Circadian rhythms confer a higher level of fitness to Arabidopsis plants

Abstract

Circadian rhythms have been demonstrated in organisms across the taxonomic spectrum. In view of their widespread occurrence, the adaptive significance of these rhythms is of interest. We have previously shown that constitutive expression of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene in Arabidopsis plants (CCA1-ox) results in loss of circadian rhythmicity. Here, we demonstrate that these CCA1-ox plants retain the ability to respond to diurnal changes in light. Thus, transcript levels of several circadian-regulated genes, as well as CCA1 itself and the closely related LHY, oscillate robustly if CCA1-ox plants are grown under diurnal conditions. However, in contrast with wild-type plants in which transcript levels change in anticipation of the dark/light transitions, the CCA1-ox plants have lost the ability to anticipate this daily change in their environment. We have used CCA1-ox lines to examine the effects of loss of circadian regulation on the fitness of an organism. CCA1-ox plants flowered later, especially under long-day conditions, and were less viable under very short-day conditions than their wild-type counterparts. In addition, we demonstrate that two other circadian rhythm mutants, LHY-ox and elf3, have low-viability phenotypes. Our findings demonstrate the adaptive advantage of circadian rhythms in Arabidopsis.

Figure 1

Circadian oscillations of transcript accumulation from clock-controlled genes in wild-type and CCA1-ox lines. Wild-type and CCA1-ox plants were transferred to constant light (LL) after entrainment in light:dark conditions (12L:12D). The relative RNA levels of Lhcb1*1 (A) and CCR2 (B) are shown relative to the maximum levels of expression. Methylene blue staining was used to check equal loading. Experiments were performed twice with similar results. The hatched bars indicate subjective dark periods in LL.

Figure 2

Diurnal oscillations of transcript accumulation from clock-controlled genes in wild-type and CCA1-ox lines. Plants were grown in photoperiods of 16 h of light and 8 h of dark (16L:8D; A–C) and 8 h of light and 16 h of dark (8L:16D; D–F). The relative RNA levels of Lhcb1*1 (A and C), CAT2 (B and E), and CCR2 (C and F) are shown relative to the maximum levels of expression. Methylene blue staining was used to check equal loading. Experiments were performed twice with similar results. The black and yellow bars beneath the graphs represent dark and light photoperiods.

Figure 3

Diurnal oscillations of LHY and CCA1 mRNAs in wild-type and CCA1-ox lines. Plants were grown in 16L:8D and 8L:16D photoperiods. Methylene blue staining was used to check equal loading. Representative northerns are shown. Relative levels of CCA1 and LHY RNA were plotted on the graphs. Experiments were performed twice with similar results. The black and yellow bars beneath the graphs represent dark and light photoperiods.

Figure 4

Flowering times of wild-type and CCA1-ox plants. Seeds of wild-type plants and two CCA1-ox lines, CCA1-ox 34 and CCA1-ox 38, were sown onto soil. Plants were grown in 16L:8D and 8L:16D photoperiods. The numbers of rosette leaves at bolting were counted. The results were plotted on a graph ± sd. White boxes represent 16L:8D and hatched boxes represent 8L:16D.

Figure 5

Diurnal oscillations of transcript accumulation in wild-type and CCA1-ox lines in very short-day conditions. Plants were grown in 4L:16D photoperiods. Methylene blue staining was used to check equal loading. Relative levels of RNA were plotted on the graphs. Experiments were performed twice with similar results. The black and yellow bars beneath the graphs represent dark and light photoperiods.

Figure 6

Viability of wild-type and CCA1-ox plants. A, Approximately 100 seeds of wild-type (Columbia) plants and a CCA1-ox line, CCA1-ox 34, were sown onto soil. Plants were grown in 16L:8D, 8L:16D, and 4L:20D photoperiods. The number of plants surviving was counted each week and expressed as a percentage of the number of seeds that had germinated by 1 week after sowing. The results were plotted on a graph ± sd. Experiments were performed four times with similar results. B, Forty seeds of wild-type plants and a CCA1-ox line, CCA1-ox 34, were sown onto Murashige and Skoog medium in magenta pots and then grown in 8L:16D and 4L:20D photoperiods for 6 weeks. C, Twenty-five seeds of wild-type plants and a CCA1-ox line, CCA1-ox 34, were sown onto soil in a 105- × 155-mm pot and grown 8L:16D (2 weeks) and 4L:20D (4 weeks) photoperiods.

Figure 7

Viability of wild-type, LHY-ox, and ELF3 plants. Approximately 100 seeds of wild-type (Columbia and Landsberg erecta) plants, LHY-ox and ELF3 lines, were sown onto soil. Plants were grown in 16L:8D and 4L:20D photoperiods. The number of plants surviving was counted each week and expressed as a percentage of the number of seeds that had germinated by 1 week after sowing. The results were plotted on a graph ± sd. Experiments were performed three times with similar results.