SIZ1 is a SUMO E3 ligase that facilitates conjugation of SUMO to protein substrates. siz1-2 and siz1-3 T-DNA insertion alleles that caused freezing and chilling sensitivities were complemented genetically by expressing SIZ1, indicating that the SIZ1 is a controller of low temperature adaptation in plants. Cold-induced expression of CBF/DREB1, particularly of CBF3/ DREB1A, and of the regulon genes was repressed by siz1. siz1 did not affect expression of ICE1, which encodes a MYC transcription factor that is a controller of CBF3/DREB1A. A K393R substitution in ICE1 [ICE1(K393R)] blocked SIZ1-mediated sumoylation in vitro and in protoplasts identifying the K393 residue as the principal site of SUMO conjugation. SIZ1-dependent sumoylation of ICE1 in protoplasts was moderately induced by cold. Sumoylation of recombinant ICE1 reduced polyubiquitination of the protein in vitro. ICE1(K393R) expression in wild-type plants repressed cold-induced CBF3/DREB1A expression and increased freezing sensitivity. Furthermore, expression of ICE1(K393R) induced transcript accumulation of MYB15, which encodes a MYB transcription factor that is a negative regulator of CBF/DREB1. SIZ1-dependent sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1A and repression of MYB15, leading to low temperature tolerance.

To investigate essential components mediating stress signaling in plants, we initiated a large-scale stress response screen using Arabidopsis plants carrying the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter. Here we report the identification and characterization of a mutant, hos9-1 (for high expression of osmotically responsive genes), in which the reporter construct was hyperactivated by low temperature, but not by abscisic acid or salinity stress. The mutants grow more slowly, and flower later, than do wild-type plants and are more sensitive to freezing, both before and after acclimation, than the wild-type plants. The HOS9 gene encodes a putative homeodomain transcription factor that is localized to the nucleus. HOS9 is constitutively expressed and not further induced by cold stress. Cold treatment increased the level of transcripts of the endogenous RD29A, and some other stress-responsive genes, to a higher level in hos9-1 than in wild-type plants. However, the C repeat͞ dehydration responsive element-binding factor (CBF) transcription factor genes that mediate a part of cold acclimation in Arabidopsis did not have their response to cold altered by the hos9-1 mutation. Correspondingly, microarray analysis showed that none of the genes affected by the hos9-1 mutation are controlled by the CBF family. Together, these results suggest that HOS9 is important for plant growth and development, and for a part of freezing tolerance, by affecting the activity of genes independent of the CBF pathway.

Carbohydrate composition changed seasonally in red osier dogwood (Cornus sericea L.) stem tissues. Starch concentration was highest in fall and decreased to a minimum in midwinter. Coincident with the breakdown of starch in fall, there was an increase in the concentrations of soluble sugars. Soluble sugars were present in highest concentrations in midwinter. Glucose, fructose, sucrose, and raffinose were the predominant soluble sugars present in both bark and wood tissues. In early spring, the soluble sugar concentration decreased and the concentration of starch increased. The seasonal interchange between sugars and starch did not simply reflect a general quantitative shift in the balance between sugars and starch because qualitative changes in soluble sugars were also noted. The most striking changes involved the trisaccharide raffinose. Raffinose was barely detectable in summer and early fall, but increased to one fifth and one third of the total soluble sugars in January samples of bark and wood tissues, respectively. The potential physiological role of raffinose in overwintering red osier dogwood tissue is discussed.

;A predominant 24-kD dehydrin-like protein was previously found to fluctuate seasonally within red-osier dogwood (Cornus sericea L.) stems. The current study attempted to determine what environmental cues triggered the accumulation of the 24-kD protein and to assess its potential role in winter survival. Controlled photoperiod and field studies confirmed that photoperiod regulates a reduction of stem water content (SWC), freeze-tolerance enhancement and accumulation of the 24-kD protein.Diverse climatic ecotypes, which are known to respond to different critical photoperiods, displayed differential reduction of SWC and accumulation of the 24-kD protein. A time-course study confirmed that prolonged exposure to short days is essential for SWC reduction, 24-kD protein accumulation, and freeze-tolerance enhancement. Water deficit induced 24-kD protein accumulation and enhanced freeze-tolerance under long-day conditions. In all instances, freeze-tolerance enhancement and 24-kD protein accumulation was preceded by a reduction of SWC. These results are consistent with the hypothesis that C. sericea responds to decreasing photoperiod, which triggers a reduction in SWC. Reduced SWC in turn may trigger the accumulation of the 24-kD protein and a parallel increase in freezetolerance.

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue-or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue-or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.Due to their sessile nature, plants have been forced to adapt to the dynamic environmental conditions that surround them. Temperature creates a selective pressure on plants growing in temperate climates and has affected their geographical distribution based upon a capacity to survive seasonal thermal fluctuations (Smithberg and Weiser, 1968;Sakai and Weiser, 1973;George et al., 1974;Becwar et al., 1981;Gusta et al., 1983). In woody plants, two distinct and fundamentally different strategies for the seasonal survival of subzero temperatures have evolved: freeze tolerance (nonsupercooling) and freeze avoidance (supercooling; Burke et al., 1976;George et al., 1982). Freezing behavior strategies employed by a woody plant vary from tissue to tissue and are species specific. For example, cortical tissues are strictly nonsupercooling; however, buds and xylem ray parenchyma may exhibit either strategy. In nonsupercooling tissues, ice formation is initiated within extracellular spaces and generates a dehydrative vapor pressure gradient between extracellular ice and intracellular water. Nonsupercooling cells readily desiccate in response to extracellular ice formation (George et al., 1982; and are capable of surviving low temperature extremes (Guy et al., 1986) due to an inherent capacity to tolerate desiccation Fujikawa et al., 1997). In supercooling tissues, ice may also initiate in extracellular spaces; however, cells are thought to resist intracellular desiccation (Burke et al., 1976;George et al., 1982;Wisniewski and Ashworth, 1985;Fujik...