A TERMINAL FLOWER1-like gene from perennial ryegrass involved in floral transition and axillary meristem identity

Abstract

Control of flowering and the regulation of plant architecture have been thoroughly investigated in a number of well-studied dicot plants such as Arabidopsis, Antirrhinum, and tobacco. However, in many important monocot seed crops, molecular information on plant reproduction is still limited. To investigate the regulation of meristem identity and the control of floral transition in perennial ryegrass (Lolium perenne) we isolated a ryegrass TERMINAL FLOWER1-like gene, LpTFL1, and characterized it for its function in ryegrass flower development. Perennial ryegrass requires a cold treatment of at least 12 weeks to induce flowering. During this period a decrease in LpTFL1 message was detected in the ryegrass apex. However, upon subsequent induction with elevated temperatures and long-day photoperiods, LpTFL1 message levels increased and reached a maximum when the ryegrass apex has formed visible spikelets. Arabidopsis plants overexpressing LpTFL1 were significantly delayed in flowering and exhibited dramatic changes in architecture such as extensive lateral branching, increased growth of all vegetative organs, and a highly increased trichome production. Furthermore, overexpression of LpTFL1 was able to complement the phenotype of the severe tfl1-14 mutant of Arabidopsis. Analysis of the LpTFL1 promoter fused to the UidA gene in Arabidopsis revealed that the promoter is active in axillary meristems, but not the apical meristem. Therefore, we suggest that LpTFL1 is a repressor of flowering and a controller of axillary meristem identity in ryegrass.

Figure 1

Comparative morphology of perennial ryegrass and Arabidopsis. A, The ryegrass vegetative apex is very compact with the SAM and the semicircular ridges that later will give rise to leaves and tillers. It is positioned on the basal crown and surrounded by developing leaves. Bar = 1.0 mm. B, The ryegrass inflorescence consists of spikelets alternately attached to the main axis (rachis). Each spikelet consists of three to 10 flowers. Bar = 1.0 mm. C, Schematic diagrams of ryegrass and Arabidopsis. During vegetative growth the SAM of ryegrass and Arabidopsis produce very closely spaced leaves in a rosette. After the floral transition the SAM of both species elongate (bolt) and floral organs (circles) are produced along the main axis. In both plants secondary shoots arise from the axils of subtending leaves. In Arabidopsis wild type, flowers mature in an acropetal order and the SAM grows indefinitely (arrowheads), whereas in the tfl1 mutant the SAM and the secondary shoots terminate in a flower. Like the tfl1 mutant, the ryegrass SAM and secondary shoots also terminate in a flower. Maturation of flowers in the ryegrass inflorescence is basipetal, and all the secondary shoots formed below the apex also develop into arrays of flowers in a cymose pattern. The collar is a special meristematic region on the leaf blade in the junction between the leaf blade and the stem (black circles). An enlargement of a floret is shown (redrawn from K. Esau, Anatomy of Seed Plants, Ed 2. Wiley and Sons, New York, 1977). Each floret consists of four whorls of organs. The outermost whorl consists of the palea and the lemma surrounding the lodicules (whorl 2), the three stamens (whorl 3), and the ovary (whorl 4), which is interpreted as syncarpous, consisting of two or three carpels forming the ovary (C).

Figure 2

Genomic organization of LpTFL1 and similarity of the deduced protein with other plant PEBPs. A, The upper bar shows the genomic organization of the gene, including the untranslated (black boxes) and the translated (white boxes) regions. A 180-bp TFL1-like DNA fragment was isolated from ryegrass by RT-PCR. B, Comparison of the deduced protein sequence for the LpTFL1 gene (accession no. AF316419) with those of TFL1 (Bradley et al., 1997; Ohshima et al., 1997), CEN (Bradley et al., 1996), SP (Pnueli et al., 1998), BNTFL1-1 and BNTFL1-3 (Mimida et al., 1999), CET1, CET2, and CET4 (Amaya et al., 1999), FDR1 and FDR2 (accession nos. AAD42896 and AAD42895, respectively), and FT (Kardailsky et al., 1999; Kobayashi et al., 1999). CLUSTAL W program was used to make the alignment and the deduced distance tree. Identical residues are in black. Dashed lines indicate gaps introduced by the program to achieve maximum alignment. Identical intron positions among all species are marked with black arrowheads. White arrowheads indicate amino acids identified to be at the ligand-binding sites by crystallography (Banfield and Brady, 2000) and asterisks indicate amino acids in which point mutations were described for Arabidopsis (Bradley et al., 1997; Ohshima et al., 1997) and tomato (Pnueli et al., 1998). C, Distance tree of different plant PEBPs. The lengths of the horizontal lines are proportional to the similarity between the predicted protein sequences.

Figure 3

LpTFL1 mRNA levels in various tissues detected by ribonuclease protection assay (A) and RT-PCR (B). Fifteen micrograms of RNA was used from each kind of tissue that was hybridized with a 350-bp LpTFL1 and a 180-bp GAPDH antisense riboprobe before RNase digestion. For a positive control, LpTFL1 antisense probe was incubated with yeast RNA. B, Five micrograms of RNA from meristems and roots from different time-points during flowering induction was used for the RT-PCR.

Figure 4

UBI-LpTFL1 dramatically alters the duration of the vegetative phase of Arabidopsis. A, RNA gel-blot analysis of primary transformants (lines 1–30) and wild-type plants (WT). Fifteen micrograms of RNA from rosette leaves was blotted and probed with a LpTFL1 cDNA probe. Transgenic lines 5, 16, 29, and 30 have single-copy insertions as detected by DNA-blot analysis (not shown). Lines 2, 7, 9, 11, and 13 were non-flowering. B, Expression of AP1 and ACTIN in a tfl1-14 mutant line, wild type, and in a UBI::LpTFL1 plant (line 17) as detected by RT-PCR on 5 μg of RNA from each plant. C, Number of cauline leaves produced on the main stem in tfl1 mutant, the complemented mutant (tfl1*), wild type (WT), and UBI-LpTFL1 primary transformants (groups A–D). Each bar represents the mean value of the plants within the specific group. Numbers above the bar indicates the total number of days from germination till the onset of the first flower. The plants were grouped according to the time to flowering: (A ≥ 75 d; B ≥ 100 d; C ≥ 150 d; D non-flowering [NF]) The number of plants in each group is tfl1, 6; tfl1*, 6; WT, 6; A, 6; B, 13; C, 5; and D, 5.

Figure 5

The effect of UBI-LpTFL1 on the morphology of Arabidopsis. A, The UBI::LpTFL1 Arabidopsis primary transformants, line 1 and 2 (right-hand side), showing extensive vegetative growth and up to fourth-order branching 4 months after germination compared with a 1-month-old flowering wild-type plant (left side). Line 2 (middle) was non-flowering after 7 months of growth. B, The SAM of most UBI::LpTFL1 Arabidopsis lines is compact, filled with leaf primordia, and covered with trichomes. C and D, Trichome distribution on the adaxial surface of the uppermost cauline leaves on the main stem of UBI::LpTFL1 (C) compared with wild-type cauline leaves at same age (D). E, In the UBI::LpTFL1 plants leafy shoots filled with trichomes are produced in place of normal flowers on the upper coflorescences.

Figure 6

LpTFL1 promoter activity in Arabidopsis revealed by GUS expression. A, Thirteen-day-old seedling; B, lower stem section bearing a coflorescence subtended by a cauline leaf 7 d after bolting,