Identification of flowering genes in strawberry, a perennial SD plant

Abstract

Background: We are studying the regulation of flowering in perennial plants by using diploid wild strawberry (Fragaria vesca L.) as a model. Wild strawberry is a facultative short-day plant with an obligatory short-day requirement at temperatures above 15 degrees C. At lower temperatures, however, flowering induction occurs irrespective of photoperiod. In addition to short-day genotypes, everbearing forms of wild strawberry are known. In 'Baron Solemacher' recessive alleles of an unknown repressor, SEASONAL FLOWERING LOCUS (SFL), are responsible for continuous flowering habit. Although flower induction has a central effect on the cropping potential, the molecular control of flowering in strawberries has not been studied and the genetic flowering pathways are still poorly understood. The comparison of everbearing and short-day genotypes of wild strawberry could facilitate our understanding of fundamental molecular mechanisms regulating perennial growth cycle in plants.

Results: We have searched homologs for 118 Arabidopsis flowering time genes from Fragaria by EST sequencing and bioinformatics analysis and identified 66 gene homologs that by sequence similarity, putatively correspond to genes of all known genetic flowering pathways. The expression analysis of 25 selected genes representing various flowering pathways did not reveal large differences between the everbearing and the short-day genotypes. However, putative floral identity and floral integrator genes AP1 and LFY were co-regulated during early floral development. AP1 mRNA was specifically accumulating in the shoot apices of the everbearing genotype, indicating its usability as a marker for floral initiation. Moreover, we showed that flowering induction in everbearing 'Baron Solemacher' and 'Hawaii-4' was inhibited by short-day and low temperature, in contrast to short-day genotypes.

Conclusion: We have shown that many central genetic components of the flowering pathways in Arabidopsis can be identified from strawberry. However, novel regulatory mechanisms exist, like SFL that functions as a switch between short-day/low temperature and long-day/high temperature flowering responses between the short-day genotype and the everbearing 'Baron Solemacher'. The identification of putative flowering gene homologs and AP1 as potential marker gene for floral initiation will strongly facilitate the exploration of strawberry flowering pathways.

Figure 1

Environmental regulation of flowering in everbearing wild strawberries. The effect of photoperiod (SD 12 h, LD 18 h) and temperature (11/18°C) on the flowering time of 'Baron Solemacher' (A) and 'Hawaii-4' (B). Seeds were germinated in LD at 18°C, and seedlings were exposed to the treatments for five weeks, when the cotyledons were opened. After treatments, plants were moved to LD at 18°C and flowering time was recorded as number of leaves in the main crown before the terminal inflorescence. Values are mean ± SD. Pairwise comparisons between the treatments were done by Tukey's test, and statistically significant differences (p ≤ 0.05) are denoted by different letters above the error bars.

Figure 2

A simplified chart showing Arabidopsis flowering pathways and corresponding gene homologs in Fragaria. Gene homologs found in cDNA libraries produced from SD and EB genotypes are surrounded by blue and red boxes, respectively. Arrows indicate positive regulation and bars negative regulation.

Figure 3

Protein alignments of Fragaria flowering integrator and identity genes. Multiple alignments of Fragaria protein sequences of full length SOC1 (A), partial LFY (B) and full-length AP1 (C) with closest protein homologs and corresponding protein sequence of Arabidopsis thaliana. Alignments were done by ClustalW (A, B) or T-Coffee (C) and modified by Boxshade program. F. vesca AP1 protein sequence was translated from GDR Fragaria EST contig 4941. PTM5 = Populus tremuloides MADS5, AFL2 = Apple FLORICAULA 2, PpAP1 = putative Prunus persica AP1.

Figure 4

Developmental regulation of gene expression in wild strawberry shoot apices. The expression of AP1 (A), LFY (B), SOC1 (C) and GA3ox (D) in the SD and EB ('Baron Solemacher') genotype of the wild strawberry. Triplicate shoot apex samples were collected from LD grown plants at one to four leaf stage. Ct values were normalized against a Ubiquitin [GenBank:DY672326] gene to get normalized ΔCt values. The expression differences between one leaf stage and later developmental stages were calculated from the formula 2^{ΔCt later developmental stage}/2^{ΔCt one leaf stage}. The expression values at one leaf stage were artificially set to 1 separately for both genotypes. Values are mean ± SD. Note that Ubiquitin was amplified ~1 cycle earlier in SD genotype, but was stable between different developmental stages. Therefore, expression values between genotypes cannot be directly compared, while the expression levels between the various developmental stages are comparable.