Cauliflower (Brassica oleracea var. italica) and Broccoli (Brassica oleracea var. botrytis) differ mainly in the stage of reproductive arrest. Cauliflower curd is an inflorescence meristem, while broccoli arrests just before anthesis. Arabidopsis studies led to the hypothesis that a mutant BoCAL allele arrested cauliflower earlier. Later, a mutant in BoAP1 was found to have similar effects. These partially redundant genes, and several identified since, are present in multiple copies in B. oleracea. Understanding their role in the arrest requires quantification of transcript abundance analysis by real-time PCR. Designing selective PCR primers is a critical first step in the process. Designs were based on alignment among the genes of interest (MADS-box genes BoCAL, BoAP1, FUL, and the non MADS-box genes LFY and TFL1) and their paralogs. The high sequence similarity (some over 95%) makes the target transcripts difficult to distinguish. Therefore, primers were designed mostly for targets in the 3'UTR region in order to gain specificity. Short amplicons, 68bp to 200bp, were required for the high PCR efficiency required to quantify these low-abundance transcripts. Primers were evaluated by conventional RT-PCR and real-time PCR. By altering temperature, Total RNA was isolated from plants that were arrested at three developmental stages, inflorescence meristem (cauliflower), floral meristem (intermediate), and floral bud (broccoli) by varying temperature. RT-PCR products were single bands of the expected size, despite the high homology between genes under study. Real-time melting curve analysis (fluorescence derivative vs. melting temperature) corroborated the presence of a single amplicon. The identity of products was confirmed by sequencing and restriction enzyme digestion.
Denise V. Duclos* and Thomas Björkman
Denise V. Duclos and Thomas Björkman
Cauliflower (Brassica oleracea var. botrytis) and broccoli (B. oleracea var. italica) differ in the developmental stage of the reproductive meristem at harvest. A cauliflower head is formed by arrest at the inflorescence meristem stage and broccoli at the flower bud stage, and the horticultural value of the crop depends on synchronous development across the head. In other plant species, gibberellin (GA) can promote floral development and is therefore a candidate for providing the early developmental cues that shape the curd morphology. This research investigated the effect of GAs on the two horticulturally important transitions of the reproductive meristem: initiation of the inflorescence meristem and initiation of floral primordia on the proliferated inflorescence meristems. GA is known to affect the former in many species, but effects on the latter have not been determined. It is also not known whether one or both active forms produced by the two GA biosynthetic pathways is involved in the reproductive transitions in this crop. GAs from the early-13 hydroxylation pathway (GA3) and the non-13 hydroxylation pathway (GA4+7) were applied to the shoot apical meristems of cauliflower and broccoli at three developmental stages: adult-vegetative, curd initiation, and curd enlargement. GAs applied during the adult vegetative stage caused the curd to form faster and after fewer additional nodes in both cauliflower and broccoli. GAs applied to the inflorescence meristem did not cause floral primordia to form nor did the expression of transition-associated genes change. Integrator genes BoLFY and SOC1 had constant expression over 24 hours, and meristem-identity genes BoAP1-a and BoAP1-c remained undetectable. However, GAs applied early during the reproductive phase increased bract development in cauliflower curds. This study shows that GAs from both pathways can trigger the vegetative-to-reproductive transition in both cauliflower and broccoli, resulting in earlier curd formation. However, GAs did not advance the inflorescence-meristem-to-floral-primordium transition; on the contrary, they increased bract incidence in cauliflower, a sign of reversion toward the vegetative stage, suggesting that another pathway is responsible for this second transition in cauliflower and broccoli.
Denise V. Duclos and Thomas N. Björkman
Brassica oleracea species differ in the developmental stage of their reproductive meristems at harvest. The stage that characterizes each variety depends on its genetic makeup, environment and the interaction between them. We tested a model of arrest in B. oleracea to determine functional redundancy among the paralogous genes CAL, AP1a, AP1c, FULa, FULb, FULc, and FULd; and to resolve the immediate effect of temperature on gene expression in meristems whose developmental fate is temperature regulated. By varying temperature during reproductive development, three stages of arrest were obtained: inflorescence meristem (cauliflower), floral meristem (intermediate) and floral bud (broccoli), the latter initiated by low temperature. Gene expression was measured by quantitative real time PCR (qRT-PCR). The LFY/TFL1 ratio increased as the reproductive development advanced, mainly due to decreased TFL1 expression; influenced by a dramatic increase in AP1c toward floral bud formation. The expression patterns of the FUL paralogs indicate different roles in reproductive development. FULa was more abundant in the floral primordia, while FULb, FULc, and FULd were associated with earlier arrest at the inflorescence meristem stage. The high expression of FULc and FULd at all stages of arrest differs from their homolog in Arabidopsis. High temperature reduced AP1 and LFY expression but the meristem did not revert from reproductive to vegetative. Floral bud formation in plants recessive for AP1a and CAL reaffirm that functional redundancy among some of these genes can complement the mutations. Varying temperature alone, at a fixed developmental stage, caused little variation in the expression of genes studied, causing small significant differences in TFL1 and AP1c.