Alterations in the endogenous ascorbic acid content affect flowering time in Arabidopsis

Abstract

Ascorbic acid (AA) protects plants against abiotic stress. Previous studies suggested that this antioxidant is also involved in the control of flowering. To decipher how AA influences flowering time, we studied the four AA-deficient Arabidopsis (Arabidopsis thaliana) mutants vtc1-1, vtc2-1, vtc3-1, and vtc4-1 when grown under short and long days. These mutants flowered and senesced before the wild type irrespective of the photoperiod, a response that cannot simply be attributed to slightly elevated oxidative stress in the mutants. Transcript profiling of various flowering pathway genes revealed a correlation of altered mRNA levels and flowering time. For example, circadian clock and photoperiodic pathway genes were significantly higher in the vtc mutants than in the wild type under both short and long days, a result that is consistent with the early-flowering phenotype of the mutants. In contrast, when the AA content was artificially increased, flowering was delayed, which correlated with lower mRNA levels of circadian clock and photoperiodic pathway genes compared with plants treated with water. Similar observations were made for the autonomous pathway. Genetic analyses demonstrated that various photoperiodic and autonomous pathway mutants are epistatic to the vtc1-1 mutant. In conclusion, our transcript and genetic analyses suggest that AA acts upstream of the photoperiodic and autonomous pathways.

Figure 1.

Flowering and senescence phenotypes of wild-type and vtc mutant plants. A, Col wild-type (Col WT) and vtc mutant plants were grown under SD and LD, and photographs were taken at 10 and 4 weeks after sowing. B, Number of rosette leaves when inflorescences were 1 cm in length. Means ± se of 10 individual plants are shown. Significant differences in comparison with the wild type are indicated with asterisks: *** P < 0.001, by Student's t test. C and D, Number of leaves and senescence phenotypes of the wild-type and vtc mutant plants grown under SD and LD.

Figure 2.

Relative transcript levels, based on ACTIN, of circadian clock and photoperiodic pathway genes in Col wild-type (Col WT) and vtc mutant plants. Plants were grown under SD (A and B) and LD (C and D). Transcript levels of the circadian clock gene LHY and the photoperiodic flowering pathway genes GI, CO, and FT were determined by reverse transcription-PCR from leaf tissue of 2- and 5-week-old plants harvested at 4 h after lights were turned on. Note the different scale to visualize FT expression in B. Expression data ± se of three individual samples per genotype are shown. Significant differences in comparison with the wild type are indicated with asterisks: * P < 0.05, ** P < 0.01, *** P < 0.001, by Student's t test.

Figure 3.

Expression analysis of FCA, FLC, and LFY in Col wild-type (Col WT) and vtc mutant plants. Relative transcript levels of FCA and FLC, which are key regulatory genes in the autonomous pathway, and the floral meristem identity gene LFY in inflorescences of 11-week-old plants grown under SD (A) and 5-week-old plants grown under LD (B) are shown. Inflorescence tissues were harvested at 4 h after lights were turned on. Transcript levels were assessed in inflorescences of three individual plants and normalized to ACTIN. Means ± se of three independent replicates are shown. Significant differences in comparison with the wild type are indicated with asterisks: * P < 0.05, *** P < 0.001, by Student's t test.

Figure 4.

Effects of l-Gal treatment on flowering time in LD-grown plants. A, Total AA content in 5-week-old Col wild-type plants sprayed with l-Gal or water. FW, Fresh weight. B, Flowering phenotype of l-Gal- and water-treated plants. C, Number of leaves and senescence phenotype of l-Gal- and water-sprayed plants. D, Expression analysis of photoreceptor (PHYA, PHYB, CRY1, and CRY2), circadian clock (LHY), photoperiodic pathway (GI, CO, and FT), autonomous (FCA and FLC), and floral meristem identity (LFY) genes in plants treated with l-Gal or water. Leaf and inflorescence tissues of 5-week-old plants were harvested at 4 h after lights were turned on. To assess the expression of FT, 33 PCR amplification cycles were run. Transcript levels were based on ACTIN. Results represent means ± se of three independent replicates. Significant differences between l-Gal and water treatments are indicated with asterisks: * P < 0.05, ** P < 0.01, *** P < 0.001, by Student's t test.

Figure 5.

Effects of the vtc1-1 mutation on flowering time and AA content in the background of photoperiodic and autonomous pathway mutants. Plants were grown under LD. A, Total AA content in 3-week-old wild-type (WT) controls and single and double mutants. Means ± se of three to six independent replicates per genotype are shown. FW, Fresh weight. B, Total rosette leaf number of all genotypes at flowering time. Results depict means ± se of nine to 17 independent plants per genotype. Shading patterns indicate plants of the same genetic background, allowing for easier statistical comparison of single and double mutants with their respective wild-type controls. Asterisks denote significant differences: * P < 0.05, ** P < 0.01, *** P < 0.001, by Student's t test.