The Cape Verde Islands allele of cryptochrome 2 enhances cotyledon unfolding in the absence of blue light in Arabidopsis

Abstract

We analyzed the natural genetic variation between Landsburg erecta (Ler) and Cape Verde Islands (Cvi) accessions by studying 105 recombinant inbred lines to search for players in the regulation of sensitivity to light signals perceived by phytochromes in etiolated seedlings of Arabidopsis. In seedlings grown under hourly pulses of far-red (FR) light, we identified three quantitative trait loci (QTLs; VLF3, VLF4, and VLF5) for hypocotyl growth inhibition and three different QTLs (VLF6, VLF7, and VLF1) for cotyledon unfolding. This indicates that different physiological outputs have selective regulation of sensitivity during de-etiolation. Ler alleles, compared with Cvi alleles, of VLF3, VLF4, VLF5, VLF7, and VLF1 enhanced, whereas the Ler allele of VLF6 reduced, the response to pulses of FR. We confirmed and narrowed down the position of some QTLs by using near-isogenic lines. VLF6 mapped close to the CRY2 (cryptochrome 2) gene. Transgenic Ler seedlings expressing the Cvi allele of CRY2 showed enhanced cotyledon unfolding under hourly pulses of FR compared with the wild type or transgenics expressing the CRY2-Ler allele. This response required phytochrome A. The cry1 cry2 double mutant lacking both cryptochromes showed reduced cotyledon unfolding under FR pulses. Because the CRY2-Cvi is a gain-of-function allele compared with CRY2-Ler, cryptochrome activity correlates positively with cotyledon unfolding under FR pulses. We conclude that the blue light photoreceptor cryptochrome 2 can modulate seedling photomorphogenesis in the absence of blue light. In addition to the nuclear loci, we identified cytoplasmic effects on seedling de-etiolation.

Figure 1.

Phenotypes of Ler, Cvi, and Cvi/Ler RILs. Histograms show the distributions of mean hypocotyl length and mean cotyledon angle in different dark/light environments. The mean (arrows) and sds (horizontal lines) of parental accessions are indicated. Photographs of representative seedlings of both parental accessions grown in different conditions are shown at the top.

Figure 2.

QTL mapping of hypocotyl growth and cotyledon angle for the Cvi/Ler RIL population grown under different light/dark environments. Mapping was based either on absolute values of hypocotyl length and cotyledon angle in darkness, hourly pulses of FR, hourly pulses of R, and continuous FR, or on the specific photobiological response (VLFR, LFR, and HIR). VLFR, Difference between darkness and pulses of FR. LFR, Difference between pulses of FR and pulses of R. HIR, Difference between pulsed and continuous FR. Rectangles, Two-log of the odds (LOD) support interval. The white arrowhead points to the position of the highest LOD score. The numbers above the arrowhead indicate the LOD score. The numbers below the white arrowhead indicate the percentage of explained variance for each QTL. Additive allele effects of each QTL are shown close to the 2-LOD support interval. The black arrowheads point upwards when the value of a given trait is increased by Ler compared with Cvi alleles and downwards when the value of a given trait is increased by Cvi compared with Ler alleles. Note that for hypocotyl growth, higher values of VLFR, LFR, or HIR mean shorter hypocotyls.

Figure 3.

VLF6 is the CRY2 gene. Cotyledon angle in seedlings of Ler, NIL-EDI, NIL 45, transgenic lines carrying genomic alleles of CRY2 from Ler or Cvi (CRY2-Ler and CRY2-Cvi, respectively) or with the amino acid 367 replaced to Val (CRY2-Cvi-367V) or with the amino acid 367 replaced to Met (CRY2-Ler-367M), the cry1, cry2, and cry1 cry2 mutants grown under hourly pulses of FR. phyA-201 and CRY2-Cvi in the phyA-201 background are also included. Data are means ± se of three independent experiments with at least three replicate boxes each. Photographs of representative seedlings are shown at the top.

Figure 4.

The Cvi allele of CRY2 enhances the VLFR but reduces the LFR. Cotyledon angle in seedlings of Ler, NIL-EDI, CRY2-Cvi, and CRY2-Ler-367M grown under hourly pulses of FR, R, or R-FR mixtures providing a series of calculated Pfr/P (3%, 10%, 33%, 61%, and 87%). The standard FR source used in the rest of the experiments corresponds to a calculated Pfr/P = 10%. Inset, Slopes (Δ cotyledon angle/Δ Pfr/P) of the VLFR (0%–10% Pfr/P) and LFR (10%–87% Pfr/P). Data are means ± se of three independent experiments with at least three replicate boxes each.

Figure 5.

Cytoplasmic effects on cotyledon unfolding. Average VLFR, LFR, and HIR of RILs with an Ler or Cvi cytoplasm. Data are means ± se of 25 (Ler) or 81 (Cvi) RILs.