MADS-box genes in plants are putative transcription factors involved in regulating numerous developmental processes, such as meristem and organ identity in inflorescences and in flowers. Recent reports indicate that they are involved in other processes than flower development such as the establishment of developing embryos, seed coat and ultimately in root and fruit development. We have identified seven tomato MADS-box genes that are highly expressed during the first steps of tomato fruit development. According to comparisons of their deduced amino acid sequences, they were classified into two groups: (1) already identified tomato MADS-box genes previously defined as flower identity genes (TAG1, TDR4 and TDR6) and (2) new tomato MADS-box genes (TAGL1, TAGL2, TAGL11 and TAGL12). With the exception of TAGL12, which is expressed near uniformly in every tissue, the other genes show an induction during the tomato fruit development phase I (anthesis) and phase II, when active cell division occurs. In situ hybridization analyses show a specific expression pattern for each gene within the fruit and embryo sac tissues suggesting an important role in the establishment of tissue identity. Yeast two-hybrid analyses indicate that some of these proteins could potentially form dimers suggesting they could act together to accomplish their proposed role.

MADS-domain proteins are important transcription factors involved in many biological processes of plants. Interactions between MADS-domain proteins are essential for their functions. In tomato (Solanum lycopersicum), the number of MIKC(c)-type MADS-domain proteins identified has totalled 36, but a large-scale interaction assay is lacking. In this study, 22 tomato MADS-domain proteins were selected from six functionally important subfamilies of the MADS-box gene family, to create the first large-scale tomato protein interaction network. Compared with Arabidopsis and petunia (Petunia hybrida), protein interaction patterns in tomato displayed both conservation and divergence. The majority of proteins that can be identified as putative orthologues exhibited conserved interaction patterns, and modifications were mostly found in genes underlining traits unique to tomato. JOINTLESS and RIN, characterized for their roles in abscission zone development and fruit ripening, respectively, showed enlarged interaction networks in comparison with their Arabidopsis and petunia counterparts. Novel interactions were also found for members of the expanded subfamilies, such as those represented by AP1/FUL and AP3/PI MADS-domain proteins. In search for higher order complexes, TM5 was found to be the preferred bridge among the five SEP-like proteins. Additionally, 16 proteins with the MADS-domain removed were used to assess the role of the MADS-domain in protein-protein interactions. The current work provides important knowledge for further functional and evolutionary study of the MADS-box genes in tomato.

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.

MADS-box genes encode transcriptional regulators that are critical for flowering, flower organogenesis and plant development. Although there are extensive reports on genes involved in flower organogenesis in model and economically important plant species, there are few reports on MADS-box genes in woody plants. In this study, we have cloned and characterized AGAMOUS (AG), SEEDSTICK (STK) and SEPALLATA (SEP) homologs from peach tree (Prunus persica L. Batsch) and studied their expression patterns in different tissues as well as in fruit pericarp during pit hardening. AG- STK- and SEP-like homologs, representative of the C-, D-, E-like MADS-box gene lineages, respectively, play key roles in stamen, carpel, ovule and fruit development in Arabidopsis thaliana. Sequence similarities, phylogenetic analysis and structural characteristics were used to provide classification of the isolated genes in type C (PPERAG), type D (PPERSTK) and type E (PPERSEP1, PPERSEP3, PPERFB9) organ identity genes. Expression patterns were determined and in combination with phylogenetic data provided useful indications on the function of these genes. These data suggest the involvement of MADS-box genes in peach flower and fruit development and provide further evidence for the role of these genes in woody perennial trees that is compatible with their function in model plant species.

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.

Post-fertilization, the expanding carpel of fleshy fruit goes through a phase change to ripening. While the role of ethylene signalling in mediating climacteric ripening is established, our knowledge regarding the regulation of ethylene biosynthesis and its association with fruit developmental programs is still lacking. A functional screen of tomato transcription factors showed that silencing of the TOMATO AGAMOUS-LIKE 1 (TAGL1) MADS-box gene results in altered fruit pigmentation. Overexpressing TAGL1 as a chimeric repressor suggested its role in controlling ripening, as transgenic tomato fruit showed reduced carotenoids and ethylene levels, suppressed chlorophyll breakdown and downregulation of ripening-associated genes. Moreover, fruits overexpressing TAGL1 accumulated more lycopene, while their sepals were inflated, accumulated high levels of the yellow flavonoid naringenin chalcone and contained lycopene. Transient promoter-binding assays indicated that part of TAGL1 activity in ripening is executed through direct activation of ACS2, an ethylene biosynthesis gene recently reported to be a target of the RIN MADS-box factor. Examination of TAGL1 transcript and its overexpression in the rin mutant background suggested that RIN does not regulate TAGL1 or vice versa. It also pointed to RIN-dependent and -independent processes that are regulated by TAGL1. We also noticed that fruit of TAGL1 loss-of-function lines exhibited a thin pericarp layer indicating an additional role of TAGL1 in carpel expansion prior to ripening. The results add a new component to the current model of the regulatory network that controls fleshy fruit ripening and its association with the ethylene biosynthesis pathway.