Molecular Regulation of Temperature-Dependent Floral Induction in Tulipa gesneriana

Abstract

The vegetative-to-reproductive phase change in tulip (Tulipa gesneriana) is promoted by increasing temperatures during spring. The warm winters of recent years interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular mechanisms would be of help, but unlike the model plant Arabidopsis (Arabidopsis thaliana), very little is known about floral induction in tulip. To shed light on the gene regulatory network controlling flowering in tulip, RNA sequencing was performed on meristem-enriched tissue collected under two contrasting temperature conditions, low and high. The start of reproductive development correlated with rounding of the shoot apical meristem and induction of TGSQA expression, a tulip gene with a high similarity to Arabidopsis APETALA1 Gene Ontology enrichment analysis of differentially expressed genes showed the overrepresentation of genes potentially involved in floral induction, bulb maturation, and dormancy establishment. Expression analysis revealed that TERMINAL FLOWER1 (TgTFL1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-like1 (TgSOC1-like1) might be repressors, whereas TgSOC1-like2 likely is an activator, of flowering. Subsequently, the flowering time-associated expression of eight potential flowering time genes was confirmed in three tulip cultivars grown in the field. Additionally, heterologous functional analyses in Arabidopsis resulted in flowering time phenotypes in line with TgTFL1 being a floral repressor and TgSOC1-like2 being a floral activator in tulip. Taken together, we have shown that long before morphological changes occur in the shoot apical meristem, the expression of floral repressors in tulip is suppressed by increased ambient temperatures, leading either directly or indirectly to the activation of potential flowering activators shortly before the commencement of the phase change.

Figure 1.

Morphology of the vegetative-to-reproductive phase change at the SAM and transcriptional changes over time. A, Morphology of the SAM inside the bulb and its surrounding tissues in spring prior to the temperature experiment. Note that the SAM is still vegetative and that one leaf primordium has developed. Bar = 1 mm. B, Morphological changes at the SAM inside the main daughter bulbs of cv Dynasty during low- and high-temperature conditions. In the first 5 weeks of both temperature conditions, only one leaf primordium developed (green) and the SAM remained flat (yellow). After 6 weeks at 18°C, the SAM got a dome-like appearance, which is the first known morphological change upon making the switch from vegetative to reproductive development. Shortly after this, the floral meristem (FM; orange) gives rise to the development of the different floral organs (tepals, cyan; stamens, violet; carpel, red). Note that the SAM of bulbs in the low-temperature condition (8°C–9°C) remains vegetative and flat for the complete period of 8 weeks. The different tissues have been artificially colored in the right image of each group. Bars = 1 mm. C, Expression pattern of TGSQA at low-temperature (8°C–9°C) and high-temperature (18°C) conditions. D, MDS plot revealing global transcriptional changes over time. The bulbs from the low-temperature (8°C–9°C) condition remain in a relatively stable transcriptional state, whereas the bulbs from the high-temperature (18°C) condition show significant transcriptional changes over time associated with the switch from the vegetative to the reproductive phase. C, Cold; W, warm; the number indicates the week after the start of the experiment. FC, Fold change.

Figure 2.

Overview of the GO enrichment analysis in the transcriptome data of the floral induction in tulip. Output GO enrichment analysis was performed by comparing each week with week 0. At left in red, the GO terms listed are specifically overrepresented in the up-regulated genes upon high temperatures. At right in blue, GO terms specifically overrepresented in the down-regulated genes are shown.

Figure 3.

Three selected clusters from the cluster analysis of all tulip transcripts that have high similarity with known transcription factors in Arabidopsis. The clusters represent transcripts of the high-temperature condition. On the x axis, the different time points are plotted, and on the y axis, the z score is shown (normalized cpm). A, Expression of the genes in cluster 17 remains stable until week 4, after which their expression decreases. The cluster includes AP2, INOSITOL 3-PHOSPHATE SYNTHASE2 (ATIPS2), AUXIN RESPONSE FACTOR22 (ARF22), JASMONATE-ZIM-DOMAIN PROTEIN1 (JAZ1), and ALBINO3 (ALB3). B, Expression of the genes in cluster 37 decreases slowly over time. This cluster includes ARABIDOPSIS CENTRORADIALIS (ATC), MYB5, GALACTURONOSYLTRANSFERASE15 (GAUT15), and PINORESINOL REDUCTASE2 (PRR2). C, Expression of the genes in cluster 238 increasing from week 0 onward and reaching a plateau around week 4. This cluster includes FLOWERING LOCUS K (FLK), FT, TATA BOX-BINDING PROTEIN2 (TBP2), and EMBRYO SAC DEVELOPMENT ARREST35 (EDA35). Transcripts with a high similarity (BLAST cutoff value of 1e-05) to a known flowering time gene in Arabidopsis are marked with red stars.

Figure 4.

Expression analysis by quantitative reverse transcription-PCR of eight putative tulip flowering time genes in the SAM region of the main daughter bulb during 8 weeks of high- or low-temperature treatment. A, Expression of TgTFL1. B, Expression of TgSOC1L1. C, Expression of TgSOC1L2. D, Expression of TgFT-like. E, Expression of TgSEP1. F, Expression of TGSQB. G, Expression of TgSPL1. H, Expression of TgSPL2.

Figure 5.

Morphological and molecular analysis of the vegetative-to-reproductive phase change in three tulip cultivars. A, Morphological analysis of the changes at the SAM in cv Purple Prince, cv Dynasty, and cv Strong Gold. I, Vegetative; II, reproductive; P1, first whorl of tepals; P2, second whorl of tepals; A2, second whorl of stamens; A2+, beginning of carpel development. FM, Floral meristem. The first visual observation of the transition from vegetative to reproductive development is marked with an asterisk. Bars = 1 mm. B, Expression of TgTFL1. C, Expression of TgSOC1L1. D, Expression of TgSOC1L2. E, Expression of TgFT-like. F, Expression of TGSQA. G, Expression of TgSEP1. H, Expression of TgSPL1. I, Expression of TgSPL2.

Figure 6.

Phenotypic and molecular analyses of Arabidopsis overexpressing different potential tulip flowering time genes. A, 35S:TgSOC1L2. B, 35S:TgTFL1. C, Wild-type flower. D, 35S:TgTFL1 flower. E, Number of days to flowering for 35S:TgSOC1L2. F, Leaf number of 35S:TgSOC1 when the inflorescence reaches a length of 1 cm. G, Expression of TgSOC1L2 in the overexpression line TgSOC1L2-2 in comparison with Columbia-0 (Col-0). H, Number of days to flowering for 35S:TgTFL1. I, Leaf number of 35S:TgTFL1 when the inflorescence reaches a length of 1 cm. J, Expression of TgTFL1 in the overexpression line TgTFL1-1 in comparison with Columbia-0. * represents <0.05 significance and ** represents <0.01 significance.

Figure 7.

Yeast two-hybrid assay of potential tulip flowering time regulators. A, Protein-protein interactions between TgSOC1L2 and Arabidopsis MADS domain proteins (synthetic drop-out medium – Leu, Trp, and His + 1 mm 3-aminotriazole). B, Protein-protein interaction of TgTFL1 and Arabidopsis FD and FDP proteins (synthetic drop-out medium – Leu, Trp, and His + 1 mm 3-aminotriazole). AtFT, AtTSF, and AtTFL1 were added to the assay as positive controls. AD, Activation domain; BD, binding domain.

Figure 8.

Proposed model of the vegetative-to-reproductive phase change in tulip. During spring, high temperature induces the floral induction in tulip by first repressing TgTFL1 and TgSOC1L1. After this suppression, the floral activators TgSOC1L2 and TgFT-like are induced, leading to direct or indirect activation of floral meristem and organ identity genes (TGSQA, TGSQB, and TgSEP1).