Elsevier

Gene

Volume 404, Issues 1–2, 1 December 2007, Pages 10-24
Gene

Isolation of the three grape sub-lineages of B-class MADS-box TM6, PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development

https://doi.org/10.1016/j.gene.2007.08.005Get rights and content

Abstract

The B class of MADS-box floral homeotic genes specifies petal and stamen identity in angiosperms. While this group is one of the most studied in herbaceous plant species, it has remained largely uncharacterized in woody species such as grapevine. Although the B class PI/GLO and AP3/DEF clades have been extensively characterized in model species, the role of the TM6 subgroup within the AP3 clade is not completely understood, since it is absent in Arabidopsis thaliana. In this study, the coding regions of VvTM6 and VvAP3 and the genomic sequence of VvPI, were cloned. VvPI and AtPI were confirmed to be functional homologues by means of complementation of the pi Arabidopsis mutant. Expression analysis revealed that VvPI and VvAP3 transcripts are restricted almost exclusively to inflorescences, although VvPI was detected at low levels in leaves and roots. VvTM6 expresses throughout the plant, with higher levels in flowers and berries. A detailed chronological study of grape flower progression by light microscopy and temporal expression analysis throughout early and late developmental stages, revealed that VvPI expression increases during pollen maturation and decreases between the events of pollination and fertilization, before the cap fall. On the other hand, VvTM6 is expressed in the last stage of anther development. Specific expression of VvAP3 and VvPI was detected in petals and stamens within the flower, while VvTM6 was also expressed in carpels. Moreover, this work provides the first evidence for expression of a TM6-like gene throughout fruit growth and ripening. Even if these genes belong to the same genetic class they could act in different periods and/or tissues during reproductive organ development.

Introduction

In angiosperms, species from different taxa have colonized almost every habitat, resulting in an enormous range of morphological diversity in reproductive organs. Therefore, it is not surprising that genes involved in the control of floral development have become an important focus in the study of plant development and evolution.

One of the most studied group of flowering genes are those belonging to the MADS-box gene family (reviewed by Jack, 2004). MADS-box genes encode transcription factors containing a 58 amino acid DNA binding motif (MADS domain) and participate in diverse eukaryotic physiological processes. They have been defined on the basis of primary sequence similarity of numerous proteins from different organisms, including yeast, plants, insects, amphibians and mammals (Shore and Sharrocks, 1995). In plants, they regulate a variety of other developmental processes, such as root architecture (Burgeff et al., 2002), dehiscence of dry fruits (Liljegren et al., 2000, Ferrandiz et al., 2000), ripening in fleshy fruits (Vrebalov et al., 2002), anthocyanin production (Lalusin et al., 2006) and seed pigmentation (Nesi et al., 2002), among many other functions. The MADS-box genes correspond to a monophyletic gene superclade. Within this clade the MIKC-type MADS-box genes have a conserved structural organization, including the MADS (M), Intervening (I), Keratin-like (K) and C-terminal (C) domains (Theissen et al., 2000, Becker et al., 2000).

Genetic studies of the MADS-box family in Arabidopsis thaliana and Anthirrinum majus lead to the proposal of the ABC model, which explains how the development of a specific floral organ takes place within a specific section of the flower (Coen and Meyerowitz, 1991). In this model, floral organ identity genes have been subdivided into five different classes, termed A, B, C, D and E class genes, which act in a combinatorial manner to determine the formation of sepals, petals, stamens, carpels and ovules (Weigel and Meyerowitz, 1994, Theissen et al., 2000, Honma and Goto, 2001). With the exception of APETALA2 (AP2), all genes belonging to these classes are MADS-box genes.

All B-function genes belong to the MADS-box family. They fall into either one of two different lineages, named APETALA3/DEFICIENS (AP3/DEF) and PISTILLATA/GLOBOSA (PI/GLO) (Theissen et al., 1996). The AP3 and PI lineage genes are thought to represent paralogous gene lineages that arose from a duplication event before the origin of the angiosperms (Kramer et al., 1998, Theissen et al., 2000). In addition, the AP3 lineage underwent another major duplication at the base of the core eudicots, giving rise to two AP3 sub lineages: the euAP3 and the Tomato MADS-box gene 6 (TM6) (Kramer et al., 1998, Kramer and Irish, 2000).

In Arabidopsis, AP3 and PI genes are both required to specify petal and stamen identities and their expression is restricted to these organs. Similar expression patterns have been reported in other core eudicots such as apple (Malus domestica, Yao et al., 2001) and petunia (Petunia hybrida, Vandenbussche et al., 2004). Homeotic changes are produced in Arabidopsis when there is a loss of function of either PI or AP3, producing flowers with two whorls of sepals and double numbers of carpels, without any stamens or petals (Bowman et al., 1989). Among the AP3 sublineages, euAP3 proteins have been studied extensively, but little is known about the function of TM6 in higher eudicots since this gene is absent in Arabidopsis (Lamb and Irish, 2003). Recently, the functional characterization of TM6 was reported in tomato (Solanum lycopersicum) and petunia (De Martino et al., 2006, Rijpkema et al., 2006). These studies demonstrated functional diversification between euAP3 and TM6, in which the former plays a role in petal development and the latter appears to play a predominant role in stamen differentiation.

Although the flowering process in herbaceous species has been well characterized, the genetic and environmental factors regulating flowering in woody species are far from being elucidated. In grapevine (Vitis species), flower organ formation begins with an uncommitted flower primordium (floral initiation) which takes place within the dormant buds of growing shoots just after bloom of the current season's flower cluster (Mullins et al., 1992). The flower primordium is capable of differentiating into either a flower cluster or a tendril, depending on the environmental conditions which affects gibberellin synthesis (Boss and Thomas, 2002). Within buds, flower clusters continue to develop from this primordium in the spring of the following season during the first stages of shoot growth (Pratt, 1971). Sepal development is minimal and five petals develop over the floral apex. The petals are interlocked at their margins by special cells, forming a protective covering known as the cap, that is also present in some balsaminoid asterids such as Marcgravia (Geuten et al., 2006) and which protects the reproductive floral organs (Srinivasan and Mullins, 1978). Upon anthesis, the flower does not open at the tip; instead the cap becomes detached at the base and falls away in a process known as dehiscence or bloom. After pollination and fertilization, each ovary from the inflorescence develops to form a berry.

The latest studies on understanding the molecular basis of flowering in Vitis species have described the cloning and characterization of flowering time genes and A, C and E-class genes. The VFL gene, an ortholog of the Arabidopsis flowering initiation LFY, was found to play a role in the development of some vegetative organs, in addition to flowering meristem specification and development (Carmona et al., 2002), and could be related to the maintenance of indeterminacy before the differentiation of apical meristems into flowers, leaves or tendrils. Some grape MADS-box genes have also been cloned. To study the early steps of flower initiation and development in grapevine, Calonje et al. (2004) isolated two grapevine MADS-box orthologs, VFUL-L and VAP1, which belong to the A-class, and analyzed their expression patterns during vegetative and reproductive development. Related to the C-class genes, VvMADS11 was identified showing homology to the Arabidopsis AG, SHP1 and SHP2 genes (Boss et al., 2001). This gene was expressed in flowers and throughout most of grape berry development, suggesting a regulatory role in flower and fruit development. Additional C-class genes include VvMADS3, related to AGAMOUS-LIKE6 and 13, and VvMADS5, related to AGAMOUS-LIKE11 (Boss et al., 2002). Associated to the E-class, VvMADS2 and VvMADS4, related to SEPALLATA genes, were also identified (Boss et al., 2002).

With the exception of VvMADS9, a PISTILLATA homologue sequence recently identified (Sreekantan et al., 2006), all of these grape genes are not members of the B-class. In addition, no grape B-class genes have being functionally characterized. In this study, we report the isolation of three B-class MADS box genes, VvTM6, VvPI and VvAP3. The functional and expression analyses undertaken may contribute to understand their function in flowering and other processes regarding, for instance, fruit development.

Section snippets

Plant materials and growth conditions

Different vegetative and reproductive tissues were collected from grapevine plants (Vitis vinifera L. cv. Cabernet-Sauvignon) growing in commercial fields in central Chile. Wild-type and mutant Arabidopsis thaliana plants were grown in hydroponic media (Gibeaut et al., 1997) in a culture chamber under 150 μmol m 2 s 1 photosynthetically active radiation and a 16/8 h light/dark photoperiod at 24 ± 2 °C.

Tissue and organ sampling, nucleic acid isolation and cDNA synthesis

Genomic DNA was isolated as described by Porebski et al., 1997. cDNA was prepared from young

Cloning of grape B-class MADS genes

With the aim of obtaining B-class MADS-box genes involved in flower development of V. vinifera, degenerate primers were designed using conserved regions of MADS-box gene sequences from Arabidopsis, apple and Antirrhinum. These primers were used in PCR reactions with cDNA and genomic DNA of V. vinifera cv. Cabernet-Sauvignon isolated from pre-bloom inflorescences. Four of the isolated cDNA clones had significant similarity with the PISTILLATA gene from Arabidopsis and 3 clones with TM6 from

A scenario for the evolution of B-class MADS-box genes in Grapes

While the B class of MADS-box genes is the most studied group in other plant species, it has remained largely uncharacterized in grapes. B-class genes comprise the PI/GLO and AP3/DEF lineages, whose members have been associated with petal and stamen organ fate determination. In addition to this important homeotic function, B class genes have been used as a model to study how sequence changes (i.e. gene duplication and frameshift mutations) have allowed sub and neofunctionalization of these

Acknowledgments

We gratefully acknowledge Dr. Beth Krizek for providing Arabidopsis pi-1 mutant seeds. Thanks to Vicente Castro for their advice in phylogenetic analysis and to Dr. Marco Lardies for his statistical analysis assistance. This work was supported by Chilean Wine Consortium 05CTE01-03, Fruit Consortium and by Fellowships awarded to MJ Poupin (CONICYT) and JT Matus (MECESUP and CONICYT AT24060171).

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