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Proc Natl Acad Sci U S A. Jul 12, 2005; 102(28): 9966–9971.
Published online Jul 1, 2005. doi:  10.1073/pnas.0503960102
PMCID: PMC1175003
Plant Biology

This article has been retractedRetraction in: Proc Natl Acad Sci U S A. 2010 July 9; 107(31): 13972    See also: PMC Retraction Policy

HOS10 encodes an R2R3-type MYB transcription factor essential for cold acclimation in plants

Abstract

We report the identification and characterization of an Arabidopsis mutant, hos10-1 (for high expression of osmotically responsive genes), in which the expression of RD29A and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The hos10-1 plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of NCED3 (the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The hos10-1 plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The HOS10 gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development.

Keywords: Arabidopsis, freezing, abscisic acid, NCED3

Exposure to low temperature, soil drying, or high levels of salt results in altered expression of a diverse range of plant genes (1). The products of these genes may either directly protect plants against stresses or further control the expression of other target genes. Although the signal transduction pathways responsible for the activation of these genes are still mostly unknown, transcriptional control of some stress-responsive genes is understood to involve transcription factor-binding and activation of cis elements in target genes, such as the RD29A (responsive to dehydration 29A) gene promoter (2), which contains the abscisic acid (ABA) responsive element (ABRE) and the dehydration-responsive element (DRE)/C-repeat (CRT) sequences (2).

Transcription factors in the ethylene response element binding protein/apetala 2 family that bind to the DRE/CRT element are termed C-repeat binding factor (CBF)/DRE-binding proteins (DREBs) (3, 4). CBF/DREB1 genes are rapidly and transiently induced by cold stress and subsequently activate the expression of target genes. DREB2 genes are also induced by osmotic stress and may confer osmotic stress induction of target stress-responsive genes (4). Ectopic expression of CBF/DREB1 genes in plants was reported to improve tolerance to cold, drought, and salt stresses (57).

Early signaling components upstream of CBF/DREB1 may be subject to specific ubiquitination-mediated degradation, as suggested by the characterization of the HOS1 gene in Arabidopsis (8). HOS1 encodes a protein with a RING finger motif similar to that present in a group of inhibitor of apoptosis proteins in animals that act as E3 ubiquitin ligases to target certain regulatory proteins for degradation. Recently, ICE1 (inducer of CBF expression 1) was identified with the CBF3/DREB1A promoter::LUC screening system (9). ICE1 is a MYC-like basic helix–loop–helix transcriptional activator, and it binds specifically to the MYC recognition sequences in the CBF3/DREB1A promoter. ICE1 is constitutively expressed, and its overexpression enhances the expression of the CBF regulon in the cold, resulting in increased freezing tolerance. These results suggest that ICE1 acts as an upstream regulator that positively controls the transcription of CBF genes in the cold (9) and may be a target for degradation by HOS1 (8).

ABRE is another major cis element in ABA-responsive gene expression. Two ABRE motifs are important in the ABA-responsive expression of the Arabidopsis gene RD29B (10). The ABRE-binding proteins (AREBs)/ABRE-binding factors (ABFs) can bind to ABRE and activate ABA-dependent gene expression (10). The AREB/ABF proteins have reduced activity in ABA-deficient aba2 mutants and the ABA-insensitive abi1 mutant and enhanced activity in the ABA-hypersensitive era1 mutant. Hence, activation of the AREB/ABF proteins has been shown to require an ABA-mediated signal (10), which probably involves ABA-dependent phosphorylation. Overexpression of ABF3 or AREB2/ABF4 caused ABA hypersensitivity, a reduced transpiration rate, and enhanced drought tolerance (11).

A number of lines of evidence suggest the existence of crosstalk among the drought, salinity, cold, and ABA signal transduction pathways (12, 13). Many transcription-factor genes, including MYBs, were found to be stress-inducible, suggesting that transcription regulation is a part of drought, cold, or salt stress signaling (13, 14). Although acclimation to cold has been associated with the CBF/DREB1 family of transcription factors (5), evidence has revealed a possible CBF-independent pathway that is also necessary for cold acclimation (15).

Here we report that the Arabidopsis mutant hos10-1 exhibits altered expression of low temperature, salt stress, and ABA-responsive genes, some of which belong to the CBF regulon. However, mutation of the HOS10 locus does not alter expression of the CBF family of genes. The hos10-1 mutant plants show dramatically reduced capacity for cold acclimation and are hypersensitive to dehydration and NaCl. The ability to increase ABA levels in response to dehydration stress also is impaired in hos10-1 mutant plants. HOS10 encodes an R2R3-type MYB transcription factor that is localized to the nucleus. It appears that HOS10 is essential for cold acclimation and may affect dehydration stress tolerance in plants by controlling stress-induced ABA biosynthesis.

Materials and Methods

Isolation of hos10-1 Mutant. Arabidopsis thaliana plants (ecotype C24) expressing the RD29A::LUC transgene (referred to as wild type) were mutagenized with an Agrobacterium tumefaciens-mediated (strain GV3101) T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) transformation with the activation tagging vector pSKI015 (12, 16). Seeds from T2 plants were used for screening mutants with altered RD29A::LUC expression in response to low temperature, ABA, and/or osmotic stress by luminescence imaging with a charge-coupled device camera as described in ref. 12.

ABA Measurement and Real-Time PCR Analysis of NCED3 Expression. ABA was quantified by using a radioimmuno assay essentially as described in ref. 17. For measurement of low water potential-induced ABA accumulation, 5-d-old seedlings grown on half-strength Murashige and Skoog medium (PhytoTechnology Laboratories, Lenexa, KS) (without addition of sugar) containing 2.5 mM Mes buffer were transferred using nylon mesh overlaid on the agar surface to –1.2-MPa, polyethylene glycol (PEG)-infused plates (18). Control seedlings were transferred to half-strength Murashige and Skoog medium plates. Samples for ABA measurement were collected at 8 h (peak ABA accumulation) and 96 h (steady-state ABA level) after the transfer. For cold treatment, 3-week-old, soil-grown hos10-1 and wild-type plants were transferred to a growth chamber at 4°C under a long-day photoperiod (16 h of light/8 h of dark). ABA content of untreated and cold-treated plants was assayed at the designated time points (see Fig. 5C).

Fig. 5.
Water loss and ABA biosynthesis defects in hos10-1 plants. (A) Transpirational water loss as indicated by loss of the initial fresh weight is greater in the hos10-1 mutant detached shoots. Error bars represent standard deviation (n = 10). (B) ABA accumulation ...

For real-time PCR analysis, total RNA was extracted from untreated or treated (–1.2-MPa, PEG-infused plates) seedlings with the RNeasy plant mini kit (Qiagen, Valencia, CA). First-strand cDNA synthesized with 1 μg of total RNA was used for the real-time PCR. Real-time PCR analysis of NCED3 expression was performed as described in ref. 19 by using a Sequence Detection System 7700 instrument (Applied Biosystems). Quantification of NCED3 copy number was conducted by amplifying a 600-bp portion of the NCED3 cDNA containing the Taqman probe-binding site. The primers used were 5′-TCCAGCTCTTCAT T TCCCTA A-3′ (forward) and 5′-CGGCCAT TGAAATAGACCAA-3′ (reverse). The amplified DNA fragment was purified, quantified spectrophotometrically, and diluted appropriately to generate a standard curve for calculation of the copy number of the NCED3 transcript.

RNA Gel Analysis and Microarray Analyses. Wild-type and hos10-1 seedlings were grown on separate halves of the same Murashige and Skoog medium agar plates for 14 d and then left untreated or treated with low temperature, ABA, or NaCl. Total RNA was extracted from whole seedlings, and RNA analysis was conducted as described in ref. 16.

Total RNA (20 μl) extracted with the RNeasy plant mini kit (Qiagen) from 21-d-old wild-type and hos10-1 seedlings after cold treatment (24 h at 0°C) was used to make biotin-labeled cRNA targets. Microarray analysis (Affymetrix GeneChip array) was performed as described in ref. 9.

Cloning of the HOS10 Gene. The genomic DNA fragment flanking the left border of the inserted T-DNA in hos10-1 plants was isolated by thermal asymmetric interlaced PCR and subcloned into the cloning vector pBluscript SK(+) (Stratagene) as described in ref. 16. The entire isolated fragment was sequenced.

The coding region of HOS10 was amplified by RT-PCR by using primer pair 5′-ACTGGAGCTCATGGGAAGATCACCATGTTGTG-3′ (forward) and 5′-ACGTTCTAGACACACGAGCTAGTAACAAGATC-3′ (reverse). The RT-PCR product was then subcloned into pGEM-T Easy Vector (Promega) and sequenced. The HOS10 was released from the resulting clone 10-133 and cloned into binary vector 99-1 between SacI and XbaI sites, resulting in an expression cassette of HOS10 under the control of the cauliflower mosaic virus 35S promoter. The construct was introduced into hos10-1 mutant plants through an A. tumefaciens-mediated (strain GV3101) T-DNA transformation. Primary transformants, which were resistant to 50 mg/l hygromycin (Invitrogen), were transferred to soil to grow to maturity. Progenies of these transformants were examined for RD29A::LUC expression and for freezing tolerance.

GFP–HOS10 Fusion Protein Construct. The coding region of HOS10 was amplified from the cDNA clone 10-133 by PCR and cloned in-frame into pEGAD vector between the EcoRI and BamHI sites, and the entire insert and the conjunction regions were sequenced. This construct was then introduced into Arabidopsis wild-type plants by floral dip transformation with Agrobaterium strain GV3101.

Results

Identification of the HOS10 Locus. Through a luciferase imaging-based screen (12), we isolated one mutant, hos10-1, that has a 3- to 4-fold higher level of RD29A::LUC expression after low temperature, ABA, and/or NaCl stress treatments (Fig. 1). All 41 F1 progeny of hos10-1 backcrossed to wild-type plants exhibited a wild-type phenotype. Approximately three-quarters of the F2 progeny of the selfed F1 plants displayed a wild-type phenotype, indicating that hos10-1 is a recessive mutation in a single nuclear gene.

Fig. 1.
Elevated RD29A::LUC expression in the hos10-1 mutant. (A) RD29A::LUC expression was quantitatively measured as luminescence intensity (counts per plant). Wild-type (right side of plate) and hos10-1 (left side of plate) seedlings and their luminescence ...

Regulation of Stress-Responsive Gene Expression in hos10-1 Plants. Northern hybridizations revealed that, without stress, there was no detectable RD29A transcript in hos10-1 or wild-type plants (Fig. 2A). Induction of the endogenous RD29A, of the introduced luciferase gene, and of COR15A, ADH, and KIN1 genes by cold was more rapid in hos10-1 than in wild type (Fig. 2 A). After ABA or NaCl treatment, the expression of the above genes was slightly higher in hos10-1 than wild type (Fig. 2 A). The steady-state transcript levels of P5CS1 (Fig. 2B) and of the cold-specific transcription factor genes CBF1, CBF2, and CBF3 (Fig. 2 B and C) were virtually not altered in hos10-1.

Fig. 2.
Gene regulation in hos10-1 and wild-type plants. (AC) Steady-state transcript abundance of several stress-regulated genes in wild-type and hos10-1 plants by Northern blot analysis. The blots were also hybridized with probes from tubulin and ...

Analysis of Affymetrix near-full genome GeneChip arrays showed that, compared with wild-type plants, the expression levels of only six genes were higher by at least 2-fold in the hos10-1 plants after 24 h of cold treatment (Table 1, which is published as supporting information on the PNAS web site). There were also only six genes with lower expression in hos10-1 by at least 2-fold compared with wild-type plants (Table 2, which is published as supporting information on the PNAS web site). The altered expression of three randomly selected genes (Atlg75830, At3g49620, and At5g01600) from the microarray tables was confirmed by RT-PCR analysis (Fig. 2D). This number of genes is unusually low compared with estimated targets of other transcription factors but could reflect the limited types of conditions used in our experiments.

hos10-1 Plants Are Defective in Cold Acclimation. Non-cold-acclimated young (≈3-week-old) plants of wild type could tolerate lower temperatures than hos10-1 plants (Fig. 3 A and B). When acclimated at 4°C for 8 d, the majority of the wild-type plants tolerated freezing temperatures as low as –8°C. However, <2% of the hos10-1 mutant plants survived freezing at –2°C, and none (nonacclimated or acclimated) survived below –2°C (Fig. 3 A and B). With or without cold acclimation, detached leaves of hos10-1 plants also showed more injury than wild type and were unable to increase their freezing tolerance significantly when measured by electrolyte leakage (15) (Fig. 3C). Thus, plants carrying the hos10-1 mutation are extremely sensitive to freezing temperatures and are unable to acclimate to the cold.

Fig. 3.
hos10-1 mutant plants are defective in cold acclimation. (A) Four-week-old plants grown at room temperature or at 4°C for 8 d were used for freezing treatments as described in ref. 15. The photographs were taken 7 d after the freezing treatments. ...

hos10-1 Mutant Plants Are Hypersensitive to NaCl. The hos10-1 seedlings form shorter roots than wild-type when grown vertically in germination medium (Fig. 4A), and their growth was also substantially inhibited by NaCl stress (Fig. 4B). The hos10-1 mutant plants accumulated essentially the same amounts of Na+ or K+ compared with wild type with or without NaCl treatment (Fig. 4 C and D) (16), indicating that increased sensitivity of hos10-1 plants to these stresses was not due to impaired Na+ homeostasis or impaired K+ acquisition.

Fig. 4.
hos10-1 mutant plants are hypersensitive to NaCl. (A) hos10-1 plants are hypersensitive to NaCl. (B) Dose–response of hos10-1 and wild-type seedlings to NaCl. Data shown are relative root growth (growth on 0 mM NaCl was considered 100%). Error ...

hos10-1 Mutant Plants Are Impaired in ABA Biosynthesis Under Dehydration Stress. The hos10-1 mutant plants lost water much faster than wild-type plants during slow dehydration (Fig. 5A). This result suggests that, during dehydration, the hos10-1 plants either may not make enough ABA, or their stomata fail to respond to waterdeficit-induced ABA. Under normal, nonstress conditions, hos10-1 and wild type accumulate similar amounts of ABA. However, there is a much smaller rise in ABA accumulation in hos10-1 than in wild-type after PEG-induced dehydration stress (Fig. 5B). In contrast, we did not find any substantial difference in ABA accumulation between wild-type and hos10-1 plants during cold acclimation (Fig. 5C) at 4°C. The transcript level of NCED3 that encodes a key enzyme in ABA biosynthesis is much less in hos10-1 than in wild-type after PEG-induced dehydration stress (Fig. 5D), suggesting that HOS10 controls expression of genes involved in ABA biosynthesis under dehydration stress.

hos10-1 Mutant Plants Flower Early. hos10-1 plants are somewhat smaller than wild type (Fig. 6 AC). Under long-day (16 h of light/8 h of dark) and short-day (8 h of light/16 h of dark) photoperiods, hos10-1 plants flower earlier than wild type (Fig. 6 B and D). The vernalization responses of hos10-1 and wild-type plants were the same (Fig. 6H). The expression of FLC, a gene encoding a critical flowering time regulator, also is not altered in hos10-1, consistent with its unchanged vernalization response (Fig. 6E). hos10-1 plants also have reduced fertility (Fig. 6 C and F), and their seeds are more round than wild-type (Fig. 6G).

Fig. 6.
hos10-1 mutant plants flower early. (A) Three-week-old wild-type (left plant) and hos10-1 (right plant) plants grown in soil. (B) Five-week-old, soil-grown, wild-type (left plant) and hos10-1 (right plant) plants. (C) Mature plants of wild-type (left ...

Identification of the HOS10 Gene. A DNA fragment flanking the T-DNA insert in hos10-1 mutant plants was obtained by thermal asymmetric interlaced PCR. This sequence was found to match that of the predicted Arabidopsis gene At1g35515. The HOS10 cDNA was cloned by RT-PCR using RNA prepared from wild-type plants. We conducted a complementation test by constitutive expression of HOS10 cDNA under the control of the cauliflower mosaic virus 35S promoter. Twenty of 29 hos10-1 plants transformed with the wild-type HOS10 cDNA cassette exhibited wild-type phenotypes in the T2 generation (Fig. 7B). Genetic analyses indicated that additional lines carrying T-DNA inserts in the At1g35515 gene (seed stock nos. SALK_122356, SALK_031231, and SALK_088230) are allelic to hos10-1 (Fig. 7A). The expression of HOS10 is disrupted in hos10-1 and the other three alleles of HOS10 (Fig. 7C). Excess electrolyte leakage induced by freezing treatments in the other hos10-1 alleles, before and after cold acclimation, showed that they were all defective in cold acclimation (Fig. 7D). In addition, plants carrying allelic mutations of hos10-1 flower earlier than do their wild-type background strain (Col-0) (Fig. 7E).

Fig. 7.
HOS10 encodes an R2R3-type MYB transcription factor. (A) Structure of the HOS10 gene and positions of T-DNA inserts in the HOS10 gene. LB and RB, T-DNA left border and right border, respectively. (B) Complementation of hos10-1 mutant plant. Shown is freezing ...

Comparison of the predicted HOS10 amino acid sequence with those of other gene products revealed that HOS10 shares greatest sequence similarities with R2R3-type MYB transcription factors from Arabidopsis, cotton, rice, and tomato within the R2 or R3 domain (Fig. 7F). MYP transcription factors comprise a large gene family in Arabidopsis that are involved in numerous functions, including response to stresses (18, 19). The HOS10 gene is constitutively expressed (Fig. 7G). HOS10 was fused in-frame to the C terminus of GFP and expressed under the control of the cauliflower mosaic virus 35S promoter. We found that the GFP–HOS10 fusion protein accumulates in the nucleus with or without low-temperature treatment, consistent with its predicted function as a transcription factor (Fig. 7H) (20).

Discussion

Tolerance and acclimation to freezing temperatures is a long-observed characteristic of plants indigenous to temperate regions (21). Cold acclimation is thought to involve many changes in cellular processes. Acclimation must include changes in metabolism that ensure maintenance of essential cellular structures/functions that are directly affected by low temperature. Often-cited examples include membrane fluidity changes caused by the behavior of lipids at low versus high temperatures and temperature-dependent protein conformation changes (22).

Much research has focused on the particular details of cellular function that fails under freezing conditions. This approach has not been very instructive in the actual mechanisms of tolerance. Guy (21) has pointed out that the large volume of reports on the when and where of freezing injury in plants has not led to any particular method or approach by which increased freezing tolerance can be achieved in plants. Essentially, this earlier work has established only a catalog of the biochemical and physiological changes that occur during cold acclimation. To go beyond this catalog requires a more analytical approach. The broad variation in the ability of plant species to acquire cold hardiness bolstered the hope that the understanding of the mechanism of acclimation and, hence, the ability to control it could be achieved by uncovering genetic differences between cold-tolerant and -sensitive species (21). However, only recently, with the introduction of a plant model system (A. thaliana) with appropriate molecular genetic attributes that could be applied to cold acclimation studies, has this genetic and biochemical dissection of the mechanism(s) of cold acclimation begun to be realized.

Thus, decades after Weiser (23) proposed an important role for altered gene expression in cold acclimation, it has now become well established that plants respond to cold treatment by changing the transcript levels of specific genes and that these changes can be linked to phenotypic adjustment. Prominent among these changes is the induction of the CBF family of transcription factors that, when overexpressed in transgenic plants, results in increased cold tolerance (5).

Notwithstanding the clear importance of the CBFs to cold acclimation, several lines of evidence have suggested that there are other signal pathways that control gene transcription in response to cold treatment and consequently contribute to freezing tolerance. First, many cold-induced genes do not contain in their promoter regions the DRE/CRT elements that are controlled by CBFs, and the apparent CBF regulon that has been determined by microarray analysis does not include all cold-induced genes (14). Importantly, some genes that are constitutively expressed, such as ESK1 and HOS9, have major effects on cold tolerance and acclimation but apparently do not require the induction of CBF genes for their activities (15, 24).

Although the hos10-1 mutation affects the expression of genes in the CBF regulon, the expression of the CBF genes themselves is not altered by the hos10-1 mutation (Fig. 2 B and C). The induction of the native RD29A gene by stress in hos10-1 was not as dramatic as that of the RD29A::LUC transgene. Similar differences between the RD29A::LUC transgene and the RD29A endogenous gene have been observed previously and are probably due to the presence of additional regulatory elements that are only present in the native RD29A gene (25). This finding may indicate the presence of negative regulators that do not recognize the RD29A::LUC transgene promoter or cannot access the chromatin structure at the RD29A::LUC site. More rapid induction of RD29A and other stress-responsive genes (Fig. 2) may be the result of the increased cold sensitivity of the hos10-1 mutation as suggested for other cold-sensitive mutants (25).

Microarray analysis revealed that HOS10 controls the expression of at least 12 genes under the conditions used (24 h of cold treatment) (Fig. 2D and Tables 1 and 2). Only two of these genes belong to the CBF regulon (26): At2g39030 encoding GCN5-related N-acetyl transferase and At1g19670 encoding coronatine-induced protein 1. RD29A, COR15A, ADH, and KIN1 are positively regulated by CBF2 (26) but these are negatively regulated by HOS10 (Fig. 2). CBF2 and HOS10 positively control the transcript level of the At2g39030 gene. In the case of At1g19670, CBF2 negatively regulates its transcript level, whereas HOS10 acts in the opposite way (Table 2) (26). These results indicate that, besides the CBFs, other transcription factors, including HOS10, also participate in the complex network controlling stress-responsive genes.

A striking feature of hos10-1 mutant plants is their complete inability to acclimate to freezing (Fig. 3). Besides the dramatic sensitivity of hos10-1 plants to freezing temperatures as high as –2°C, both visual and ion leakage assays revealed no acclimation at all after 4°C treatment for 8 d (Fig. 3). hos10-1 mutant plants are also sensitive to NaCl stress, and this sensitivity does not involve altered ion accumulation (Fig. 4). This finding suggests that hos10-1 plants have impaired ability to adjust to osmotic stress and/or injury responses (27). In fact, the water balance of hos10-1 plants is changed, and mutant shoots lose water more rapidly during dehydration (Fig. 5A). Measurement of the ABA content of hos10-1 and wild-type plants revealed that hos10-1 plants are impaired in their ability to increase ABA level in response to dehydration stress (Fig. 5B).

The impaired ABA biosynthesis response of hos10-1 plants raises the issue of whether ABA plays an important role in cold tolerance and the ability of plants to acclimate to freezing. Freezing of plant tissues may be viewed biologically in two distinct phases. Almost universally, for many reasons that have been reviewed by Guy (21), ice crystals form first in the extracellular space or apoplast of plant cells. Formation of ice crystals in the cytosol or growth of crystals through the plasma membrane into the cytosol is essentially a lethal process. Maintenance of an intact plasma membrane and the slow cooling rate of many natural environments will restrict ice crystals to the apoplast. The formation of ice crystals greatly depresses the water potential of the apoplast by the intense concentration of solutes and the large decline of the vapor pressure of water as it enters the frozen state. The reduced water potential imposes a large dehydration force on the intracellular solution (28) and explains why freezing and dehydration stresses share a physical–chemical property and involve similar biological responses.

Thus, the freezing sensitivity of hos10-1 plants coupled with their salt sensitivity and water imbalance argues that the primary physiological lesion of this mutation is the inability to adjust to dehydration stress. Such impairment could understandably be manifested through the inability of hos10-1 plants to normally accumulate ABA in response to dehydration as seen in Fig. 5B. Increased ABA accumulation during cold treatment has been correlated with acclimation ability between species (29, 30). In addition, mutants impaired in ABA biosynthesis have moderately altered freezing tolerance (31, 32), although Thomashow (22) has argued that this could be the result of pleiotropic effects. Measurement of bulk ABA in plants did not reveal any changes in ABA content in hos10-1 plants different from wild-type plants during cold acclimation (Fig. 5C). However, the low level of ABA during cold acclimation makes accurate measurement difficult. Furthermore, the bulk measurements would obscure differences in ABA content in specific tissues or cells that may be critical for freezing tolerance.

Reduction in dehydration-induced ABA accumulation coupled with the inability of hos10-1 plants to fully induce NCED3 expression strongly implies that HOS10 may encode a transcription factor that controls the increased expression of ABA biosynthesis genes during stress (33). As such, HOS10 could be a crucial gene controlling an important aspect (ABA amplification) of stress responses. It is well established that partial dehydration can act like a low-temperature treatment and induce cold acclimation in plants that may link ABA to cold acclimation (21). In fact, treatment with exogenous ABA is also able to lead to freezing tolerance, sometimes very rapidly, and to levels near those induced by cold treatment (21). In view of these observations, the hos10-1 mutation could prove very useful in further studies of the connection between freezing tolerance and ABA. Because hos10-1 plants appear to be impaired only in the ability to increase ABA in response to dehydration stress and may actually be deficient only in an ABA-mediated amplification cycle (34), important insights into the complex role of ABA in several stress responses may be gained with additional experiments with hos10-1. A particular allele of NCED3 (35) also displays reduced ability to accumulate ABA after osmotic stress, but its ability to tolerate or acclimate to cold is unknown. These special mutant alleles of genes specifically controlling ABA accumulation after stress clearly show that mutants with altered ABA accumulation are not phenotypically the same and many more such mutants are needed to fully understand the roles of ABA in growth and development during stress responses.

Supplementary Material

Supporting Tables:

Acknowledgments

We thank the researchers who have generated and donated the SALK T-DNA insertional lines used in this work (36). This work was supported by National Science Foundation Grants DBI9813360, IBN-0212346, and MCB-0241450 and by United States Department of Agriculture National Research Initiative Grant 2003-00751. This is Purdue University Agricultural Program Paper 2005-17644.

Notes

Abbreviations: ABA, abscisic acid; ABRE, ABA responsive element; CRT, C-repeat; CBF, CRT binding factor; DRE, dehydration-responsive element; DREB, DRE-binding protein; PEG, polyethylene glycol.

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