Functional analysis of PHYB polymorphisms in Arabidopsis thaliana collected in Patagonia

Abstract

Arabidopsis thaliana shows a wide range of natural genetic variation in light responses. Shade avoidance syndrome is a strategy of major adaptive significance that includes seed germination, elongation of vegetative structures, leaf hyponasty, and acceleration of flowering. Previously, we found that the southernmost Arabidopsis accession, collected in the south of Patagonia (Pat), is hyposensitive to light and displays a reduced response to shade light. This work aimed to explore the genetic basis of the shade avoidance response (SAR) for hypocotyl growth by QTL mapping in a recently developed 162 RIL population between Col-0 and Pat. We mapped four QTL for seedling hypocotyl growth: WL1 and WL2 QTL in white light, SHADE1 QTL in shade light, and SAR1 QTL for the SAR. PHYB is the strongest candidate gene for SAR1 QTL. Here we studied the function of two polymorphic indels in the promoter region, a GGGR deletion, and three non-synonymous polymorphisms on the PHYB coding region compared with the Col-0 reference genome. To decipher the contribution and relevance of each PHYB-Pat polymorphism, we constructed transgenic lines with single or double polymorphisms by using Col-0 as a reference genome. We found that single polymorphisms in the coding region of PHYB have discrete functions in seed germination, seedling development, and shade avoidance response. These results suggest distinct functions for each PHYB polymorphism to the adjustment of plant development to variable light conditions.

Keywords: Arabidopsis; PHYB; QTL mapping; natural genetic variation; shade avoidance response.

Figure 1

QTL mapping for shade avoidance response in Col-0 and Pat RIL population. (A) SAR index for Col-0 and Pat seedlings calculated as the ratio of hypocotyl length in simulated shade and WL. The photo shows representative Col-0 (left) and Pat (right) seedlings in WL and simulated shade. (B) QTL mapping for hypocotyl growth in WL, simulated shade, and SAR index in the Col-0 and Pat RIL population. LOD score is shown for the accumulated distance of the five chromosomes separated by vertical dashed lines. The horizontal dashed line indicates the LOD significance threshold value. (C) LOD score is shown for the absolute distance of chromosome II (Mb). PHYB is the candidate gene mapped into the confidence interval of SAR1 QTL. For SAR1 QTL, is indicated the LOD, % explained variability, additive effects (positive additive effects mean a higher contribution of Col-0 alleles) and confidence interval.

Figure 2

PHYB polymorphisms between Col-0 and Pat accessions. (A) PHYB polymorphisms in the coding region between Col-0 and Pat. The diagram shows the four polymorphisms in the N-and C-termini domains (M1 = deletion, M2 = I143L, M3 = V980I, and M4 = L1072V). (B) Col-0 and Pat amino-acid sequences show the GGGR deletion of Pat in the amino termini of PHYB protein. (C) The two indels polymorphisms in the promoter of PHYB gene between Col-0 and Pat tested in this study. The deletion of 8 nucleotides at position −341/−334, and the insertion of 2 nucleotides at position −318/−319 in the Pat promoter up to ATG starting codon. (D) Natural variation for hypocotyl SAR index calculated as the ratio of hypocotyl length in simulated shade and WL between accessions sharing the same PHYB-Pat polymorphism at the positions M1, M2, M3, and M4. Significant differences between means are shown with different letters by one-way ANOVA followed by Bonferroni post-test (p < 0.05). Each bar represents mean ± SE (n = 6).

Figure 3

Functional variation of PHYB polymorphisms in shade avoidance response (SAR) and seedling de-etiolation response. (A) SAR index calculated as the ratio of hypocotyl length in simulated shade and WL. Each bar represents Mean ± SE (n = 4). Significant differences between means are shown with different letters by one-way ANOVA followed by Bonferroni post-test (p < 0.05). (B) Inhibition of hypocotyl length in red continuous light (relative to darkness) during seedling de-etiolation. Each bar represents Mean ± SE (n = 4). Significant differences between means are shown with different letters by one-way ANOVA followed by Bonferroni post-test (p < 0.05).

Figure 4

Functional variation of PHYB polymorphisms in seed germination. (A) Germination (%) with an R or FR saturated pulse and then incubated in darkness for 4 days at 25°C before germination was counted. The seeds were imbibed in water for 3 days at 7°C before the light pulse. (B) R/FR reversible response (%) calculated as the germination with a red pulse minus the germination with a far-red pulse. Mean ± SE (n = 6). Significant differences between means are shown with different letters by Two-way ANOVA (A) or One-way ANOVA (B) followed by Bonferroni post-test (p < 0.05).

Figure 5

Functional analysis of PHYB polymorphisms on the promoter region by LUC analysis. Luc bioluminescence (units) for transgenic plants carrying the promotor of PHYB-Col or PHYB-Pat in the Col and Pat backgrounds. LL continuous light treatment. Each point represents the media of n ≥ 4.