Florigen is involved in axillary bud development at multiple stages in Arabidopsis

Abstract

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.

Regulation of axillary bud development is crucial for the plant architecture, which in turn affects assimilate production and flower/seed fecundity. In Arabidopsis (Arabidopsis thaliana), axillary buds are formed on the base of foliage leaves, and develop into an inflorescence shoot after floral transition of the plant. Our recent work¹ showed that florigen protein, FT, moves from leaves into the subtended axillary bud and promotes the phase transition of the axillary meristem, while BRC1, an axillary bud-specific factor, suppresses it through interaction with FT and another florigen protein TSF. Interaction between BRC1 and FT (and possibly TSF as well) is not mediated by 14–3-3 protein, which is necessary for FT to form a complex with FD.² In the axillary buds of brc1 mutant, FT-downstream genes are over-induced, and ectopic expression of BRC1 in the shoot apical meristem causes delay in floral transition. These findings suggest that modulation of florigen signal is conferred by BRC1 through direct interaction with FT or TSF possibly present in the florigen complex (FT/14–3-3/FD or TSF/14–3-3/FD) in axillary buds. Genetic interactions between BRC1 and FT and other flowering-pathway genes also support the model that BRC1 inhibits the activity of florigen in axillary buds.

It was also demonstrated that the 2 florigen genes are involved in the promotion of axillary shoot elongation.³ In axillary shoots of ft, tsf, and ft tsf mutants, the onset of elongation is delayed and the growth rate is reduced. It was also confirmed that these alterations are independent of the florigen’s effect on the floral transition in the primary shoot. These findings imply that florigen action is not restricted to the promotion of floral transition, but is also involved in coordinated systemic changes of the plant upon flowering. This fits well with a recent notion of multi-faceted physiological roles of florigen in diverse plant species (for a review, see ref. 4).

Here, we report another possible role of the 2 florigen genes in axillary bud development revealed by ectopic overexpression analysis. It was previously reported that double overexpression of FT (or TSF) in combination with LEAFY (LFY) under the control of cauliflower mosaic virus (CaMV) 35S promoter drastically accelerates flowering, resulting in formation of the whole shoot system with a few leaves and a single terminal flower.^5-9 Closer observation revealed that ectopic overexpression of FT or TSF with LFY induced ectopic axillary buds at the base of cotyledonary petiole in 35S:LFY/-; 35S:FT/- or 35S:LFY/-; 35S:TSF/- plants, which do not usually appear in the wild-type plants of laboratory accessions such as Columbia (Col) and Landsberg erecta (Ler) (Fig. 1 and Table 1; see also Figure 3E of ref. 6). LFY or TSF single overexpression also induced ectopic buds at the cotyledonary axils, but at much lower frequency than the double overexpression, which resulted in ectopic bud formation at no less than one-fourth of cotyledonary axils (Table 1). The cotyledonary buds of the double overexpression plants usually consisted of a single floral bud with peduncle, while those of the LFY or TSF single overexpression plants remained without macroscopic floral organs, indicating the single overexpression of either gene is insufficient to support further development (Fig. 1B). These observations suggest that FT and TSF, combined with LFY, promote initiation or early development of axillary buds in addition to floral transition and elongation of axillary buds.

Figure 1. Representative image of transgenic plants. 35S:LFY (homozygous) or 35S:LFY/- (hemizygous) (A, B), 35S:FT #1 (weak line) (C), 35S:LFY/-; 35S:FT #1/- (D), 35S:FT #11 (strong line) (E), 35S:LFY/-; 35S:FT #11/- (F), 35S:TSF #2 (strong line) (G), 35S:LFY/-; 35S:TSF #2/- (H), 35S:TSF #4 (very weak line) (I), 35S:LFY/-; 35S:TSF #4/- (J) are shown. Plants were grown on 1/2 MS agar plates for 15 d under 16h light/8h dark long day conditions at 22 °C. Arrowheads indicate primary inflorescences or flowers. Arrows indicate ectopic axillary buds at cotyledonary axils. Transgenic lines (all in Col background) were described previously.^6,8

Table 1. Frequency of ectopic buds at cotyledonary axils.

Genotype	Number of axillary buds at cotyledonary axils	Frequency of axillary bud formation per cotyledonary axil	Number of plants
35S:LFY or 35S:LFY/-	2	0.04	25
35S:FT #1	0	0	41
35S:FT #11	0	0	42
35S:TSF #2	1	0.01	38
35S:TSF #4	0	0	45
35S:LFY/-; 35S:FT #1/-	6	0.38	8
35S:LFY/-; 35S:FT #11/-	9	0.25	18
35S:LFY/-; 35S:TSF #2/-	11	0.42	13
35S:LFY/-; 35S:TSF #4/-	0	0	14

Plants were grown under same conditions as in Figure 1 for 15 d and the number of axillary buds at cotyledonary axils was counted under a dissecting microscope. Note that the combination between 35S:TSF #4 (very weak transgene) and 35S:LFY did not result in axillary bud formation at cotyledonary axils.

Ectopic buds at the cotyledonary axils in these plants are reminiscent of brc1 mutant, which sometimes develops axillary shoots on the cotyledonary axils.¹⁰ Although how phenotypes of these plants are related in terms of molecular interactions remains to be examined, it is envisaged that BRC1 acts antagonistically to FT and TSF at cotyledonary axils in a similar manner as we previously reported for axillary buds of foliage leaves. Besides brc1 mutant, some mutants such as shoot meristemless (stm) also develops ectopic cotyledonary buds, suggesting that Arabidopsis has potential to form buds at the cotyledonary axils, although initiation or early development of the buds is suppressed in the wild-type background of accessions such as Col and Ler and/or under normal laboratory growth conditions.¹¹ It was previously suggested that LFY stimulates meristematic activity in Arabidopsis and other plants.^12-15 This may explain why LFY single overexpression can initiate ectopic buds at the cotyledonary axils although the frequency is very low. That FT plays a role in axillary bud initiation was also previously proposed. In certain mutant backgrounds of Arabidopsis, such as stm-10, further loss of FT function causes reduction in the number of axillary buds, implying FT has a role in the initiation of axillary meristems redundantly with other factors.^14,16 Our observation that ectopic overexpression of FT alone was not sufficient to induce cotyledonary meristems also indicates that FT requires other factors to promote the formation of axillary buds (Table 1). Thus, our present work provides support for the previously proposed role of LFY and FT in axillary meristem initiation or development.

In conclusion, florigen (FT and TSF) in Arabidopsis regulates meristem initiation or early development of axillary buds. Taken together with our recent findings and previous reports by others,^1,3,14,16 we propose that florigen is involved in multiple steps of axillary bud development, likely to coordinate axillary shoot development with flowering. In other species, FT homologs are also associated with regulation of growth and maturation in various organs such as dormant buds in poplar, compound leaves in tomato, and tubers in potato.^17-19 The modulation of florigen activity by BRC1 suggests the existence of specific modes of action and modulation of the florigen complex depending on organs. It is an important problem to elucidate detailed molecular mechanisms enabling multi-faceted roles of florigen.

Glossary

Abbreviations:

BRC1

BRANCHED1

CaMV

cauliflower mosaic virus

Col

Columbia

FT

FLOWERING LOCUS T

Ler

Landsberg erecta

LFY

LEAFY

stm

shoot meristemless

TSF

TWIN SISTER OF FT