A PLENA-like gene of peach is involved in carpel formation and subsequent transformation into a fleshy fruit

Abstract

MADS-box genes have been shown to play a role in the formation of fruits, both in Arabidopsis and in tomato. In peach, two C-class MADS-box genes have been isolated. Both of them are expressed during flower and mesocarp development. Here a detailed analysis of a gene that belongs to the PLENA subfamily of MADS-box genes is shown. The expression of this PLENA-like gene (PpPLENA) increases during fruit ripening, and its ectopic expression in tomato plants causes the transformation of sepals into carpel-like structures that become fleshy and ripen like real fruits. Interestingly, the transgenic berries constitutively expressing the PpPLENA gene show an accelerated ripening, as judged by the expression of genes that are important for tomato fruit ripening. It is suggested that PpPLENA might interfere with the endogenous activity of TAGL1, thereby activating the fruit ripening pathway earlier compared with wild-type tomato plants.

Fig. 1.

Phylogenetic tree of several plant MADS-box proteins. The peach proteins have been highlighted by a grey background. A peach MADS-box protein highly similar to Arabidopsis seedstick has also been included (PpSTK: ABQ85556). Protein sequences from other plants have been retrieved from public databases. Their GenBank accession numbers are as follows: Malus×domestica (apple), ‘MdAG’ AF401637, ‘Md10’ CAA04324, and ‘Md15’ CAC80858; Rosa rugosa, ‘RrD1’ AB025643, and ‘Rr BAA90745’; Solanum lycopersicum (tomato), ‘AGL1’ AAM33101, ‘TAG1’ AAM33099, ‘Le Rin’ AAM15775, and ‘Le MC’ AF448521; Antirrhinum majus (snapdragon), ‘AmPLE’ AAB25101, ‘AmFAR’ CAB42988, and ‘AmSQUA’ CAA45228; Vitis vinifera, ‘Vitis MADS 1’ AAK58564; Fragaria×ananassa, ‘FaAG’ AAD45814; Arabidopsis, ‘AG’ NP_567569, ‘SHP1/AGL1’ NP_191437, ‘SHP2/AGL5’ NP_850377, ‘STK/AGL11’ NP_001078364, ‘SEP1/AGL2’ AAA32732, ‘SEP2/AGL4’ AAA32734, ‘SEP3/AGL9’ AAB67832, and ‘AP1’ CAA78909.

Fig. 2.

Relative expression profiles of two peach C-type MADS-box genes in flower and fruit. Grey bars represent the values obtained for PpFAR (462), while black bars indicate those of PpPLE (794). Values (means of the normalized expression) have been obtained by real-time qRT-PCR analyses. Stages S1–S4II encompass the development (S1–S3I) and ripening (S3II–S4II) of peach fruits. Bars are the standard deviations from the means.

Fig. 3.

In situ analysis of PpFAR and of PpPLE expression in peach flower buds. Panels in the top half of the figure show hybridization with a PpFAR antisense probe (A–C) and sense probe (D), while those in the bottom half show hybridisation with a PpPLE antisense probe (E–G) and sense probe (H). Scale bars in A, D, F and I represent 100 μm; in B and G, 20 μm; and in C and E, 50 μm. o, ovule; c, carpel; a, anther; f, filament.

Fig. 4.

Tomato plants constitutively expressing the peach PpPLE (794) cDNA. (A) An ESEM (environmental scanning electron microscopy) picture of a transgenic flower bud with sepals completely fused up to the blossom end of the calyx from which only the style/stigma emerges. (B) Partial removal of the fused calyx permits the partially deformed anthers to be seen. (C) An inflorescence of a transgenic line with a mild phenotype. As in A, the calyx is fused, although not as far as the top, thus allowing the petal tips to become visible at anthesis. For comparison, a wild-type flower is shown in D. Transgenic fruits at various stages of development are shown in E, where a white arrow indicates the fleshy sepals undergoing ripening. A wild-type ripe fruit with leafy sepals still attached is shown in F.

Fig. 5.

Relative expression profiles of ripening-related genes in ripe fleshy sepals of tomato plants overexpressing the peach PpPLE cDNA (794). Grey bars represent the values obtained for transgenic tomato sepals, while white bars indicate those of the wild type. The analysed genes are: PpPLE (794) (peach PpPLE cDNA), ACO1 (climacteric ACC oxidase), PSY1 (tomato chromoplast phytoene synthase), and PG (tomato ripening endopolygalacturonase). Values (means of the normalized expression) have been obtained by means of real-time qRT-PCR. Bars are the standard deviations from the means.

Fig. 6.

Relative expression profiles of ripening-related genes in fruit of tomato plants either wild type or overexpressing the peach PpPLE cDNA (794). Grey bars represent values obtained for the transgenic fruit, white bars indicate those of the wild type. (A) pPLE (794) (peach PpPLE cDNA), ACO1 (climacteric ACC oxidase), PSY1 (tomato chromoplasts phytoene synthase), CAB (a chlorophyll a/b-binding protein-encoding gene used as a marker of the fruit ‘green’ state). (B) Softening-related genes: β-GAL (β-galactosidase), PL (pectate lyase), PME (pectin methyl esterase), PG (endopolygalacturonase), XET (xyloglucan endotransglycosylase), EXP (expansin). (C) Tomato transcription factor-encoding genes: TAGL1 (a C-type MADS-box gene), RIN (a SEPALLATA-type MADS-box gene), NOR (a NAM-like gene), and CNR (an SBP-box gene). Values (means of the normalized expression) have been obtained by means of real-time qRT-PCR. Bars are the standard deviations from the means of three independent replicates.