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Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis.

Espinoza C, Degenkolbe T, Caldana C, Zuther E, Leisse A, Willmitzer L, Hincha DK, Hannah MA - PLoS ONE (2010)

Bottom Line: Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation.However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms.These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Molecular Plant Physiology, Potsdam, Germany.

ABSTRACT
In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C) affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

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Experimental design of diurnal and circadian time series.Whole rosettes were harvested at the indicated ZT (zeitgeber time, in hours) and used for transcript and metabolite profiling. Temperature and light conditions are indicated. White, black and grey bars indicate the corresponding day, night and subjective night periods.
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pone-0014101-g001: Experimental design of diurnal and circadian time series.Whole rosettes were harvested at the indicated ZT (zeitgeber time, in hours) and used for transcript and metabolite profiling. Temperature and light conditions are indicated. White, black and grey bars indicate the corresponding day, night and subjective night periods.

Mentions: Metabolite and transcript profiling were performed on soil-grown plants (standard compost mix – see Methods), sampled from four different conditions. Time series were harvested under light-dark cycles (L/D; 16/8 h) and continuous light (L/L, 24 h) at control temperature (20°C) and after transfer to 4°C for cold acclimation. Long-day conditions were used for consistency with previous studies of metabolic changes associated with cold acclimation [4], [5], [7], [14] and previous work on interactions between cold and the circadian clock [1], [23], [24]. These studies, including our own work, indicate that under these experimental conditions Arabidopsis cold acclimates, metabolic changes occur and the expression of clock components is disrupted. The experiment was started 2 h before (subjective) dusk and leaves were sampled at time 0 (ZT14) and after 2 h (to coincide with (subjective) light-dark transitions) and then every 4 h until 58 h (ZT72) (Figure 1). Previous analyses already demonstrated that gene expression becomes arrhythmic under L/L in the cold [1]. Therefore, the L/L 4°C time series was not analyzed by transcript profiling, while all four conditions were analyzed by metabolite profiling, yielding a total of seven time course datasets. To identify transcripts and metabolites under circadian control we used the subset of our L/L 20°C condition from 10 h to 58 h (corresponds to Zeitgeber Time, ZT24 – ZT72), in agreement with previous studies [19]. It should be noted that in the context of circadian regulation our 4°C conditions include the adjustment phase, where cycles observed from 0 h to 24 h could still be attributed to free-running cycles established before transfer. Transcript profiling was performed using Affymetrix ATH1 arrays and metabolite profiling of polar metabolites by GC-MS. Following normalization and filtering (see Methods) our final dataset included transcripts corresponding to 14874 genes and 50 metabolites. In addition, starch content was determined by an enzymatic assay.


Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis.

Espinoza C, Degenkolbe T, Caldana C, Zuther E, Leisse A, Willmitzer L, Hincha DK, Hannah MA - PLoS ONE (2010)

Experimental design of diurnal and circadian time series.Whole rosettes were harvested at the indicated ZT (zeitgeber time, in hours) and used for transcript and metabolite profiling. Temperature and light conditions are indicated. White, black and grey bars indicate the corresponding day, night and subjective night periods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990718&req=5

pone-0014101-g001: Experimental design of diurnal and circadian time series.Whole rosettes were harvested at the indicated ZT (zeitgeber time, in hours) and used for transcript and metabolite profiling. Temperature and light conditions are indicated. White, black and grey bars indicate the corresponding day, night and subjective night periods.
Mentions: Metabolite and transcript profiling were performed on soil-grown plants (standard compost mix – see Methods), sampled from four different conditions. Time series were harvested under light-dark cycles (L/D; 16/8 h) and continuous light (L/L, 24 h) at control temperature (20°C) and after transfer to 4°C for cold acclimation. Long-day conditions were used for consistency with previous studies of metabolic changes associated with cold acclimation [4], [5], [7], [14] and previous work on interactions between cold and the circadian clock [1], [23], [24]. These studies, including our own work, indicate that under these experimental conditions Arabidopsis cold acclimates, metabolic changes occur and the expression of clock components is disrupted. The experiment was started 2 h before (subjective) dusk and leaves were sampled at time 0 (ZT14) and after 2 h (to coincide with (subjective) light-dark transitions) and then every 4 h until 58 h (ZT72) (Figure 1). Previous analyses already demonstrated that gene expression becomes arrhythmic under L/L in the cold [1]. Therefore, the L/L 4°C time series was not analyzed by transcript profiling, while all four conditions were analyzed by metabolite profiling, yielding a total of seven time course datasets. To identify transcripts and metabolites under circadian control we used the subset of our L/L 20°C condition from 10 h to 58 h (corresponds to Zeitgeber Time, ZT24 – ZT72), in agreement with previous studies [19]. It should be noted that in the context of circadian regulation our 4°C conditions include the adjustment phase, where cycles observed from 0 h to 24 h could still be attributed to free-running cycles established before transfer. Transcript profiling was performed using Affymetrix ATH1 arrays and metabolite profiling of polar metabolites by GC-MS. Following normalization and filtering (see Methods) our final dataset included transcripts corresponding to 14874 genes and 50 metabolites. In addition, starch content was determined by an enzymatic assay.

Bottom Line: Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation.However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms.These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Molecular Plant Physiology, Potsdam, Germany.

ABSTRACT
In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C) affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

Show MeSH
Related in: MedlinePlus