Chapter 3 shows that the acute cold induction of key components in the CBF pathway described earlier for laboratory strains of Arabidopsis (Gilmour et al. 1998; Ishitani et al. 1998; Liu et al. 1998; Jaglo et al. 2001; Zarka et al. 2003) occurs in diverse natural accessions (Fig. 3.4) and that this pathway is also active in roots of plants raised at low temperature (Fig. 3.6). Evidence suggests that cold acclimation in laboratory strains of Arabidopsis depends on the activation of multiple low-temperature regulatory pathways (Kreps et al. 2002; Seki et al. 2001, 2002; Vogel et al. 2005). Downstream CBF-pathway genes are not expressed in the constitutively freezing-tolerant eskimo1 mutant (Xin & Browse 1998) and certain cold responsive genes do not depend on the CBF pathway (Fowler & Thomashow 2002). The function of additional cold perception pathways might provide an explanation for the weak cold induction of downstream COR15a and RD29A genes in the high-altitude accessions 3661 and 3658.
Cold induction of the CBF-pathway genes (Fig 3.6A, 3.6C) and root (Fig. 3.1) elongation was usually more rapid and pronounced in 21 °C/10 °C plants than in plants grown continuously at 10 °C. This indicates that temperature early in seedling development affects the subsequent response of roots to chronic cold treatment. Similar effects of temperature shifts have been reported for physiological responses of Arabidopsis leaves. Whereas mesophyll cells of leaves formed at low temperature showed increased cytoplasmic volume and density, these changes were minimal in temperature shifted leaves. Leaves formed at a low temperature exhibited greater changes in the activity of Calvin Cycle enzymes and accumulation of sucrose than did temperature shifted leaves(Strand et al. 1999).
CBF2 was the only CBF gene appreciably induced in roots in response to chronic cold (Fig. 3.6A, 3.6C). CBF2 is believed to serve a dual function: it appears to down regulate CBF1 and CBF3, and transcriptionally activates genes downstream in the CBF pathway ((Novillo et al. 2004). Growth is inhibited by overexpression of CBF1, CBF2, or CBF3 in transgenic Arabidopsis (Gilmour et al. 2004). These findings together with my results suggest that expression of CBF2 in chronic cold regulates the expression of
CBF1 and CBF3 and that this affects the balance between growth and cold tolerance. Chronic-cold induced of CBF2 may thus, not only explain cold tolerance but also contribute to the overall slow growth rate at low temperature.
Root elongation at low temperature was correlated with expression of RD29A mRNA suggests that chronic activation of the CBF pathway could help facilitate growth in cold environments. Nevertheless, we found neither appreciable constitutive expression of CBF genes nor a correlation of expression levels in accessions from cold habitats. Therefore, while there is natural variation in different accessions, constitutive activity of the CBF-pathway and chronic cold activation of this pathway do not appear to play a major role in determining the distribution of the accessions tested in cold habitats.
I also examined the possibility that cold-induced expression of cell-cycle genes has a role in regulating root growth at low temperatures. The elongation of primary roots depends on a combination of cell production in the meristem and expansion of cortical cells in the elongation zone (Beemster et al. 2002). CDKA;1 and CYCD2;1 mRNAs are constitutively expressed in the meristem, the elongation zone, and mature zone of warm grown Col-0 root (Birnbaum et al. 2003; Beemster et al. 2005). Cell production rates in different warm-grown Arabidopsis accessions are strongly correlated with the activity of CDKA;1 in roots suggesting that CDKA;1 activity, but not CDKA;1 mRNA is a factor limiting root growth in natural populations. In most accessions I studied, including the Col-0, Ler-0, and Est-0 accessions investigated by Beemster et al. (2002), neither acute or chronic cold treatment increased CDKA;1 or CYCD2;1 mRNA expression. The only exception was the high altitude accessions 3661 and 3658, which exhibited a consistent induction of CDKA;1 mRNA by chronic cold treatment (Fig 3.8A). In no case, however, were expression levels of CDKA;1 or CYCD2;1 mRNA correlated either with root elongation rates or the habitat temperature of the accessions. Thus, expression of CDKA;1 and CYCD2;1 mRNAs are not likely to be growth-limiting factors in the accessions I studied.
CYCB1;1 mRNA is an established marker for cell proliferation and a limiting factor for growth of Arabidopsis roots (Doerner et al. 1996; Beemster et al. 2002; Inzé & De Veylder 2006). My most interesting finding was that CYCB1;1 mRNA consistently induced in roots by chronic but not acute cold in all the accessions studied (compare Fig. 3.8 with Fig. 3.7). Levels of CYCB1;1 mRNA were not, however, significantly correlated with the rate of root elongation at either 10 °C or 21 °C. This is in contrast to recent studies of developing maize leaves showing that low (4 °C) night temperature reduced
cycle in association with reduced growth and cell production (Rymen et al. 2007). While organ-specific differences in cold response cannot be ruled out, one possible explanation is that maize is a chilling-sensitive plant, whereas Arabidopsis has the capacity to acclimatize to cold (Allen & Ort 2001). Accumulation of CYCB1;1 mRNA in the entire root system depends on the expression level of CYCB1;1 in proliferating cells and the size of the meristematic compartment. CYCB1;1 mRNA accumulation was not correlated with the elongation rate of cold-grown roots relative to that of warm-grown roots. Thus, it is unlikely that the cold induction I observed reflects solely an increase in the relative amount of meristematic tissue in shorter, cold-grown roots. Growth at low, non-freezing temperatures is known to prolong the duration of the cell cycle (Francis & Barlow 1988), which appears to be compensated, at least in part, by an increase in the number of cells entering the cell cycle (Creber et al. 1993; Rymen et al. 2007). We speculate that cold is perceived by a signaling pathway that acts down stream of CYCB1;1 to inhibit root elongation. This increases CYCB1;1 expression by an as yet unknown mechanism, and, hence the size of the meristematic compartment. According to this view, induction of CYCB1;1 mRNA could be part of the compensation mechanism that helps maintain proliferation at low, non-freezing temperatures.