Effects of chronic cold treatment on root elongation and gene expression in "Arabidopsis thaliana"

Low temperature is a major limitation of plant growth. Cold adaptation is important for the survival and distribution of plant species at high elevations and high latitudes. Much is known about the molecular basis for cold acclimation and freezing tolerance, which are triggered by acute cold treatment. The causes of growth limitation at low, non-freezing temperatures are largely unexplored. To better understand the mechanisms limiting plant growth in cold environments, I studied the elongation-growth of roots and patterns of gene expression in Arabidopsis accessions from diverse habitats. Arabidopsis thaliana (L.) Heyhn is a small, annual weed that is widely distributed in different growth environments and is well-suited for molecular genetic studies. My initial study of 23 accessions failed to detect ecotypic differentiation for root elongation rates at low, nonfreezing temperatures (10 °C); however, evidence was obtained implicating the cell-cycle gene CYCB1;1 as part of a compensatory mechanism for maintaining proliferation under these conditions. I used microarray technology to obtain a global picture of cold-responsive gene expression in the temperate Col-0 accession and the high-altitude (3400 m) Sha accession, which is expected to be adapted for a cold environment. I compared the effects of acute-cold treatment (4 h at 10 °C) and chronic-cold treatment (6 weeks at 10 °C) using plants grown at 21 °C as a control. Cold-treatment had major effects on gene expression at the mRNA level: 11% of the 24,000 genes represented on the Affymetrix ATH1 GeneChip responded by at least 2-fold to either or both cold treatments. A substantial fraction of cold-responsive genes, 35%, responded specifically to chronic cold treatment. This suggests there are fundamental differences in the response of plants to acute-cold treatment and growth at low, nonfreezing temperatures. Datasets of annotated genes were screened for significant, non-redundant enrichment for Gene Ontology (GO) terms to identify functional groups and processes. GO-term enrichment provided a rough picture of major trends in gene expression associated with coldresponses, which were then verified by examining the expression patterns of individual genes. Flavonoid biosynthesis, particularly the activation of anthocyanin biosynthesis, was the only major function induced by both acute- and chronic-cold treatment. In contrast, genes concerned with electron transport and light-reactions in photosynthesis were repressed by both cold treatments. This is consistent with the well-documented, general reduction of these functions associated with growth at low temperatures. Thus,
regulation at the mRNA level appears to be an important mechanism for down-regulating
energy metabolism in cold environments.
Acute-cold treatment induced numerous genes concerned with responses to
pathogen infection, cold, drought, salt stress, and UV damage. The breadth of these
stress responses emphasizes that brief exposure to cold, even at temperatures as high
as 10 °C, is perceived by plants as a form of stress. Unexpectedly, global induction of
stress-related genes was restricted primarily to the acute-cold response. This strongly
suggests that in contrast to “cold shock,” growth at low, non-freezing temperatures is not
recognized by Arabidopsis plants as a stress per se. Therefore, mechanisms exist for
suppressing prolonged stress responses in the cold. This implies that general stress
responses are not essential for growth of Arabidopsis at low temperatures.
Several other processes and pathways responded primarily to chronic- cold
treatment and are likely to be relevant to growth at low temperatures. Sha-specific,
chronic-cold induction of genes encoding ion transporters; genes concerned with
compensation for Pi deprivation; and, genes required for formation of root hairs,
comprised the only major functional group showing ecotypic differentiation. Induction of
genes encoding primary wall constituents and enzymes concerned with cell enlargement
and pectin metabolism were induced specifically by chronic-cold treatment, while those
genes important for secondary wall formation such as those encoding cellulose synthase
and laccase required for lignification were repressed. These findings and the coldrepression
of genes concerned with fiber and vascular tissue formation suggest as a
working hypothesis that chronic cold treatment increases the flexibility of roots and cell
wall extensibility as a compensatory response to the reduced root growth in the cold. In
summary, the present study identified several functional groups of genes showing novel
regulation by chronic cold treatment. These findings provide the starting point for future
studies using informative mutants and biochemical profiling to establish causal
relationships between gene expression and adaptations for growth in cold environments.