www.czu.cz ; www.raupp.info

The genetic basis for plant cold tolerance, that is, which genes are needed
to convey cold tolerance to plants, has been a long standing goal of plant
scientists. Two general molecular genetic approaches toward achieving this
goal have been implemented in the past decade or two using the model plant
Arabidopsis, October 2004 by Ray A. Bressan, Jianhua Zhu, Paul M. Hasegawa .

First, directly screening for plants with altered freezing tolerance has
yielded several mutants such as the sfr mutants which are sensitive to
freezing (1) and the eskimo mutant (2) that displays increased freezing
tolerance. So far, few of the genes responsible for these altered freezing
phenotypes have been identified. Only recently has one of the SFR mutant
genes, sfr2, been cloned (3). Another approach utilizing the identification
of genes that have altered expression after exposure to cold temperatures
such as the kin (cold induced), lti (low temperature induced) and cor (cold
regulated) genes has resulted in the identification and cloning of more
genes that are required for cold tolerance.

After the identification of the cold/desiccation induction recognition
sequence motifs called CRT/DRE (C repeat/dehydration responsive element) in
the promoters of cor target gene (4), Stockinger et al. (5) identified the
CBF1 (cor binding factor) gene that encodes an APETELA2 /ethylene response
element class transcription factor that regulates the expression of cor
target genes by binding to the CRT/DRE promoter region. Later, using yeast
one-hybrid screening, other members of the CBF transcription factor family
were identified and called DRE binding (DREB) proteins (6).

Overexpression of a number of the CBF transcription factor genes in
Arabidopsis has resulted in increased stress tolerance (7). A modification
of this approach toward identifying cold stress tolerance genes has utilized
the CRT/DRE-containing promoter of the cor gene RD29A fused to the
LUCIFERASE marker gene (8). Genes that affect cold tolerance, such as HOS1,
LOS2, and HOS2 (9), have been identified by using this luciferase marker
strategy. Some of the genes that affect acclimation to cold appear to
function as regulators of transcription. HOS1 is a negative regulator of
genes that are targeted by CBF (9) and appears to act through a
transcription factor called ICE which controls CBF gene expression by
binding to the CBF promoter (10). The central role of the CBF family of
genes in cold tolerance has been highlighted by the absence of any known
genes (identified sequences) that affect cold tolerance without affecting
expression of the CBF gene family. Because the CBF genes are not expressed
before cold treatment (7), the identification of mutants in genes such as
ESKIMO that directly increase cold tolerance without cold treatment and
subsequent induction of expression of CBF genes, and the known occurrence of
cold-induced target genes outside of the CBF regulon (11), have indicated
that there should exist cold tolerance signal pathway(s) independent of the
CBF mediated regulon. Using the RD29A::LUCIFERASE screening strategy, Zhu et
al. (11) have recently reported the existence of such a pathway that is
mediated by the HOS9 gene product.

HOS9 encodes a putative homeodomain family transcription factor. Zhu et al.
showed that the protein encoded by HOS9 when fused to GFP protein is
localized to the nucleus. Using microarray analysis they also demonstrated
that the HOS9 gene controls the expression of about 175 gene targets that
appear not to be in the CBF regulon. Also, 41 of the genes targeted by HOS9
were reported to be cold-induced. Thus, HOS9 appears to encode a nuclear
factor that acts, at least in part, separately from the CBF family and its
target genes to mediate cold-induced cold acclimation in Arabidopsis. The
hos9 mutation led to the alteration of other characteristics including
trichome development, growth rate, and flowering time. Interestingly, both
growth rate and flowering time are also altered in CBF-overexpressing
transgenic plants (7).

Although it is known that CBF affects growth through a gibberillic
acid-mediated mechanism, the mechanism by which HOS9 controls growth is
unknown. Thus it appears that both CBF-mediated and HOS9-mediated pathways
control cold acclimation, growth rate and flowering time. Growth rate, and
flowering time are phenological traits that are associated with stress
avoidance mechanisms (12). The consistent alteration in growth rate and
flowering time in mutants with altered stress tolerance indicates that these
are likely to be important traits that also affect cold acclimation and
tolerance and need to be coordinated by cross-talking signal pathways. Like
CBF genes, HOS9 has low expression in flower tissues that are particularly
vulnerable to cold temperature, especially in valuable fruit crops such as
citrus and stonefruit species.

The strategy of screening mutants in a genetic background containing a
cold-inducible promoter (R929A or CBF) fused to the luciferase marker has
been remarkably successful in finding genes that control cold acclimation
and should result in the discovery of other components of the cold
acclimation signal pathway system.

References

1. Warren G, McKown R, Martin AL, Teutonico V (1996) Isolation of mutations
affecting the development of freezing tolerance in Arabidopsis thaliana (L.)
Heynh. Plant Physiol 111:1011-1019

2. Xin Z, Browse J (1998) eskimo1 mutants of Arabidopsis are constitutively
freezing-tolerant. Proc Natl Acad Sci USA 95: 7799-7804

3. Thorlby G, Fourrier N, Warren G (2004) The SENSITIVE TO FREEZING2 gene,
required for freezing tolerance in Arabidopsis thaliana, encodes a
?-glucosidase. Plant Cell 16: 2192-2203

4. Yamaguchi-Shinozaki K, 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

5. Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1
encodes an AP2 domain-containing transcription 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

6. Liu Q et al. (1998) Two transcription factors, DREB1 and DREB2, with an
EREBP/AP2 DNA binding domain, separate two cellular signal transduction
pathways in drought- and low temperature-responsive gene expression,
respectively, in Arabidopsis. Plant Cell 10: 1391-1406

7. Thomashow MF (2001) So what's new in the field of plant cold acclimation?
Lots! Plant Physiol 125: 89-93

8. Ishitani M et al. (1998) HOS1, a genetic locus involved in
cold-responsive gene expression in Arabidopsis. Plant Cell 10: 1151-1161

9. Viswanathan C, Zhu J-K (2002) Molecular genetic analysis of
cold-regulated gene transcription. Phil Trans R Soc Lond B 357: 877-886

10. Chinnusamy V et al. (2003) ICE1: a regulator of cold-induced
transcriptome and freezing tolerance in Arabidopsis. Genes & Dev 17:
1043-1054

11. Zhu J t al. (2004) An Arabidopsis homeodomain transcription factor gene,
HOS9, mediates cold tolerance through a CBF-independent pathway. Proc Natl
Acad Sci (USA) 101: 9873-9878

12. Maggio A, Joly RJ, Hasegawa PM, Bressan RA (2003) Can the quest for
drought tolerant crops avoid Arabidopsis any longer? In: (Goyal SS, Sharma
SK, Rains DW, eds) rnal of Crop Production The Haworth Press, Inc. Vol 7,
No. 1/2, pp. 99-129

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