Mesophyll-localized phytochromes gate stress- and light-inducible anthocyanin accumulation in Arabidopsis thaliana

Figures & data

Figure 1. Development and anthocyanin content of wild-type and phytochrome-deficient seedlings under continuous far-red light. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), and phyA (Salk_014575) seedlings were grown at 22 °C for 4 d or 12 °C for 10 d on basal (B), nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium with 1% sucrose under continuous FR (FRc) illumination of 5 μmol m^-2 s^-1. (A, B) Images of seedlings. Scale bar represents 1 cm. (C, D) Anthocyanin content. Bars represent the mean (± SD) of at least 3 independent measurements. Numbers above each bar represent the percent of anthocyanins accumulated relative to WT grown on the same medium. Fold-increase values for anthocyanin levels for seedlings grown on nutrient-deficient medium relative to growth of the same line on basal medium are shown below the bar graph. Two-tailed, unpaired Mann-Whitney U-test analyses were performed to compare the means of anthocyanin contents per mg of fresh weight for a particular line on basal medium relative to respective nutrient limitations. Letters above bars: a, P < 0.001; b, P < 0.01; c, P ≤ 0.05.

Table 1. Fold-difference values for anthocyanin levels determined for seedlings grown at 12 °C vs. 22 °C under different light conditions. No-0 wild-type (No-0 WT), CAB3::pBVR1, CAB3::pBVR2, Col-0 wild-type (Col-0 WT), phyA, phyB, cry1, and cry2 seedlings were grown on basal (B), nitrogen-limited (-N), or phosphate-limited (-P) medium at 12 °C or 22 °C under continuous far-red (FRc), red (Rc) or blue (Bc) illumination. Fold-difference values for anthocyanin content for seedlings grown at 12 °C relative to 22 °C are indicated (based on averages for 3 independent experiments)

Plant line	Fold difference of anthocyanin content (12 °C /22 °C)
	FRc			Rc			Bc
	B	-N	-P	B	-N	-P	B	-N	-P
No-0 WT	0.91	2.70	2.70	2.78	3.19	1.33	0.47	0.63	1.36
CAB3::pBVR1	0.73	0.47	0.50	0.84	1.05	1.09	0.34	0.60	1.07
CAB3::pBVR2	0.78	0.45	0.55	0.56	1.63	0.53	0.45	0.82	2.65
Col-0 WT	0.57	1.91	0.27	6.05	9.15	2.94	0.68	1.13	1.43
phyA	0.71	1.30	0.56	NA^a	NA	NA	0.48	1.16	1.31
phyB	NA	NA	NA	1.98	26.7	2.10	NA	NA	NA
cry1	NA	NA	NA	NA	NA	NA	0.37	1.06	1.53
cry2	NA	NA	NA	NA	NA	NA	0.17	0.69	0.42

^a NA, not applicable. The indicated line was not grown under the indicated light condition.

Figure 2. Development and anthocyanin content of wild-type and phytochrome-deficient seedlings under continuous red light. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), and phyB (Salk_022035) seedlings were grown at 22 °C for 4 d or 12 °C for 10 d on basal (B), nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium with 1% sucrose under continuous R (Rc) illumination of 50 μmol m^-2 s^-1. (A, B) Images of seedlings. Scale bar represents 1 cm. (C, D) Anthocyanin content. Bars represent the mean (± SD) of at least 3 independent measurements. Numbers above each bar represent the percent of anthocyanins accumulated relative to WT grown on the same medium. Fold-increase values for anthocyanin levels for seedlings grown on nutrient-deficient medium relative to growth of the same line on basal medium are shown below the bar graph. Two-tailed, unpaired Mann-Whitney U-test analyses were performed to compare the means of anthocyanin contents per mg of fresh weight for a particular line on basal medium relative to respective nutrient limitations. Letters above bars: a, P < 0.001; b, P < 0.01; c, P ≤ 0.05.

Figure 3. Development and anthocyanin content of wild-type, phytochrome-deficient and cryptochrome mutant seedlings under continuous blue light. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), phyA, cry1, and cry2 seedlings were grown at 22 °C for 4 d or 12 °C for 10 d on basal (B), nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium with 1% sucrose under continuous B (Bc) illumination of 25 μmol m^-2 s^-1. (A, B) Images of seedlings. Scale bar represents 1 cm. (C, D) Anthocyanin content. Bars represent the mean (± SD) of at least 3 independent measurements. Numbers above each bar represent the percent of anthocyanins accumulated relative to WT grown on the same medium. Fold-increase values for anthocyanin levels for seedlings grown on nutrient-deficient medium relative to growth of the same line on basal medium are shown below the bar graph. Two-tailed, unpaired Mann-Whitney U-test analyses were performed to compare the means of anthocyanin contents per mg of fresh weight for a particular line on basal medium relative to respective nutrient limitations. Letters above bars: a, P < 0.001; b, P < 0.01; c, P ≤ 0.05.

Figure 4. Light-dependent primary root lengths of wild-type, phytochrome chromophore–deficient, phytochrome apoprotein and cryptochrome apoprotein mutants. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), phyA, phyB, cry1, and cry2 seedlings were grown vertically on basal (B), nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium containing 1% (w/v) Suc for 7 d at 22 °C under (A) far-red continuous (FRc) light of 5 µmol m⁻² s⁻¹, (B) red continuous (Rc) light of 50 µmol m⁻² s⁻¹, or (C) blue continuous (Bc) light of 25 µmol m⁻² s⁻¹. Bars represent the mean (± S.D.) of primary root lengths in mm (n = 5 to 10 for 2 independent biological experiments). For statistical significance tests comparisons were made for a particular line on basal medium relative to the respective nutrient limitations: a, P < 0.001; b, P < 0.01; c, P ≤ 0.05.

Figure 5. Root development of wild-type and phytochrome-deficient seedlings under continuous far-red light. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), and phyA seedlings were grown vertically at 22 °C for 7 d on basal, nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium with 1% Suc under far-red continuous (FRc) illumination of 5 µmol m^-2 s^-1. Representative images of seedlings from 2 independent biological replicates are shown. Scale bars represent 0.5 cm.

Figure 6. Root development of wild-type and phytochrome-deficient seedlings under continuous red light. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), and phyB seedlings were grown vertically at 22 °C for 7 d on basal, nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium with 1% sucrose under red continuous (Rc) illumination of 50 µmol m^-2 s^-1. Representative images of seedlings from 2 independent biological replicates are shown. Scale bars represent 0.5 cm.

Figure 7. Root development of wild-type, phytochrome-deficient and cryptochrome mutant seedlings under continuous blue light. No-0 wild-type (No-0 WT), CAB3::pBVR1 (CAB-1), CAB3::pBVR2 (CAB-2), Col-0 wild-type (Col-0 WT), phyA, cry1, and cry2 seedlings were grown vertically at 22 °C for 7 d on basal, nitrogen-limited (-N), or phosphate-limited (-P) Phytoblend medium with 1% sucrose under blue continuous (Bc) illumination of 25 µmol m^-2 s^-1. Representative images of seedlings from 2 independent biological replicates are shown. Scale bars represent 0.5 cm.