the expression of RD22 was not altered substantially in the mutant. As for the wild type, no expression of either RAB18, RD22, or RD29B was detected in the mutant under cold treatment (Figure 3).

As controls, the transcript levels of the LUC transgene and the actin gene were determined. Figure 3 shows that the LUC transcript was induced by cold to a much greater extent in the hos1-1 mutant than in the wild type. However, its induction by ABA, NaCl, or PEG was not changed in the hos1-1 mutant (Figure 3). The expression of the actin gene was not affected by the hos1-1 mutation when plants were not treated or were treated with cold, ABA, NaCl, or PEG (Figure 3).

RD29A–LUC Expression during Cold Acclimation and Deacclimation

Figure 5A shows the time course of RD29A–LUC expression when plants were subjected to cold treatment. At all time points after cold treatment, RD29A–LUC expression was significantly higher in the mutant (Figure 5). In both the wild type and hos1-1 mutant, maximum levels of RD29A–LUC expression were reached after 2 days of cold treatment. The enhanced RD29A–LUC expression in hos1-1 persisted throughout the cold treatment for as long as 7 days (data not shown). Time-course analysis as well as dosage experiments on RD29A–LUC expression under ABA or high-salt treatment were also performed but failed to detect any substantial differences between hos1-1 and wild-type plants (Figures 5B and 5C).

To determine whether the hos1-1 mutation affects RD29A–LUC expression during recovery from cold (i.e., deacclimation), we treated seedlings at 0°C for 24 hr and then incubated them at 22°C for various time periods. Figure 6 shows that although RD29A–LUC expression was higher at all time points during the deacclimation (Figure 6A), the rate of decline in RD29A–LUC expression is the same in hos1-1 and wild-type plants (Figure 6B). Cold-induced luciferase activity decayed rapidly during the first few hours after plants were removed from the cold and was reduced to preacclimation levels by 48 hr after transfer to room temperature.

RD29A–LUC Expression Can Be Induced at Higher Temperatures in hos1-1 Plants

Wild-type and hos1-1 plants were treated for 24 hr at different temperatures ranging from −10°C to 22°C. Figures 7A and 7B show that maximal RD29A–LUC expression in both hos1-1 and the wild-type plants was achieved at 0°C. At temperatures >0°C, RD29A–LUC expression declined sharply. The wild-type plants did not exhibit any expression at 10°C or higher. Although RD29A–LUC expression was also much lower in hos1-1 plants at temperatures >0°C, expression was detectable at temperatures as high as 19°C. At 22°C, hos1-1 plants also failed to show RD29A–LUC expression. These results suggest that cold-responsive genes can be induced at higher temperatures in the mutant.

hos1-1 Mutation Decreases Freezing Tolerance

To evaluate the effect of the hos1-1 mutation on plant freezing tolerance, an electrolyte leakage test (Sukumaran and Weiser, 1972; Ristic and Ashworth, 1993) was conducted with hos1-1 and wild-type plants. The hos1-1 mutation appeared to decrease freezing tolerance of nonacclimated plants; however, little difference was observed between the mutant and the wild type when both were pretreated for 48 hr at 4°C (Figure 8). Temperatures at 50% ion leakage (LT50) were estimated to be −3.0 and −4.1°C for nonacclimated hos1-1 and wild-type plants and −5.6 and −5.8°C for cold-acclimated hos1-1 and cold-acclimated wild-type plants, respectively (Figure 8). The ion leakage experiments were repeated independently three times by using different batches of plants, and similar results were obtained. Based on these results, we conclude that without acclimation, hos1-1 plants are less cold hardy than are the wild-type plants.

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5.

Time Course of RD29A–LUC Expression in hos1-1 and Wild-Type Plants in Response to Low Temperature, ABA, or NaCl Treatment.

(A) Low-temperature treatment. One-week-old hos1-1 and wild-type seedlings grown on the same plate were placed in an incubator at 0°C. Plates were removed from the incubator at different time points; luminescence images were taken immediately, and the intensities were determined.

(B) ABA treatment. hos1-1 and wild-type seedlings grown on the same plate were sprayed with 100 μM ABA and incubated under light for the indicated time periods before luciferase imaging.

(C) NaCl treatment. Plants were transferred on filter paper saturated with a solution of MS salts plus 300 mM NaCl under light for the indicated time periods before luciferase imaging.

Error bars represent standard deviation (n = 20). WT, wild type.

hos1-1 Mutant Flowers Early

Under the growth conditions used in this study (16 hr of light at 22°C; 8 hr of dark at 18°C), hos1-1 plants flowered ~2 weeks after imbibition, compared with 3 weeks for wild-type plants. We selected from >100 hos1-1 plants from a segregating F2 population (selfed from a cross between the wild type and hos1-1); thus far, none has been an exception to this early-flowering phenotype. Because flowering time in Arabidopsis can be influenced by periods of low-temperature treatment (i.e., vernalization), we determined the vernalization responses of hos1-1 and wild-type plants. To accurately determine the flowering phenotype of hos1-1 plants, we measured the total leaf number (LN) at flowering. As shown in Figure 9, the LN value of hos1-1 is smaller than that for wild-type plants, without vernalization or with various periods of vernalization. Both hos1-1 and wild-type plants responded to vernalization. Without vernalization, the LN values for hos1-1 and wild-type plants were 8.0 ± 0.9 and 13.2 ± 1.9, respectively. After 8 weeks of vernalization, the LN value for hos1-1 plants decreased to 5.0 ± 0.4, whereas the LN value for the wild type dropped to 7.7 ± 0.9. It is interesting that the LN value for nonvernalized hos1-1 was nearly the same as the LN value for wild-type plants after 8 weeks of vernalization.

Both hos1-1 and wild-type seeds germinated during the long periods of vernalization. Because the vernalization was performed with dim influorescent light in a cold room, the seedlings were etiolated. When the seedlings were moved to a growth chamber after 8 weeks of vernalization, all wild-type plants deetiolated rapidly and resumed normal growth. In contrast, hos1-1 seedlings deetiolated slowly, and only 30% survived. The hos1-1 seedlings appeared to have sustained damage during the long period of vernalization, and most withered and died before deetiolation.

The appearance of hos1-1 plants is shown in Figure 10. In general, hos1-1 plants are smaller and appear less vigorous than wild-type plants. Whereas the wild-type plants typically reach a height of 25 to 30 cm, hos1-1 plants usually do not grow taller than 18 cm. Mature hos1-1 plants possess fewer leaves than do wild-type plants. The leaves on hos1-1 plants do not appear as green as those of the wild type. Siliques on hos1-1 are typically less than two-thirds the length of wild-type siliques.

Figure 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 6.

RD29A–LUC Expression of hos1-1 and Wild-Type Plants during Deacclimation.

hos1-1 and wild-type (WT) plants on the same agar plate were placed at room temperature under cool-white light after they were treated at 0°C for 24 hr. Luminescence intensities were determined at different time points. Error bars represent standard deviation (n = 20).

(A) Decrease in luminescence intensities with time after removal from cold.

(B) Percentage of decrease in luminescence intensity relative to nondeacclimated plants.

DISCUSSION

hos1-1 is a plant mutant whose gene regulation is altered under low-temperature conditions. This recessive mutation enhances cold induction of stress genes. Gene regulation by osmotic stress or ABA is not affected significantly in this mutant. The recessive hos1-1 mutation thus reveals an important negative regulator of low-temperature signal transduction in plants. Our results indicate that the HOS1 locus not only plays a significant role in cold regulation of gene transcription but also influences other cold-regulated processes, such as freezing tolerance and time to flower.

It is clear that not all of the cold-responsive genes are affected to the same degree by the hos1-1 mutation (Figure 3). The difference between hos1-1 and the wild type is less dramatic for COR15A and KIN1, although the expression of these two genes was still reproducibly higher in hos1-1 plants. The reason for this differential effect is not clear. It is possible that these two genes may use signaling pathways that overlap with but differ from the ones used by RD29A, COR47, and ADH, whose expression is more dramatically affected by the hos1-1 mutation. Alternatively, COR15A and KIN1 transcripts could be degraded more rapidly in hos1-1 plants, thereby preventing their overaccumulation.

Figure 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 7.

RD29A–LUC Expression in hos1-1 and Wild-Type Plants under Different Temperature Treatments.

hos1-1 and wild-type (WT) plants were planted on the same agar plates and allowed to grow for 1 week under constant lighting at room temperature (22 ± 2°C). The plates were then treated at the indicated temperatures (±0.1°C) for either 3 hr (−5 or −10°C treatment) or 24 hr (all other temperature treatments), and luminescence images were taken (see Methods for details). Error bars represent standard deviation (n = 20).

(A) Lower temperature range.

(B) Higher temperature range.

Figure 8.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 8.

Leakage of Electrolytes in hos1-1 and Wild-Type Plants When Treated at Temperatures below Freezing.

For cold acclimation treatment, plants were incubated at 4°C for 48 hr under white fluorescent light. Triangles, nonacclimated wild type; squares, nonacclimated hos1-1; (+), acclimated wild type; asterisks, acclimated hos1-1.

Expression of the RD29A gene is induced by cold, ABA, and osmotic stress. Cold and osmotic signaling pathways converge to activate gene transcription through the cis-DNA element DRE/C repeat (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997). Because only cold regulation is altered in hos1-1 plants, HOS1 is likely in a step before the cold and osmotic signaling pathways converge. Figure 11 presents a model of HOS1 function in the activation of stress gene transcription. The model depicts HOS1 as a negative regulator for a cold-specific pathway. In addition, results presented in Figure 3 indicate that HOS1 plays a positive role in the upregulation of RAB18, RD29B, COR15A, and KIN1 by osmotic stress or ABA.

Little is known about the initial perception of cold in living organisms. In the cyanobacterium Synechocystis sp PCC6803, changes in membrane fluidity have been suggested as the basis for low-temperature perception (Vigh et al., 1993). It was demonstrated that reduction of cyanobacterial cell membrane fluidity by hydrogenation at room temperature could trigger the expression of the desA gene, as does low temperature (Vigh et al., 1993). The importance of membrane lipid fluidity for plant chilling tolerance is well known (Murata et al., 1992; Miquel et al., 1993). However, it is unclear whether there exists a connection between membrane fluidity and gene regulation by low temperature in plants. In plant cells, an increase in cytosolic calcium level has been suggested to be important in cold acclimation (Knight et al., 1996) and cold induction of gene expression (Monroy and Dhindsa, 1995). The HOS1 locus could play a role in either the initial perception of cold stress or its subsequent intracellular signaling.

It is intriguing that hos1-1 plants are less tolerant to freezing stress (Figure 8). The results may suggest membrane alterations in the mutant. For example, certain lipids in the plasma membrane of hos1-1 cells may have a slightly higher degree of saturation, which may result in increased membrane leakage during freezing stress. By analogy to the cyanobacterium system, an elevated level of membrane lipid saturation also might cause expression of cold-responsive genes at higher temperatures. We have observed that RD29A gene transcription can be induced by temperatures as high as 19°C (Figure 7). We also have observed that hos1-1 plants had a much reduced survival rate after long periods of cold treatment (i.e., vernalization). This latter observation indicates that hos1-1 plants are chilling sensitive, unlike wild-type plants, which are chilling tolerant. The level of membrane lipid saturation is known to correlate with plant chilling sensitivity (Miquel et al., 1993).

hos1-1 plants show a clear early-flowering phenotype. The early-flowering phenotype always cosegregated with the high RD29A–LUC expression observed after cold treatment. Because the total leaf number of nonvernalized hos1-1 plants is approximately the same as that of wild-type plants after 8 weeks of vernalization, the mutant may be considered as constitutively vernalized. Induction of RD29A by temperatures near 20°C (Figure 7B) indicates that “cold” signaling operates in the mutant under normal growth conditions (i.e., 18°C nighttime temperature). Therefore, the mutant may experience a vernalization effect under normal growth conditions. It is clear from the results shown in Figure 9 that hos1-1 plants can be further vernalized by exposure to low temperature, resulting in flowering at an LN of only 5. Recently, Chandler et al. (1996) identified several Arabidopsis mutants with reduced vernalization responses. Expression of cold-induced transcripts was not altered in these mutants (Chandler et al., 1996). Based on these results, the authors suggested that vernalization may be mediated through a pathway that is different from those for cold acclimation and gene regulation, and the perception of low temperature may be different for vernalization and acclimation (Chandler et al., 1996). Our results with hos1-1 plants suggest, however, that cold acclimation and vernalization may share some common mechanisms.

Figure 9.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 9.

hos1-1 Plants Flower Earlier than Wild-Type Plants.

Seeds of hos1-1 and the wild-type (WT) plants were sown in pot media and placed in a cold room (4°C) for the indicated time periods before moving to a growth chamber. When their first flowers were at anthesis, 10 to 20 plants were counted for total leaf numbers.

Figure 10.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 10.

Morphology of hos1-1 (right) Compared with a Wild-Type RD29A–LUC Plant (left).

The mutant and the wild-type plants were planted at the same time and allowed to grow in a growth chamber for 5 weeks before being photographed. Note that the hos1-1 plant is smaller and flowered earlier; its leaves are not as green as those of the wild-type plant. Some leaves on the hos1-1 plant were already dead and not visible when photographed. The bar insert at right represents 5 cm.

METHODS

Plant Materials and Growth Conditions

Plants (Arabidopsis thaliana ecotype C24) were transformed with the chimeric gene RD29A–luciferase (LUC), and the seeds from selfed progenies of the resulting transgenic plants containing homozygous transgene (referred to as wild type) were mutagenized with ethyl methanesulfonate (Ishitani et al., 1997). M2 seedlings grown on 0.8% agar plates containing a Murashige and Skoog (MS) salt base (JRH Biosciences, Lenexa, KS) were screened for altered RD29A–LUC expression in response to low temperature, exogenous abscisic acid (ABA), or osmotic stress by using a video-imaging system, as described by Ishitani et al. (1997). Plants for imaging analysis were usually 1-week-old seedlings grown on MS agar plates under constant white fluorescent light at room temperature. Plants for genetic analysis, vernalization, or freezing tolerance assays were grown in pot media (Metro-Mix 350; Scott-Sierra Horticultural Products Co., Marysville, OH) in growth chambers with 16 hr of light at 22°C and 8 hr of dark at 18°C and 70% relative humidity.

Cold Acclimation, Vernalization, and Other Stress Treatments

For mutant screening and primary characterization, low-temperature treatment was conducted at 0°C for 48 hr in the dark. For detailed characterization of RD29A–LUC expression in response to low-temperature treatment, we placed agar plates with 1-week-old hos1-1 and wild-type seedlings in an incubator set at the designated temperature (±0.1°C) for 24 hr, as stated in Results. At the end of the treatment, plates were removed from the incubator, and luminescence images were taken as described in Ishitani et al. (1997). Because longer treatment at −5 or −10°C results in freezing of the agar plates, these treatments only lasted for 3 hr. After treatment, the plates were placed at room temperature for 2 hr to thaw before taking luminescence images. For ABA treatment, 100 μM ABA in water (mixed isomers) was sprayed on leaves of the seedlings, and luminescence images were taken 3 hr after treatment. NaCl treatment was conducted on filter paper saturated with an MS salt solution that was supplemented with 300 mM NaCl, and luminescence images were taken 5 hr after the treatments.

To determine vernalization effect, seeds of the mutant and wild-type plants were sown in pot media, and the pots were kept at 4°C for different times (days), as indicated in Results. After vernalization, the pots were placed in a growth chamber until flowering occurred. At the emergence of the first flower, the rosette and cauline leaves were counted.

Figure 11.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 11.

Proposed Model of HOS1 Function.

HOS1 negatively regulates low-temperature–induced expression of many stress-responsive genes. It is also a positive factor for specific gene regulation by osmotic stress or ABA.

Freezing Tolerance

For the freezing-tolerance assay, seeds of the hos1-1 and wild-type plants were sown in pot media. For cold-acclimation treatment, plants at the rosette stage were placed in a cold room at 4°C under white fluorescent light for 48 hr before we sampled the leaves for the freezing-tolerance assay.

Fully developed rosette leaves were used to determine freezing-caused electrolyte leakage, essentially as described by Sukumaran and Weiser (1972) and Ristic and Ashworth (1993). Briefly, for each treatment, one excised leaflet was placed in a test tube containing 100 μL of deionized H2O, and the tube was placed in a refrigerated circulator (freezing bath) (model 1187; VWR Scientific, San Francisco, CA) with the temperature set at 0°C. There were three replicates for each temperature treatment. The temperature of the bath was programmed to decrease to −12°C, with 1°C decrement after 30 min. The tubes were removed from the bath when the designated temperature was reached, and they were placed immediately on ice to allow gradual thawing. The leaflets then were transferred carefully to another tube containing 25 mL of deionized water and shaken overnight, and the conductivity of the solution was measured. The tubes with the leaflets were then autoclaved. After cooling down to room temperature, conductivities of the solutions were measured again. The percentage of electrolyte leakage was calculated as the percentage of the conductivity before autoclaving over that after autoclaving.

RNA Analysis

Ten-day-old seedlings grown on MS agar plates were subjected to low temperature, ABA, or polyethylene glycol (PEG). Respective treatment conditions were as stated in Results. Total RNA from control or stressed plants was extracted as described by Liu and Zhu (1997). The RD29A gene–specific probe was from the 3′ noncoding region (Liu and Zhu, 1997). COR15 and COR47 cDNAs (Gilmour et al., 1992; Lin and Thomashow, 1992) were kindly provided by M.F. Thomashow (Michigan State University, East Lansing, MI). DNA probes for RD22 and RD29B (Yamaguchi-Shinozaki et al., 1992) were cloned from genomic DNA of wild-type Columbia plants by polymerase chain reaction by using the following primer pairs: 5′-AACGCACCGATGCAGAAGTACA-3′ and 5′-CCGGATCCGCAACACAAGATACAGAC-3′; and 5′-CCCGGATCCTTGCTGTTACACGTA-3′ and 5′-ATGCTCGAGCTGACGCTATGATTTTG-3′, respectively. The probe for KIN1 (Kurkela and Franck, 1990) was a 0.4-kb EcoRI fragment of the Arabidopsis expressed sequence tag (EST) clone YAP368T7. The probe for RAB18 (Lång and Palva, 1992) was a 0.8-kb SalI-NotI fragment of the Arabidopsis EST clone 246K10T7. The probe for ADH (Jarillo et al., 1993) was a 1.6-kb SalI-NotI fragment of the Arabidopsis EST clone 199P20T7.

ACKNOWLEDGMENTS

We thank Dr. Michael F. Thomashow for kindly providing COR15a and COR47 cDNA probes. We also thank Drs. Robert T. Leonard and Frans Tax for critical reading of the manuscript. This work was supported by grants from the National Science Foundation Integrative Plant Biology Program and the United States Department of Agriculture National Research Initiative Competitive Grants Program to J.-K.Z.

Footnotes

  • ↵1 These authors contributed equally to this work.

  • Received January 7, 1998.
  • Accepted May 5, 1998.
  • Published July 1, 1998.

REFERENCES

  1. ↵
    1. Chandler J.,
    2. Wilson A.,
    3. Dean C.
    (1996). Arabidopsis mutants showing an altered response to vernalization. Plant J. 10, 637–644.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Chen H.-H.,
    2. Li P.H.,
    3. Brenner M.L.
    (1983). Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71, 362–365.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Cherry J.H.
    1. Dhindsa R.S.,
    2. Monroy A.F.
    (1994). Low temperature signal transduction, gene expression, and cold acclimation: Multiple roles of low temperature. In Biochemical and Cellular Mechanisms of Stress Tolerance in Plants, Cherry J.H., ed (Berlin: Springer-Verlag), pp. 501–514.
  4. ↵
    1. Gilmour S.J.,
    2. Thomashow M.F.
    (1991). Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17, 1233–1240.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Gilmour S.J.,
    2. Artus N.N.,
    3. Thomashow M.F.
    (1992). cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana. Plant Mol. Biol. 18, 13–32.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Guy C.L.
    (1990). Cold acclimation and freezing stress tolerance: Role of protein metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 187–223.
    OpenUrlCrossRef
  7. ↵
    1. Guy C.L.,
    2. Haskell D.
    (1988). Detection of polypeptides associated with the cold acclimation process in spinach. Electrophoresis 9, 787–796.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Cherry J.H.
    1. Guy C.L.,
    2. Anderson J.V.,
    3. Haskell D.W.,
    4. Li Q.-B.
    (1994). Caps, cors, dehydrins, and molecular chaperones: Their relationship with low temperature responses in spinach. In Biochemical and Cellular Mechanisms of Stress Tolerance in Plants, Cherry J.H., ed (Berlin: Springer-Verlag), pp. 479–499.
  9. ↵
    1. Hajela R.K.,
    2. Horvath D.P.,
    3. Gilmour S.J.,
    4. Thomashow M.F.
    (1990). Molecular cloning and expression of cor (cold regulated) genes in Arabidopsis thaliana. Plant Physiol. 93, 1246–1252.
  10. ↵
    1. Ishitani M.,
    2. Xiong L.,
    3. Stevenson B.,
    4. Zhu J.-K.
    (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: Interactions and convergence of abscisic acid–dependent and abscisic acid–independent pathways. Plant Cell 9, 1935–1949.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Jarillo J.A.,
    2. Leyva A.,
    3. Salinas J.,
    4. Martinez-Zapater J.M.
    (1993). Low-temperature induces the accumulation of alcohol dehydrogenase mRNA in Arabidopsis thaliana, a chilling-tolerant plant. Plant Physiol. 101, 833–837.
    OpenUrlAbstract
  12. ↵
    1. Jonak G.,
    2. Kiegerl S.,
    3. Lighterink W.,
    4. Barker P.J.,
    5. Huskisson N.S.
    (1996). Stress signaling in plants: A mitogen-activated protein kinase pathway is activated by cold and drought. Proc. Natl. Acad. Sci. USA 93, 11274–11279.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Knight H.,
    2. Trewavas A.J.,
    3. Knight M.R.
    (1996). Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8, 489–503.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Knight M.R.,
    2. Campell A.K.,
    3. Smith S.M.,
    4. Trewavas A.J.
    (1991). Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352, 524–526.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kurkela S.,
    2. Borg-Franck M.
    (1992). Structure and expression of kin2, one of two cold- and ABA-induced genes of Arabidopsis thaliana. Plant Mol. Biol. 19, 689–692.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kurkela S.,
    2. Franck M.
    (1990). Cloning and characterization of a cold- and ABA-inducible Arabidopsis gene. Plant Mol. Biol. 15, 137–144.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Lång V.,
    2. Palva E.T.
    (1992). The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Mol. Biol. 20, 957–962.
    OpenUrl
  18. ↵
    1. Lång V.,
    2. Mäntylä E.,
    3. Welin B.,
    4. Sundberg B.,
    5. Palva E.T.
    (1994). Alterations in water status, endogenous abscisic acid content, and expression of rab18 gene during the development of freezing tolerance in Arabidopsis thaliana. Plant Physiol. 104, 1341–1349.
    OpenUrlAbstract
  19. ↵
    1. Levitt J.
    (1980). Responses of Plants to Environmental Stress, Vol. 1. Chilling, Freezing, and High Temperature Stress. (New York: Academic Press).
  20. ↵
    1. Lin C.,
    2. Thomashow M.F.
    (1992). DNA sequence analysis of a complementary DNA for cold-regulated Arabidopsis gene cor15 and characterization of the COR15 polypetide. Plant Physiol. 99, 519–525.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Liu J.,
    2. Zhu J.-K.
    (1997). Proline accumulation and salt-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol. 114, 591–596.
    OpenUrlAbstract
  22. ↵
    1. Lynch D.V.,
    2. Steponkus P.L.
    (1987). Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv Puma). Plant Physiol. 83, 761–767.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Miquel M.,
    2. James D. Jr..,
    3. Dooner H.,
    4. Browse J.
    (1993). Arabidopsis requires polyunsaturated lipids for low-temperature survival. Proc. Natl. Acad. Sci. USA 90, 6208–6212.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Mohapatra S.S.,
    2. Poole R.J.,
    3. Dhindsa R.S.
    (1988). Abscisic acid–regulated gene expression in relation to freezing tolerance in alfalfa. Plant Physiol. 87, 468–473.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Monroy A.F.,
    2. Dhindsa R.S.
    (1995). Low-temperature signal transduction: Induction of cold acclimation–specific genes of alfalfa by calcium at 25°C. Plant Cell 7, 321–331.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Murata N.,
    2. Ishizaki-Nishizawa O.,
    3. Higashi S.,
    4. Hayashi H.,
    5. Tasaka Y.,
    6. Nishida I.
    (1992). Genetically engineered alteration in the chilling sensitivity of plants. Nature 356, 710–713.
    OpenUrlCrossRef
  27. ↵
    1. Nordin K.,
    2. Vahala T.,
    3. Palva E.T.
    (1993). Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh. Plant Mol. Biol. 21, 641–653.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Cherry J.H.
    1. Palva E.T.,
    2. Welin B.,
    3. Vahala T.,
    4. Olson A.,
    5. Nordin-Henriksson K.,
    6. Mäntylä E.,
    7. Lång V.
    (1994). Regulation of low temperature–induced genes during cold acclimation of Arabidopsis thaliana. In Biochemical and Cellular Mechanisms of Stress Tolerance in Plants, Cherry J.H., ed (Berlin: Springer-Verlag), pp. 527–542.
  29. ↵
    1. Ristic Z.,
    2. Ashworth E.N.
    (1993). Changes in leaf ultrastructure and carbohydrates in Arabidopsis thaliana (Heynh) cv. Columbia during rapid cold acclimation. Protoplasma 172, 111–123.
    OpenUrlCrossRef
  30. ↵
    1. Stockinger E.J.,
    2. Gilmour S.J.,
    3. Thomashow M.F.
    (1997). Arabidopsis thaliana CBF1 encodes an AP2 domain–containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA 94, 1035–1040.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Sukumaran N.P.,
    2. Weiser C.J.
    (1972). An excised leaflet test for evaluating potato frost tolerance. HortScience 7, 467–468.
    OpenUrl
  32. ↵
    1. Meyerowitz E.M.,
    2. Somerville C.R.
    1. Thomashow M.F.
    (1994). Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In Arabidopsis, Meyerowitz E.M., Somerville C.R., eds (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 807–834.
  33. ↵
    1. Vigh L.,
    2. Los D.A.,
    3. Horvath I.,
    4. Murata N.
    (1993). The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc. Natl. Acad. Sci. USA 90, 9090–9094.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Yamaguchi-Shinozaki K.,
    2. Shinozaki K.
    (1993). Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet. 236, 331–340.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Yamaguchi-Shinozaki K.,
    2. Shinozaki K.
    (1994). A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6, 251–264.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Yamaguchi-Shinozaki K.,
    2. Koizumi M.,
    3. Urao S.,
    4. Shinozaki K.
    (1992). Molecular cloning and characterization of nine cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: Sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol. 33, 217–224.
    OpenUrlAbstract/FREE Full Text
PreviousNext
Back to top

Table of Contents

Print
Download PDF
Email Article

Thank you for your interest in spreading the word on Plant Cell.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
HOS1, a Genetic Locus Involved in Cold-Responsive Gene Expression in Arabidopsis
(Your Name) has sent you a message from Plant Cell
(Your Name) thought you would like to see the Plant Cell web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
HOS1, a Genetic Locus Involved in Cold-Responsive Gene Expression in Arabidopsis
Manabu Ishitani, Liming Xiong, Hojoung Lee, Becky Stevenson, Jian-Kang Zhu
The Plant Cell Jul 1998, 10 (7) 1151-1161; DOI: 10.1105/tpc.10.7.1151

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
HOS1, a Genetic Locus Involved in Cold-Responsive Gene Expression in Arabidopsis
Manabu Ishitani, Liming Xiong, Hojoung Lee, Becky Stevenson, Jian-Kang Zhu
The Plant Cell Jul 1998, 10 (7) 1151-1161; DOI: 10.1105/tpc.10.7.1151
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • METHODS
    • ACKNOWLEDGMENTS
    • Footnotes
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

In this issue

The Plant Cell Online: 10 (7)
The Plant Cell
Vol. 10, Issue 7
Jul 1998
  • Table of Contents
  • About the Cover
  • Index by author
View this article with LENS

More in this TOC Section

  • Substrate Specificity of LACCASE8 Facilitates Polymerization of Caffeyl Alcohol for C-Lignin Biosynthesis in the Seed Coat of Cleome hassleriana
  • Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide
  • Temporal Regulation of the Metabolome and Proteome in Photosynthetic and Photorespiratory Pathways Contributes to Maize Heterosis
Show more RESEARCH ARTICLES

Similar Articles

Our Content

  • Home
  • Current Issue
  • Plant Cell Preview
  • Archive
  • Teaching Tools in Plant Biology
  • Plant Physiology
  • Plant Direct
  • Plantae
  • ASPB

For Authors

  • Instructions
  • Submit a Manuscript
  • Editorial Board and Staff
  • Policies
  • Recognizing our Authors

For Reviewers

  • Instructions
  • Peer Review Reports
  • Journal Miles
  • Transfer of reviews to Plant Direct
  • Policies

Other Services

  • Permissions
  • Librarian resources
  • Advertise in our journals
  • Alerts
  • RSS Feeds
  • Contact Us

Copyright © 2021 by The American Society of Plant Biologists

Powered by HighWire