Abstract
Stress acclimation in Arabidopsis involves rapid stress-induced expression of the genes coding for the dehydration responsive element binding factor (DREB) transcription activators. The members of this factor family (DREB1 and DREB2) then activate the expression of targeted genes (e.g. COR78) that ultimately increase stress tolerance. This discovery has led to some fundamental questions about stress tolerance regarding conservation of the regulatory sequences and its extent, and environmental control of the factors in other dicotyledonous and monocotyledonous plant systems. Plants were grown under control environments or acclimated to cold (6/2ºC), salt (daily exposure to 75 or 150 mM NaCl) or drought.

Southern analyses using DREB1B (a member of the DREB1B family) as probe indicated hybridizing signals from rice, wheat, barley, A. desertorum, L. elongatum, oilseed rape, Brussels sprout, cauliflower and spinach (Spinacia oleracea) showing that counterparts of the DREB1B of the sequences may be present in these species. In contrast only cauliflower, oilseed rape, and wheat showed signals when DREB2A (a member of the DREB2 family) was used as probe. These results indicate that the DREB1B sequences may be more widely distributed across the monocot and dicot boundaries than DREB2A sequences or that sequence divergence is greater in genes of the latter type.

Sequences corresponding to the Arabidopsis DREB1B gene were cloned from rice, wheat, barley, A. desertorum, oilseed rape, cauliflower and spinach after amplification of the target fragment by PCR. The clone obtained from rice did not show any similarity whereas all other clones were nearly identical to the reference gene. This kind of similarity could be due to contamination. Consequently, a genomic library of A. desertorum was screened and six putative clones were isolated. Further analysis of the clones did not show any positive result.

In Arabidopsis, expression studies carried out by RT-PCR indicated that the DREB1B gene was invariably up-regulated by cold but expression was also seen from some samples acclimated to salt and drought stress, particularly from crowns. In leaf samples, the up-regulation was found to follow a bimodal pattern, the first peak in two-hour (higher level) and the second in three-day cold-acclimated samples. In the crowns the pattern of expression was unimodal and peak expression occurred at six hours and one day of acclimation to cold, indicating a slower response to cold in the crowns compared to the leaves. Constitutive expression of the gene was also observed from the crowns. Transcripts of DREB1B were detected by northern analysis in leaf and crown samples acclimated to cold for 2 hours. Apparently the peak expression was found in the crowns acclimated for six hours. A down-regulation of the target mRNA species was found in plant samples (leaf and crown) acclimated to cold while covered with cling film compared to the samples acclimated to cold uncovered. The other transcription factor gene tested, DREB2A, also showed differential expression in different plant organs. Roots showed a high constitutive expression whereas salt-acclimated leaves and drought-acclimated crowns showed the highest transcript levels. Except for two-hour cold-acclimated crowns, the DREB2A mRNA sequences were detected in all the test organs (roots, crowns and leaves) acclimated to cold. It was found by northern analysis that the root, leaf and crown samples, either acclimated to salt or drought, showed an up-regulation of the gene, although there were differences in the level of expression. However, a higher level of transcripts was shown by six-hour cold-acclimated crown and leaf samples compared to the samples acclimated to salt and drought. Thus crowns appear as responding organs to cold acclimation in Arabidopsis. For DREB1B a down-regulation of the transcripts was found in plant samples (leaf and crown) acclimated to cold while covered with cling film. Thus covering the plants with cling film has an impact in down-regulating both DREB1B and DREB2A and it may be a due to reduced dehydration of the covered plants.

Northern analyses indicated that sucrose up-regulates expression of the functional genes nsLTP and adsr36 in cultured barley crowns. At 10 g/l sucrose the level of expression was highest at warm (25/15ºC) and cold (6/2ºC) temperatures in the barley crowns, respectively, compared to other treatments (non-acclimated control, 1 g/l or 30 g/l). Therefore, this sugar may have a role in inducing the expression of genes for the stress acclimation process in crowns.