). † These authors contributed equally to the work. SummaryThe properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.

In plants, low temperature causes massive transcriptional changes, many of which are presumed to be involved in the process of cold acclimation. Given the diversity of developmental and environmental factors between experiments, it is surprising that their influence on the identification of cold-responsive genes is largely unknown. A systematic investigation of genes responding to 1 d of cold treatment revealed that diurnal-and circadian-regulated genes are responsible for the majority of the substantial variation between experiments. This is contrary to the widespread assumption that these effects are eliminated using paired diurnal controls. To identify the molecular basis for this variation, we performed targeted expression analyses of diurnal and circadian time courses in Arabidopsis (Arabidopsis thaliana). We show that, after a short initial cold response, in diurnal conditions cold reduces the amplitude of cycles for clock components and dampens or disrupts the cycles of output genes, while in continuous light all cycles become arrhythmic. This means that genes identified as cold-responsive are dependent on the time of day the experiment was performed and that a control at normal temperature will not correct for this effect, as was postulated up to now. Time of day also affects the number and strength of expression changes for a large number of transcription factors, and this likely further contributes to experimental differences. This reveals that interactions between cold and diurnal regulation are major factors in shaping the cold-responsive transcriptome and thus will be an important consideration in future experiments to dissect transcriptional regulatory networks controlling cold acclimation. In addition, our data revealed differential effects of cold on circadian output genes and a unique regulation of an oscillator component, suggesting that cold treatment could also be an important tool to probe circadian and diurnal regulatory mechanisms.

Low temperature negatively affects plant growth and metabolism. Plant responses to cold involve massive transcriptional changes, and much effort has been made to identify these changes and their contribution to freezing tolerance. However, the influence of differences in environmental and developmental factors between experiments had not been investigated. We found that diurnal-and circadian-regulated genes are responsible for the majority of variation between experiments. Moreover, we demonstrated that the cyclic expression pattern of circadian clock components is affected by cold and that the cold induction of many transcription factors is dependent on the time of day. This means that genes identified so far as cold responsive are dependent on the time of day the experiment was performed and that paired diurnal controls are not sufficient to correct for this effect. Ongoing work to dissect the biological relevance of cold-diurnal regulatory interactions demonstrated that some circadian mutants have altered freezing tolerance but that time-of-day appears not to affect freezing tolerance. Influence of Circadian and Diurnal Regulation on the Identification of Cold-Responsive GenesExtending our previous analyses of cold responsive transcripts, 1,2 we used microarray data from public sources as well as from our own experiments and identified massive differences in cold-responsive genes between independent studies. Diurnally regulated genes were the dominant source of these variations, highlighting that measures taken to minimize or eliminate the effect of diurnal or circadian regulation are insufficient.To gain broader insights into the molecular basis of the interaction between cold and the circadian clock, we analyzed the expression of clock components and clock output genes during cold treatments (Fig. 1). The majority of oscillator components, after an initial cold response, showed diurnal cycles with dramatically reduced amplitude but similar peak expression in cold as under control conditions. Under circadian conditions in the cold, cycles stopped. Paired controls are insufficient to avoid circadian or diurnal variations because genes in control samples show normal amplitude cycles, thus diurnal and circadian regulated genes will clearly exhibit differences in relative changes in gene expression. Interestingly, LUX expression was maintained at the same amplitude under normal and cold conditions pointing to a unique regulation among the measured components. The probable importance of LUX is supported by the atypical arrhythmic phenotype of the single mutant. 3,4 Temperature compensation of circadian clocks allows the maintenance of robust rhythms over a broad range of physiological temperatures. It has been shown that a balance between CCA1 and GI has a relevant role on this mechanism at lower temperatures down to 12°C. 5 In our cold treatment, GI diurnal cycles decrease their amplitude almost completely, and in circadian conditions GI expression increases and stops to cycle. Since GI was implicated in both co...