CIRCADIAN CLOCK ASSOCIATED1 ( CCA1) and the Circadian Control of Stomatal Aperture

Abstract

The endogenous circadian (∼24 h) system allows plants to anticipate and adapt to daily environmental changes. Stomatal aperture is one of the many processes under circadian control; stomatal opening and closing occurs under constant conditions, even in the absence of environmental cues. To understand the significance of circadian-mediated anticipation in stomatal opening, we have generated SGC (specifically guard cell) Arabidopsis (Arabidopsis thaliana) plants in which the oscillator gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1) was overexpressed under the control of the guard-cell-specific promoter, GC1. The SGC plants showed a loss of ability to open stomata in anticipation of daily dark-to-light changes and of circadian-mediated stomatal opening in constant light. We observed that under fully watered and mild drought conditions, SGC plants outperform wild type with larger leaf area and biomass. To investigate the molecular basis for circadian control of guard cell aperture, we used large-scale qRT-PCR to compare circadian oscillator gene expression in guard cells compared with the "average" whole-leaf oscillator and examined gene expression and stomatal aperture in several lines of plants with misexpressed CCA1 Our results show that the guard cell oscillator is different from the average plant oscillator. Moreover, the differences in guard cell oscillator function may be important for the correct regulation of photoperiod pathway genes that have previously been reported to control stomatal aperture. We conclude by showing that CONSTANS and FLOWERING LOCUS T, components of the photoperiod pathway that regulate flowering time, also control stomatal aperture in a daylength-dependent manner.

Figure 1.

CCA1 is overexpressed and arrhythmic in stomatal guard cells of CCA1-oxSGC plants. A, SGC24 (a line of CCA1-oxSGC) plants were grown for 2 weeks in LD and then imaged 6 h after lights-on by confocal microscopy. Light field, DAPI, YFP, and YFP + DAPI are shown. B and C, SGC24 and wild-type plants were grown for 4 weeks in LD before being transferred to LL. B, CCA1 levels in stomatal guard cells of SGC24 and the wild type. C, CCA1 levels also shown plotted for the wild type alone. B and C, The average of three independent biological repeats; the highest peak of CCA1/GDPH in the wild type of each repeat was normalized to 1. sd is shown, and the white and hatched bars represent subjective day and night, respectively.

Figure 2.

SGC plants do not show circadian rhythms of stomatal aperture regulation. A, Wild-type, SGC24, SGC2, and CCA1-ox plants were grown for 4 to 5 weeks in LD before samples were taken at ZT-0.5 (anticipation) or ZT2 (control, opening solution, and ABA) and treated as described in “Materials and Methods,” and stomatal aperture was measured. B to D, Wild-type, SGC24, and SGC2 plants were grown for 4 to 5 weeks in LD and either kept in LD (B and C) or transferred to LL (D). B and D, Stomatal aperture measurements. The white, hatched, and black bars represent light, subjective night, and dark, respectively. C, Leaf temperature measurements; each point represents the average leaf temperature/aluminum foil standard with the highest value normalized to 1, as described in “Materials and Methods,” for a single time point. A 12-point moving average is shown for each genotype. The gray and white regions represent dark and light. ***P < 0.001 (Student’s two-tailed t test).

Figure 3.

Nonstomatal circadian-controlled processes are less affected in SGC plants. A, CCA1-ox, SGC2, SGC24, and wild-type plants were grown for 5 d under low (25 µE m⁻² s⁻¹) white light or in the dark, and hypocotyl lengths were measured (n for each group = 12). B, CCA1-ox, SGC2, SGC24, and wild-type plants were grown in LD or SD, and flowering time was determined by counting the numbers of leaves at bolting (n for each group = 18–20). C, SGC2, SGC24, and wild-type plants were grown for 7 d in LD before being transferred to LL, and leaf movements were measured (n for each group = 6–16). Averages and se are shown.

Figure 4.

Under optimal conditions SGC plants grow larger than wild type. A to D, SGC2, SGC24, and wild-type plants were grown at 23°C for 4 weeks in LD on soil under optimal watering conditions. B, Fresh weight, C, dry weight, and D, photosynthetic area per plant were measured as described in “Materials and Methods.” B and C, The graphs are averages of two independent biological experiments. D, The results from three independent biological experiments were averaged and normalized to one by the wild type, and the se is shown. Student’s t test, **P < 0.01 (n for each genotype in each repeat 10–12).

Figure 5.

Growth parameters of SGC lines under mild drought stress. SGC2, SGC24, and wild-type plants were grown in fully watered conditions for 1 week then watered to 50% FC, 30% FC, or kept in fully watered conditions (control). A, The dry weights of the plants normalized to 100% by their fully watered controls. The percentage weight loss is calculated by comparison with the fully watered controls for each line. B, The average dry weights were calculated. The average for five biological independent experiments is shown with the se (n for each genotype in each repeat = 10–15). Student’s t test, *P < 0.05, ***P < 0.001.

Figure 6.

Expression of most oscillator genes is altered in guard cells. A to E, Wild-type plants were grown for 4 weeks in LD before being transferred to LL. The expression of (A) control genes KAT1, KAT2, and At4g14480, (B) CCA1, PRR7, PRR3, CHE, PRR5, RVE8, BOA, FKF1, and JMJD5, and (E) CO and GI were monitored in large-scale qRT-PCR arrays. E, FT expression was analyzed by small-scale RT-PCR. Expression levels were normalized to reference gene expression. The rest of the normalization results and the other circadian genes analyzed are shown in Supplemental Figure S7. C and D, the maximum expression level at the first peak for each of the rhythmic genes in both guard cells and whole leaves was calculated for all three reference genes (UBQ10, TUB, and GDPH) and averaged. The ratio of average levels in guard cells to average levels in whole leaves is shown together with the sem. C, The results for each gene. D, The allocation of genes into morning, afternoon, and evening categories was based on Mockler et al. (2007), and the average ratios of expression in guard cells to whole leaves was calculated for the genes in each category. Student’s t test, *P < 0.05. F, CO and FT expression in LD was analyzed by small-scale RT-PCR. The averages of two biological repeats and sd were calculated for each sample. The white, hatched, and black bars represent light, subjective night, and dark, respectively.

Figure 6.

Expression of most oscillator genes is altered in guard cells. A to E, Wild-type plants were grown for 4 weeks in LD before being transferred to LL. The expression of (A) control genes KAT1, KAT2, and At4g14480, (B) CCA1, PRR7, PRR3, CHE, PRR5, RVE8, BOA, FKF1, and JMJD5, and (E) CO and GI were monitored in large-scale qRT-PCR arrays. E, FT expression was analyzed by small-scale RT-PCR. Expression levels were normalized to reference gene expression. The rest of the normalization results and the other circadian genes analyzed are shown in Supplemental Figure S7. C and D, the maximum expression level at the first peak for each of the rhythmic genes in both guard cells and whole leaves was calculated for all three reference genes (UBQ10, TUB, and GDPH) and averaged. The ratio of average levels in guard cells to average levels in whole leaves is shown together with the sem. C, The results for each gene. D, The allocation of genes into morning, afternoon, and evening categories was based on Mockler et al. (2007), and the average ratios of expression in guard cells to whole leaves was calculated for the genes in each category. Student’s t test, *P < 0.05. F, CO and FT expression in LD was analyzed by small-scale RT-PCR. The averages of two biological repeats and sd were calculated for each sample. The white, hatched, and black bars represent light, subjective night, and dark, respectively.

Figure 7.

CCA1 levels affect FT expression and stomatal aperture. A and B, Wild-type, SGC24, and CCA1-ox plants were grown for 4 weeks in LD. C, cca1 lhy, cca1, and wild-type plants were grown for 3 weeks in LD. The expression of (A–C) CCA1 and FT in guard cells was analyzed by small-scale RT-PCR and normalized to levels of the reference gene GDPH. The averages of three biological repeats and se were calculated for each sample. The white and black bars represent light and dark, respectively. D, Stomatal apertures were measured at lights-on in plants grown for 3 weeks in LD. The averages and se were calculated for each sample.

Figure 8.

Photoperiod-dependent regulation of guard cell gene expression and stomatal aperture. A and B, Wild-type plants were grown for 4 weeks in LD or SD. The expression of (A) CO and (B) FT in guard cells was analyzed by RT-PCR and normalized to levels of the reference gene GDPH. The averages of three biological repeats and sem were calculated for each sample. C, Wild-type plants were grown for 4 to 5 weeks in SD. Stomatal apertures were measured as described in “Materials and Methods,” and the averages and se were calculated for each sample. The white and black bars represent light and dark, respectively.