Whole transcriptome profiling of the vernalization process in Lilium longiflorum (cultivar White Heaven) bulbs

Villacorta-Martin et al. BMC Genomics (2015) 16:550 DOI 10.1186/s12864-015-1675-1 RESEARCH ARTICLE Open Access Whole transcriptome profiling of the vernalization process in Lilium longiflorum (cultivar White Heaven) bulbs Carlos Villacorta-Martin1, Francisco F. Núñez de Cáceres González2,3, Jorn de Haan1, Kitty Huijben1, Paul Passarinho1, Maya Lugassi-Ben Hamo2 and Michele Zaccai2* Abstract Background: Vernalization is an obligatory requirement of extended exposure to low temperatures to induce flowering in certain plants. It is the most important factor affecting flowering time and quality in Easter lily (Lilium longiflorum). Exposing the bulbs to 4 °C gradually decreases flowering time up to 50 % compared to non-vernalized plants. We aim to understand the molecular regulation of vernalization in Easter lily, for which we characterized the global expression in lily bulb meristems after 0, 2, 5, 7 and 9 weeks of incubation at 4 °C. Results: We assembled de-novo a transcriptome which, after filtering, yielded 121,572 transcripts and 42,430 genes which hold 15,414 annotated genes, with up to 3,657 GO terms. This extensive annotation was mapped to the more general GO slim plant with a total of 94 terms. The response to cold exposure was summarized in 6 expression clusters, providing useful patterns for dissecting the dynamics of vernalization in lily. The functional annotation (GO and GO slim plant) was used to group transcripts in gene sets. Analysis of these gene sets and profiles revealed that most of the enriched functions among genes up-regulated by cold exposure were related to epigenetic processes and chromatin remodeling. Candidate vernalization genes in lily were selected based on their sequence similarity to known regulators of flowering in other species. Conclusions: We present a detailed analysis of gene expression dynamics during vernalization in Lilium, covering several time points and accounting for biological variation by the use of replicates. The resulting collection of transcripts and novel isoforms provides a useful resource for studying the changes occurring during vernalization at a fine level. The selected potential candidate genes can shed light on the regulation of this process. Background Lilium longiflorum (Easter lily) is a leading bulbous crop worldwide and is produced as cut flower, potted plant, garden plant and as dry cell bulb [1]. Like many other ornamental bulbs [2], L. longiflorum flowering requires cooling of the bulbs to meet the obligatory vernalization requirement of this plant species [3, 4]. L. longiflorum, (cultivar White Heaven) plants developing from nonvernalized bulbs grown at a constant temperature of 25 °C produced only leaves and did not flower over a period of more than 15 months (Ram et al., in preparation), confirming the obligatory cold requirement of this cultivar. * Correspondence: 2 Department of Life Sciences, Ben Gurion University of the Negev, P.O. Box 653, Beersheva 84105, Israel Full list of author information is available at the end of the article Vernalization is also the main parameter involved in flowering time regulation in Easter lily and therefore has been the focus of a considerable amount of research related to physiological aspects of this species’ development, in order to reach flowering at specific dates [3, 5]. Typically, cold exposure of L. longiflorum bulbs at 2 to 10 °C quantitatively hastens flowering time while decreasing height, leaf and flower number, up to a saturation point of 6 weeks, after which additional cold exposure will not have a further effect on these parameters [3, 5–10]. In a previous study on L. longiflorum cultivar White Heaven [11], we found that bulb exposure to 4 °C for one week induced a decrease of about 20 % in the time from planting to floral transition and to flowering. Additional cold exposure led to a gradual © 2015 Villacorta-Martin et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Villacorta-Martin et al. BMC Genomics (2015) 16:550 decrease up to about 80 % and 55 % for floral transition and flowering, respectively, after nine weeks at 4 °C. Despite the importance of vernalization in Lilium flowering, the molecular regulation of this mechanism is largely unknown in this species and other ornamental flowering bulbs. Most of the information available on molecular control of vernalization comes mainly from work performed on Arabidopsis, cereals and sugar beet and revealed that, while the general mechanism of vernalization is conserved among distant species, the sequence of the main regulatory genes is not [12–17]. In Arabidopsis, FLOWERING LOCUS C (FLC), a MADS-box gene encoding a potent repressor of flowering, is active in meristems in autumn. Flowering repression by FLC is mediated by its binding to major genes that promote flowering, such as FLOWERING LOCUS T and D (FT and FD, respectively) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) [18, 19]. While FLC represses genes that induce meristems to form flowers, it relies on FRIGIDA (FRI) to elevate its autumnal expression to a level that prevents flowering [19, 20]. During winter, vernalization causes the acquisition of meristem competence to flower by repressing FLC expression. Once it has been repressed by vernalization, FLC remains off for the rest of the plant’s life cycle after the return of warm conditions, i.e. the repression is epigenetic in the sense that it is mitotically stable in the absence of the inducing signal (cold exposure). The mechanism of epigenetic repression of FLC involves histone modifications that convert FLC into a heterochromatin-like state. A key player in the vernalization-mediated silencing of FLC is VERNALIZATION INSENSITIVE 3 (VIN3), which is required for all FLC chromatin modifications associated with vernalization-mediated silencing and as a measure of the cold period [21]. Recently, it was shown that all members of the VIN3 family act together to repress FLC family members during vernalization [22]. In addition, the non-coding (nc) antisense transcript COOLAIR and the intronic long ncRNA COLDAIR are upregulated at different points during cold exposure and are apparently playing a role in the epigenetic regulation of FLC [23–25]. Altogether, this measure of gradual cold acquisition ensures that only a prolonged cold exposure (the winter season) will lead to activation of the vernalization process. In winter cereals, which require vernalization, a system similar to that in Arabidopsis exists. Specifically, a flowering repressor prevents flowering prior to (...truncated)