Dev Genes Evol (2004) 214:122–127 DOI 10.1007/s00427-004-0388-2 SHORT COMMUNICATION Chun-Neng Wang · Michael Mller · Quentin C. B. Cronk Altered expression of GFLO, the Gesneriaceae homologue of FLORICAULA/LEAFY, is associated with the transition to bulbil formation in Titanotrichum oldhamii Received: 24 September 2003 / Accepted: 21 January 2004 / Published online: 13 February 2004 Springer-Verlag 2004 Abstract Titanotrichum oldhamii inflorescences switch GFLO and FLO, and indicates that the gene may be useful from flower to bulbil production at the end of the for phylogenetic reconstruction at the genus or family flowering season. The structure of the bulbiliferous shoots level. resembles the abnormal meristematic organization of the Antirrhinum mutant, floricaula. Gesneriaceae-FLORI- Keywords Bulbil · Gemma · Evolution of development · CAULA (GFLO) is thus a candidate gene in the regulation Floral induction · GFLO expression of bulbil formation. To investigate this hypothesis, part of the GFLO gene (between the second and third exon) was isolated using degenerate primers designed in regions Introduction conserved between Antirrhinum, Nicotiana and Arabidop- sis, followed by genome walking to obtain the complete The induction of flowering is one of the most important gene and flanking sequences. RT-PCR results showed that developmental transitions for sexually reproducing an- the GFLO homologue is strongly expressed in inflores- giosperms. When plants enter the floral transition stage, cence apical meristems and young flowers. However, in shoot apices switch from vegetative to reproductive meristems that had switched to bulbil formation, GFLO mode, often in response to environmental or endogenous transcription was greatly reduced. The down-regulation of signals (Simpson et al. 1999). GFLO in bulbil primordia indicates that this gene is However, in Titanotrichum, a reversal of this transi- connected to, or part of, the bulbil-flower regulatory tion, from flowering to bulbiliferous meristems, occurs at pathway. Phylogenetic analysis confirms the orthology of the end of the flowering season (Fig. 1). When plants are exposed to long-day (LD) conditions or in summer Edited by G. Jrgens flowers are initiated, while under short-day (SD) condi- tions or in autumn bulbil primordia are formed, replacing C.-N. Wang ()) · M. Mller · Q. C. B. Cronk all “floral” meristems at the top of the inflorescence. In Royal Botanic Garden, addition, numerous new “bulbiliferous shoots” (newly 20A Inverleith Row, Edinburgh, EH3 5LR, UK formed shoots containing only bulbils) are initiated within e-mail: [email protected] Tel.: +1-604-8229666 the inflorescence (Wang and Cronk 2003). These bulbil Fax: +1-604-8222016 shoots initiate in the axils of most bracts, and the existing inflorescence starts to branch vigorously. Usually, bulbils C.-N. Wang · Q. C. B. Cronk develop in clusters of 50–60 at each node of the Institute of Cell and Molecular Biology, inflorescence in the place of flowers. These clusters are The University of Edinburgh, Edinburgh, EH9 3JH, UK reminiscent of a compressed lateral branch. Tens of thousands of V-shaped bulbils can thus be produced from Present address: a single plant. When bulbils start forming, pollinated C.-N. Wang, UBC Botanical Garden flowers in the lower part of an existing inflorescence can and Centre for Plant Research, still form viable seeds, but the seed set is generally low. A University of British Columbia, characteristic fertilization failure occurs in any residual 6804 SW Marine Drive, Vancouver, B.C., V6T 1Z4, Canada flowers at the top of the inflorescence, possibly due to Present address: resource competition between bulbil and ovule develop- Q. C. B. Cronk, UBC Botanical Garden and Centre ment (Wang and Cronk 2003). for Plant Research, The transformation from floral meristems to bulbil University of British Columbia, primordia is an occasional phenomenon in flowering 6804 SW Marine Drive, Vancouver, B.C., V6T 1Z4, Canada 123 Fig. 1 Inflorescence transition in Titanotrichum oldhamii (A–C) low-light environment, bulbil primordia are generated in the axil of and SEM pictures of the equivalent primordia shown below (D–F), the bulbil shoot (D). Occasionally, a multi-bracteole unit is formed after Wang and Cronk (2003). The bulbil shoot (A, D), or bracteose at the axil of inflorescence node (E). B Bulbil primordia, Br bract, branching (B, E) develop from a floral inflorescence (C, F). In a C petals, S stamen, St staminode, Te bracteoles plants (e.g. Polygonum viviparum and Mimulus gemmi- Here we investigate the hypothesis that the Gesneri- parus; Diggle 1997; Moody et al. 1999) perhaps based on aceae FLO/LFY homologue (GFLO) is involved in the a common genetic mechanism in these plants. Bulbil regulation of bulbil formation and describe the isolation production in such plants is often related to environmental of the FLO/LFY homologue from Titanotrichum. To test conditions, as well as intrinsic factors such as position on whether GFLO is expressed, an RNA transcript RT-PCR the inflorescence (Diggle 1997). The inflorescence of analysis was performed to check the expression pattern Titanotrichum is an indeterminate raceme, as in Antir- among stages of bulbil and flower development in rhinum and Arabidopsis, and the shoot apical meristem Titanotrichum. The sequence of GFLO was also com- (SAM) continues growing, with flowers formed in a spiral pared to several available FLO/LFY sequences from phyllotaxy until the apex eventually senesces. When GenBank to investigate GFLO evolution. We hope this shoot apices of inflorescences are removed, even in study may provide potential lines of investigation for individuals growing under LD conditions, bulbil forma- further studies on the mechanism of bulbil development in tion commences from all axillary meristems immediately plants. after the physical manipulation of the SAM (Wang and Cronk 2003). Therefore, not only environmental condi- tions but also hormone regulation may be involved in Materials and methods bulbil initiation. In Antirrhinum, the floricaula mutant (flo) results in Primer design the transformation of flowers into indeterminate axillary Part of GFLO (from the 30 end of second exon to the middle of the inflorescence shoots bearing a spiral of single bracts third exon) was amplified with a pair of highly degenerate primers (Coen et al. 1990). A similar phenotype occurs in the designed from several FLO/LFY homologues across angiosperms Arabidopsis mutant leafy (lfy), in which most of the (M. Frhlich, Natural History Museum, London, personal commu- flowers are replaced by sepal-like structures bearing nication). The amplified products were then extensively cloned (to saturation) into vectors and more than 20 clones were sequenced trichomes (Weigel et al. 1992). The bulbiliferous inflo- according to procedures recommended by the manufacturer for the rescence and “bracteose branching” phenotype (an inter- QIAGEN PCR CloningPlus kit and QIAGEN Spin Miniprep kit mediate state between flowering and bulbiliferous inflo- (Qiagen, Dorking, United Kingdom). To extend into the first, rescence) in Titanotrichum are reminiscent of these second and third exon regions of GFLO, we designed two pairs of mutant phenotypes (see Wang and Cronk 2003). Since degenerate primers using published sequences of FLO (GenBank no. M55525), NFL1 (AH006598), NFL2 (AH006599), ALF the initiation of bulbils in Titanotrichum is day-length (AF030171) and LFY (M91208), and our first GFLO sequence sensitive and involves the conversion of floral primordia fragment. The region between the end of exon 2 and the end of into vegetative structures with no floral organ formation, exon 1, including intron 1, was amplified using our newly designed 0 the FLO/LFY homologue in Titanotrichum is a possible primers LFY-F1 (forward: 5 -GCYCTTGAYGCTCTYTCHCAA- GAA-30) and LFY-Y1R (reverse: 50-CTTRGYKGGRCATTTYT- candidate gene for the regulation of the meristem tran- CRCC-30). The region between the beginning of exon 3 and the 30 sition. end of the exon 3 was amplified using the newly designed prim- ers LFY-WZF1 (forward: 50 CCARGTGTTYAGRTACGCRAAG- 124 AA-30) and LFY-Z1R (reverse: 50-GRAGCYTGGTGGGSACAT- trees. To further evaluate the evolution of Titanotrichum GFLO,we ACCA-30). PCR profiles for the above two sets of primers started amplified GFLO second exon sequences from two other Gesner- with an initial denaturing step of 94C for 3 min, followed by iaceae species, Besleria labiosa (GenBank: AY523620) and Strep- 35 cycles of 94C for 45 s, 57C for 45 s and 72C for 3 min, and tocarpus rexii (GenBank: AY523621). was terminated by a 10-min final extension step at 72C. With the exception of the primers (MWG-Biotech, Milton Keynes, United Kingdom), all PCR reagents, including Taq polymerase, were obtained from Bioline (Bioline, London, United Kingdom). The Results and discussion PCR products were cloned into vectors and sequenced. Sequence of Titanotrichum GFLO Genome walking The complete FLO/LFY homologue (together with 50 and 0 The genome walking protocol used (G. Ingram and K. Coenen, 3 flanking region) was isolated from Titanotrichum. The University of Edinburgh, personal communication) was adapted gene (exons and introns) comprises a total of 1,687 bp from the original of Silbert et al. (1995). First, 2.5 mg genomic (GenBank: AY523619). The degenerate primers used DNA was digested with 6-bp blunt-end cutting restriction enzymes FLO LFY Antirrhinum (e.g. EcoRV, PvuII, PmlI and SmaI) in a 100-ml reaction according