Down-regulation of TM29, a tomato SEPALLATA homolog, causes parthenocarpic fruit development and floral reversion

Abstract

We have characterized the tomato (Lycopersicon esculentum Mill.) MADS box gene TM29 that shared a high amino acid sequence homology to the Arabidopsis SEP1, 2, and 3 (SEPALLATA1, 2, and 3) genes. TM29 showed similar expression profiles to SEP1, with accumulation of mRNA in the primordia of all four whorls of floral organs. In addition, TM29 mRNA was detected in inflorescence and vegetative meristems. To understand TM29 function, we produced transgenic tomato plants in which TM29 expression was down-regulated by either cosuppression or antisense techniques. These transgenic plants produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Petals and stamens were green rather than yellow, suggesting a partial conversion to a sepalloid identity. Stamens and ovaries were infertile, with the later developing into parthenocarpic fruit. Ectopic shoots with partially developed leaves and secondary flowers emerged from the fruit. These shoots resembled the primary transgenic flowers and continued to produce parthenocarpic fruit and additional ectopic shoots. Based on the temporal and spatial expression pattern and transgenic phenotypes, we propose that TM29 functions in floral organ development, fruit development, and maintenance of floral meristem identity in tomato.

Figure 1

Sequence analysis of TM29. A, An alignment of predicted protein sequences from related MADS box genes using ClustalW analysis. Gaps were introduced to maximize alignment. The MADS box, K box, I region, and C region are identified. The five amino residues at the 3′ terminal were conserved among TM29, DEFH49, AGL2, and AGL4. B, Phylogenetic analysis of selected MADS box proteins using sequences from the MADS box, the I region, and the K box regions. Bootstrap values are shown on branches. Branches with less than 50% of bootstrap support are collapsed. Accession numbers: AG, P17839; FBP2, JQ1690; SEP1, P29382; SEP2, P29384; SEP3, O22456; AP3, P35632; DEF, P23706; DEFH49, S78015; GLO, Q03378; PI, P48007; TAG1, Q40168; and TM5, Q42464.

Figure 2

Northern analysis of TM29 expression. Northern blots were probed with a TM29 cDNA fragment. Loading levels of RNA samples are shown by the gel photograph of stained rRNA bands. A, Total RNA extracted from tomato flower buds (fb), 1- to 7-DPA fruits (fr), young leaves (lf), shoot tips (sh), and roots (rt). B, RNA extracted from ovary at pre-anthesis (pa) and anthesis (a); fruit at 3, 6, 14, and 21 DPA; and young leaves (lf).

Figure 3

In situ analysis of TM29 expression in different tissues in wild-type (WT) tomato. A, Sympodial bud in the axil of a leaf showing TM29 expression at the tip. B, Bifurcating structure with a floral (fm) and an inflorescence (im) meristem. TM29 is expressed uniformly in the floral meristem and strongly at the tip of the inflorescence meristem. Transcripts are also seen in the vascular bundles (vb). C, Tissue section as in B probed with sense RNA as negative control. D, Section showing floral meristems with emerging sepal primordia at different stages. E, Tissue section as in D probed with sense RNA as negative control. F, A floral meristem with elongated sepals. TM29 expression in the sepals is markedly reduced at this stage. G, Flower bud with all four floral organ primordia. TM29 expression is reduced in the petals and is localized to the tip of the stamens where the anthers (an) are formed. Expression continues to be seen in the center of the ovary (ov). H, Flower bud at about 4 d pre-anthesis. TM29 expression is localized to the pericarp (per) region of the ovary and in the tapetum (tap) regions of the stamens. an, Anther region; fm, floral meristem; im, inflorescence meristem; lf, leaf; ov, ovary primordium; per, pericarp region; sb, sympodial bud; se, sepal primordium; st, stamen; tap, tapetal region; vb, vascular bundle. Scale bar = 1.5 mm.

Figure 4

Phenotypes of transgenic tomato plants. A, WT tomato flower at anthesis showing yellow petals and yellow stamens. B, WT flower at 3 DPA with fused stamens. C, Antisense transgenic flower at anthesis showing bigger sepals, green petals, and green stamens forming a loose cone. D, Transgenic flower displaying dialytic stamens. E, Ectopic shoot emerging from the fruit. F, Poorly developed ectopic shoot. G, Ectopic shoot produced flowers and leaf-like struc- tures. H, Ectopic flowers reiterated the development of the primary flower. I, Ectopic shoot showing successive generations (1–4) of ectopic flowers. el, Ectopic leaf; es, ectopic shoot; fr, fruit;. p, petals, se, sepals, st, stamens. Bars = 2 mm.

Figure 5

Scanning electron microscopy (SEM) photographs of floral and leaf tissues of WT and transgenic plants. A, Abaxial surface of WT petal. B, Abaxial surface of transgenic petal showing stomata (sm). C, Abaxial surface of WT stamen. D, Abaxial surface of transgenic stamen. E, Lateral hairs on WT stamens are tightly interweaved. F, Lateral hairs on transgenic stamens showing poor interweaving. G, WT flower showing hairless ovary. H, Transgenic ovary covered with hairs. I, Epidermal surface of WT ovary. J, Epidermal surface of transgenic ovary showing stomata, glandular, and non-glandular hairs. K, SEM of WT leaflet. L, SEM of ectopic leaf of a transgenic plant. M, Abaxial surface of WT leaflet. N, Abaxial surface of ectopic leaf. O, Adaxial surface of WT leaflet. P, Adaxial surface of ectopic leaf. ov, ovary; pe, petal; se, sepal; st, stamen, sm, stoma(ta). Bars in A through F, I, J, and M through P = 50 μm; in K and L, bars = 0.5 mm.

Figure 6

Early development of ectopic shoot. A, Longitudinal section of WT tomato fruit at 4 DPA. B, Transgenic fruit (line AS/45) at 3 DPA showing ectopic shoot development. C, WT fruit at 10 DPA. D, Transgenic fruit at 6 DPA. The ectopic shoot displaces the placenta and ovules within the fruit. es, Ectopic inflorescence; ov, ovule; per, pericarp; pl, placenta; sd, seed. Bars = 500 μm.

Figure 7

The expressions of sense and antisense TM29 transcripts in transgenic lines. A, Total RNA extracted from six sense transgenic plants and the WT plant was probed with antisense probe. Lane 1 contained RNA from the line S/05 showing altered phenotype. Lanes 2 through 6 contained RNA samples from five plants showing no altered phenotypes. B, Total RNA extracted from six antisense transgenic plants and the WT plant was hybridized with antisense and sense probes separately to detect endogenous sense and transgene-expressed antisense transcripts, respectively. The loading levels are shown by hybridization with rRNA gene probe. For symbols of the phenotype, refer to Table I.

Figure 8

The expressions of TM5 and TAG1 in antisense transgenic plants. Total RNA extracted from flowers of four antisense transgenic plants (AS38, 45, 69, and 70) and the WT plant was sequentially probed with TM5, TAG1, and 18S rRNA probes.