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. 2012 Jun;24(6):2443-69.
doi: 10.1105/tpc.112.097188. Epub 2012 Jun 26.

Metabolism and Growth in Arabidopsis Depend on the Daytime Temperature but Are Temperature-Compensated Against Cool Nights

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Metabolism and Growth in Arabidopsis Depend on the Daytime Temperature but Are Temperature-Compensated Against Cool Nights

Eva-Theresa Pyl et al. Plant Cell. .
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Abstract

Diurnal cycles provide a tractable system to study the response of metabolism and growth to fluctuating temperatures. We reasoned that the response to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature affects use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 °C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and an even larger inhibition of growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content. Low night temperature had no effect on protein content, starch turnover, or growth. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation is completely temperature compensated against even sudden changes in temperature, and polysome loading increases when the night temperature is decreased.

Figures

Figure 1.
Figure 1.
Rosette FW, Rosette DW, Protein Amounts, DWC , and SLA of Three Accessions Grown in Five Different Thermocycles. Rosette FW (A), rosette DW (B), protein amounts (C), DWC (D), and SLA (E). Bu-2, orange bars; Col-0, green bars; Lip-0, blue bars. Plants were grown in five different thermocycles: 12°C/12°C (day/night), 24°C/12°C, 16°C/16°C, 24°C/16°C, and 24°C/24°C. Data represent the mean ± sd (n = 4). For (C) and (D), light colors are for ED and dark ones for EN. One-way ANOVA was used to identify potential candidates for a statistically significant difference between treatments separately for each of the three accessions and two time points. After ANOVA P value correction using Holm’s method (P < 0.05), individual contrasts were then identified in a post-hoc Tukey HSD test (P < 0.05). Significant differences are indicated by different letters within the same time point (for [C] and [D], lowercase for ED and uppercase for EN). Original data are provided inSupplemental Data Set 2 online.
Figure 2.
Figure 2.
Metabolite Levels on a DW Basis in Plants Grown in Five Different Thermocycles. Bu-2, orange bars; Col-0, green bars; and Lip-0, blue bars. Determinations were made at ED (light color) and EN (dark color) for starch (A), total sugars (B), amino acids (C), organic acids (D), and total C turnover (E). Total C turnover is the sum of the C in starch, sugars, organic acids, and amino acids turned over during the night. Data represent the mean ± sd (n = 4). Error bars are absent for (E) as the calculation was based on average values. One-way ANOVA was used to identify potential candidates for a statistically significant difference between treatments separately for each of the three accessions and two time points. After ANOVA P value correction using Holm’s method (P < 0.05), individual contrasts were then identified in a post-hoc Tukey HSD test (P < 0.05). They are indicated by different letters within the same time point (ED, lowercase; EN, uppercase). Original data are provided inSupplemental Data Set 2 online.
Figure 3.
Figure 3.
Photosynthesis, Respiration, and Modeled Growth Rate in the Daytime and the Night on a DW Basis. (A) Photosynthesis (A, pale color) and respiration (R, dark color) were measured at the growth temperature in five different thermocycles. The rates are shown on a per day basis, after correcting for the length of the light period (8 h) and the night (16 h). The net diurnal C gain is the difference between A and R. (B) The RGR was estimated from the difference between biomass at harvest (Figure 2B) and biomass at transfer to the thermocycle treatments at 21 d. (C) Correlation coefficient between the net diurnal C gain and RGR. Note that C accounts for ∼42% of the DW, so the numbers on the y axis must be multiplied by 0.42 to allow a comparison of the absolute rate of use of C for growth. (D) Estimated rate of growth in the daytime and the night. The rate of growth in the day is estimated as A minus the sum of C accumulated in starch, sugars, organic acids, and amino acids (Figure 2E), divided by 0.42 (the proportion of C in DW). The rate of growth at night is estimated as the sum of C accumulated in starch, sugars, organic acids, and amino acids minus R, divided by 0.42. Data represents the mean ± sd (n = 4), with one replicate comprising five pooled rosette plants. Orange, Bu-2; green, Col-0; blue, Lip-0. For details of the calculations, see Figure 2A; seeTable 1 andSupplemental Data Set 3 online. One-way ANOVA was used to identify potential candidates for a statistically significant difference in A and R between treatments separately for each of the three accessions and two time points. After ANOVA P value correction using Holm’s method (P < 0.05), individual contrasts were then identified in a post-hoc Tukey HSD test (P < 0.05). They are indicated by different letters within the same time point (ED, lowercase; EN, uppercase). Error bars and significance tests are absent for (B) and (D), where the calculations are based on average values for A, R, and summed C.
Figure 4.
Figure 4.
Enzyme Activities and Protein Levels in Col-0 Grown in Different Thermocycles. (A) Activities of four representative primary metabolism enzymes, determined at ED (open bars) and EN (closed bars). Activities are given on a protein basis (mean ± sd, n = 4). Significant differences between treatments were identified by ANOVA, followed by P value correction using Holm’s method (P < 0.05) and selection in a post-hoc Tukey HSD test (P < 0.05), and are indicated by different letters within the same time point (ED, lowercase; EN, uppercase). Comparisons between ED and EN using a paired t test at a given thermocycle are indicated by an asterisk if significant (P < 0.05). The complete data set containing 17 enzymes is available in theSupplemental Figure 3 online and all data are available inSupplemental Data Set 2 online. (B) Qualitative variation in proteins comprised in seven major MapMan functional classes in rosettes of Col-0 plants grown in five different thermocycles. Data for each time point (ED, open bars; EN, closed bars) and thermocycle are expressed in mol % of proteins detected by LC-MS/MS. To get the relative abundance of each functional class at each time point and thermocycle, the relative abundance of each protein was summed. To calculate the standard deviations for each protein within a category, the global average across all thermocycles and harvest time points was calculated and all molar fractions were then expressed relative to this global average. By averaging these relative abundances (mol %) of all proteins belonging to a functional class, the standard deviation across all proteins within this functional class was determined and used to test for significant differences between all thermocycles and time points. Significant changes were identified as in (A) and are indicated by different letters within the same time point (ED, lowercase; EN, uppercase). Comparisons between ED and EN using a paired t test at a given thermocycle gave no significant differences. Graphs of three additional functional classes are shown in theSupplemental Figure 4 online. All raw data and information about the proteins analyzed are available inSupplemental Data Set 4 online. PS, photosynthesis.
Figure 5.
Figure 5.
Response of Transcripts Encoding Enzymes Involved in the Cycle of Glucan Phosphorylation and Dephosphorylation to a Decrease of the Night Temperature. GWD1 (A), GWD3/PWD (B), SEX4/PTPKIS1 (C), and LSF1/PTPKIS2 (D). Transcripts were measured by qRT-PCR in samples harvested at ED (open bars) and EN (closed bars) from plants growing in a 24°C/12°C, 16°C/16°C, 24°C/16°C, and 24°C/24°C thermocycle. Absolute quantification of transcripts was achieved by adding seven artificial RNA species at different concentrations before RNA purification. The results are the mean ± sd (n = 3) of the determined copy number per g DW. One-way ANOVA was used to identify potential candidates for a statistically significant difference between treatments separately for each of the two time points. After ANOVA P value correction using Holm’s method (P < 0.05), individual contrasts were then identified in a post-hoc Tukey HSD test (P < 0.05). They are indicated by different letters within the same time point (ED, lowercase; EN, uppercase). Comparisons between ED and EN using a paired t test at a given thermocycle are indicated by an asterisk if significant (P < 0.05). More data for further transcripts involved in starch metabolism is shown inSupplemental Figure 5 online.
Figure 6.
Figure 6.
Starch Metabolism after a Sudden Change in the Night Temperature. (A) to (C) Changes of starch and other metabolic parameters at EN and ED on successive days after transfer from 24°C/24°C to 12°C/12°C. Black and white bars at the top indicate light and dark periods, respectively. Col-0 (orange bars), Lip-0 (green bars), and Bu-2 (blue bars) growing in a 24°C/24°C thermocycle were transferred at the ED to a 12°C/12°C thermocycle. Samples were harvested at EN and ED on the days before the transfer, at EN on the first night after the transfer, and at ED and EN on the first, third, and seventh day after transfer and analyzed for starch (A), Suc (B), and total protein (C). SeeSupplemental Figure 6 online for displays of changes in other metabolites and enzyme activities andSupplemental Data Set 6 online for the original data. The results are given as mean ± sd (n = 3, each sample contains five individual plants). (D) and (E) Kinetics of starch depletion in Col-0 plants in the first night after reciprocal transfer between 24°C/24°C and 24°C/12°C. Plants were grown in a 24°C/24°C thermocycle and some transferred to 12°C at the ED (D). Other Col-0 plants were grown in a 24°C/12°C thermocycle (E) and some were transferred to 24°C at the ED (E). Plants were harvested from the control (nonshifted, shown as closed symbols) and shifted plants (open symbols) at various times during the night to determine the starch level. The results are on a DW basis given as the mean ± sd (n = 4, each sample contains five individual plants).
Figure 7.
Figure 7.
Starch and Phytoglycogen Breakdown in the isa1 Mutant. The isa1 mutant was grown in a 24°C/24°C, 24°C/16°C, or 24°C/12°C thermocycle and harvested at various times during the night to determine the level of phytoglycogen (A) and starch (B). The results are given as the mean ± sd (n = 4, each sample contains three individual plants). Black and white bars at the top indicate light and dark periods, respectively.
Figure 8.
Figure 8.
Polysome Loading Analysis. Polysome gradients were performed for Col-0 plants grown in five different thermocycles, harvested at ED (white bars) and EN (dark-gray bars). (A) Examples of the distribution of ribosomes in fractions collected from a density gradient obtained from plants harvested at EN and grown in a 24°C/24°C (left panel) and 24°C/12°C (right panel) thermocycle. RNA was measured as absorbance at 254 nm (A254). Free ribosomes and monosomes are on the left-hand side and increasingly large polysomes toward the right-hand side of the display. (B) Estimated proportion of ribosomes in polysomes. This is calculated as (PS)/(NP + PS). The percentage is given as numbers in the figure panel. The results are the mean ± sd (n = 3), except in the case of 12°C/12°C at EN, where n = 1. (C) Ribosome content, estimated from the sum of the ribosome profile at A254. The average of the ribosome number at ED and EN was calculated for each treatment. The results are given as mean ± sd (n = 4). One-way ANOVA was used to identify potential candidates for a statistically significant difference between treatments. After ANOVA P value correction using Holm’s method (P < 0.05), individual contrasts were then identified in a post-hoc Tukey HSD test (P < 0.05). They are indicated by different letters within the same time point (ED, lowercase; EN, uppercase). In the case of 12°C/12°C at EN, this analysis was not possible due to lack of replication (n = 1). Comparisons between ED and EN using a paired t test at a given thermocycle are indicated by an asterisk if significant (P < 0.05).
Figure 9.
Figure 9.
Schematic Representation of the Effect of Growth Temperature on the Metabolism and Growth of Arabidopsis. Pathways for C flow are indicated with solid lines, processes affected by a general increase in the protein content are indicated by dashed lines, and processes by clock-dependent regulation networks are indicated by dotted lines. Processes affected when the daytime (A) or the night (B) temperature is decreased are depicted in blue. External arrows indicate the direct effect of the decrease in temperature, and internal arrows the effect of acclimation of the protein content and adjustment of regulatory networks. The thickness of the lines, or the size of the letters, qualitatively depict the intensities. For simplicity, the effect of decreased maintenance respiration is not shown.

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