Pivotal Roles of Cryptochromes 1a and 2 in Tomato Development and Physiology

Abstract

Cryptochromes are flavin-containing blue/UVA light photoreceptors that regulate various plant light-induced physiological processes. In Arabidopsis (Arabidopsis thaliana), cryptochromes mediate de-etiolation, photoperiodic control of flowering, entrainment of the circadian clock, cotyledon opening and expansion, anthocyanin accumulation, and root growth. In tomato (Solanum lycopersicum), cryptochromes are encoded by a multigene family, comprising CRY1a, CRY1b, CRY2, and CRY3 We have previously reported the phenotypes of tomato cry1a mutants and CRY2 overexpressing plants. Here, we report the isolation by targeting induced local lesions in genomes, of a tomato cry2 knock-out mutant, its introgression in the indeterminate Moneymaker background, and the phenotypes of cry1a/cry2 single and double mutants. The cry1a/cry2 mutant showed phenotypes similar to its Arabidopsis counterpart (long hypocotyls in white and blue light), but also several additional features such as increased seed weight and internode length, enhanced hypocotyl length in red light, inhibited primary root growth under different light conditions, anticipation of flowering under long-day conditions, and alteration of the phase of circadian leaf movements. Both cry1a and cry2 control the levels of photosynthetic pigments in leaves, but cry2 has a predominant role in fruit pigmentation. Metabolites of the sterol, tocopherol, quinone, and sugar classes are differentially accumulated in cry1a and cry2 leaves and fruits. These results demonstrate a pivotal role of cryptochromes in controlling tomato development and physiology. The manipulation of these photoreceptors represents a powerful tool to influence important agronomic traits such as flowering time and fruit quality.

Figure 1.

Seed and seedling phenotypes of tomato wild-type and cryptochrome mutants. A, Seed weight (mg per 100 dry seeds). Data are means ± sd of six pools of 100 seeds. B, Optical microscopy of seed sections. Pictures separated by a white lane have been digitally extracted for comparison. C, Scanning electron microscopy of seed sections. All pictures have been digitally extracted for comparison. D, Hypocotyl length of 7-d-old seedlings grown in MSB5 1/2 synthetic medium under continuous blue, red, green, and white light and in the dark. E, Primary root length of 7-d-old seedlings grown in MSB5 1/2 in the dark. F, Primary root length of 7-d-old seedlings grown in MSB5 1/2 under 2 µmol m⁻² s⁻¹ of continuous blue, red, or green, or under 40 µmol m⁻² s⁻¹ of continuous white light, normalized to primary root length of seedlings of the same genotype grown in the dark. Data are means ± sd of at least 25 seedlings. Asterisks indicate significant differences compared with that in MM (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post-hoc honestly significant difference mean-separation test). Scale bars = B, 1 mm; C, 500 μm.

Figure 2.

Fluence-rate response of hypocotyl inhibition and primary root development in wild-type and cryptochrome mutant plants. Measurement of 7-d-old seedlings grown in MSB5 1/2 synthetic medium under continuous blue light with fluence rates of 0.1 to 20 µmol m⁻² s⁻¹. A, Hypocotyl length. B, Primary root length. C, Phenotypic comparison. Pictures have been digitally extracted for comparison. Data are means ± sd of at least 25 seedlings. Scale bars = 1 cm.

Figure 3.

Cotyledon and hypocotyl phenotypes in wild-type and cryptochrome mutant plants. Measurement of cotyledons and hypocotyls of 7-d-old seedlings grown in MSB5 1/2 synthetic medium under 2 µmol m⁻² s⁻¹ of continuous blue light. A, Relative concentration of total chlorophylls with respect to that in MM expressed as percentage. B, Cotyledon phenotype. C, Hypocotyl phenotype. Data are means ± sd of three pools of 25 seedlings. Asterisks indicate significant differences compared with that in MM (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post-hoc honestly significant difference test). Pictures have been digitally extracted for comparison. Scale bars = 5 mm.

Figure 4.

Shoot development of wild-type and cryptochrome mutant plants. Measurement of 25-d-old plants grown in MSB5 1/2 synthetic medium under 40 µmol m⁻² s⁻¹ of LD white light. Data are means ± sd of 12 plants. Asterisks indicate significant differences compared with that in MM (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post-hoc honestly significant difference test). Asterisks above the histogram refer to total stem length, whereas asterisks in the histogram sections refer to sections’ length.

Figure 5.

Pigment concentration in leaves and fruits of wild-type and cryptochrome mutant plants. A and B, Chlorophyll (A) and carotenoid (B) composition in tomato leaves. C and D, Chlorophyll (C) and carotenoid (D) composition in tomato fruits at the MG ripening stage. E, Carotenoid levels in fruits at the 10-DPB ripening stage. Representative images of analyzed tissues are depicted on the right. Amounts of the different compounds are plotted as stacked bars. Data are the average of three biological replicates and are expressed as μg/g DW. Asterisks indicate significant differences compared with that in MM (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post-hoc honestly significant difference test). Detailed data are shown in Supplemental Table S5.

Figure 6.

Metabolite analysis of wild-type and cryptochrome mutant plants. A and B, Heat-maps of nonpolar (A) and semipolar (B) metabolites profiled in tomato leaves and fruits at the MG and 10-DPB ripening stage. Representative images of analyzed tissues are depicted at the top. Red and green indicate up- and down-regulated metabolites, respectively (scale at bottom left). Gray indicates metabolites that are below detection. Fold-change values were log2-transformed. Different metabolite classes are marked with squares of different colors. Data are the average of three biological replicates. Detailed data are shown in Supplemental Tables S6 and S7.

Figure 7.

Effects of cryptochrome mutations on circadian phase and period. A and B, Mean circadian period (A) and phase (B) estimates ± sd of at least 44 plants per genotype. The letters on top of each bar indicate the significance groups as determined by one-way ANOVA and Tukey’s post-hoc honestly significant difference test (P < 0.05). The experiment was repeated in three independent trials.

Figure 8.

Flowering time in wild-type and cryptochrome mutant plants. A and B, Flowering time measured as number of days between cotyledons opening and anthesis of the first flower (A) and as number of leaves before the first inflorescence (B) of plants grown in soil under 40 and 100 µmol m⁻² s⁻¹ of LD white light. Data are means ± sd of 15 plants. Asterisks indicate significant differences compared with that in MM (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post-hoc honestly significant difference test).

Figure 9.

Diurnal gene expression pattern in wild-type and cryptochrome mutant plants. Gene expression measured in leaves of 35-d-old plants grown in LD (16-h light, 8-h dark) conditions under 40 μmol m⁻² s⁻¹ of white light. Time points are measured in hours from dawn (ZT). Yellow and dark bars along the horizontal axis represent light and dark periods, respectively. Transcript levels of the analyzed genes were measured through RT-qPCR and were normalized to the expression of the housekeeping ACTIN gene. Data are means ± sd of three biological replicates. Asterisks indicate significant differences compared with that in MM (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post-hoc honestly significant difference test).