Cloning and characterisation of grapevine (Vitis vinifera L.) MADS-box genes expressed during inflorescence and berry development☆
Introduction
Grape (Vitis sp.) flowers are unusual in their structure and development. The inflorescences are initiated during spring in latent buds formed in the axil of each leaf. These buds are not axillary to the leaf, but are formed at the basal node of the axillary shoot of each leaf (Fig. 1A). Limited inflorescence development occurs in these buds during the rest of the growing season until the buds enter dormancy and over-winter in a quiescent state (for a review see [1]). In the following spring bud burst occurs and the inflorescence undergoes further maturation and development. During this time the formation of the floral organs on the floral initial begins with the appearance of the calyx. The other floral organs begin development sequentially from the outer whorl to the inner whorl [2]. Sepal development is minimal and as the petals develop they push through the calyx and eventually form a structure (the ‘cap’) protecting the reproductive floral organs (Fig. 1B). The cap is the result of the petals interlocking with one another via special cells formed at the margins [2], [3]. At anthesis, the cap dehisces at the base of the flower and the petals separate and curl up, finally releasing the stamens and allowing pollination (Fig. 1C). Following pollination the ovaries expand as fruit formation is initiated (Fig. 1D). Some early growth is due to cell division, but most of the growth of the berry is the result of expansion of the mesocarp cells [4]. Ripening proceeds during the second phase of berry expansion while the berries are still attached to the vine. Most Vitis species are dioecious, producing either male or female flowers on individual plants. However, cultivated grapevine, Vitis vinifera L., has hermaphroditic flowers and it is thought that this character was selected early during domestication.
One family of genes with a key role in flower and fruit development is the MADS-box gene family. These genes are recognised by a highly conserved region, called the MADS domain, present in the N-terminal region of the proteins they encode. Plant MADS domain proteins also possess a less conserved region known as the K domain, that is separated from the MADS domain by the variable I region. The C terminal regions of MADS domain proteins are not well conserved. Much research has been directed at understanding the role MADS-box genes play in the differentiation of the floral meristem into the various floral organs. This has led to the development of the ‘ABC’ model of floral organ development, which has been refined more recently to include the SEPALLATA MADS-box genes [5], [6], [7], [8]. MADS-box genes also appear to be involved in fruit development in Arabidopsis [9], [10], [11]. This is not surprising as the fruit of Arabidopsis develops from the inner whorl of the flower following pollination. As well as being involved in floral organ and fruit development, Arabidopsis MADS-box genes are also involved in the control of the timing of flowering (e.g. [12], [13], [14]) and the stimulation of root growth in response to nutrients [15].
Flowering and fruit development in grapevine has been extensively studied with regards the physiology of these processes. Given the importance of MADS-box genes in these processes in other plants, we have begun to clone and analyse MADS-box genes from inflorescences and fruit in grapevine. In this study we describe cloning and expression analyses of four MADS-box genes, two isolated from young inflorescences and two from immature berries.
Section snippets
Plant material
The ‘inflorescence 5–8’ and ‘inflorescence 8–12’ samples were dissected from developing shoots on rooted Shiraz canes grown in a glasshouse at CSIRO, Adelaide, and the root sample was obtained from mature canes rooted in Perlite. All the other samples from Shiraz grapevines were obtained from vines grown on a commercial vineyard in Willunga, South Australia. The inflorescences and berry samples (seeds removed) used for the construction of the cDNA libraries were from the cultivar Cabernet
Isolation and sequence analysis of four grapevine MADS-box genes
Using both PCR-based cloning techniques and random EST sequencing we have isolated four grapevine MADS-box cDNAs that are different from a grapevine MADS-box gene (VvMADS1) previously characterised [17]. Two were originally isolated as PCR fragments generated during amplification of MADS-box-like sequences from developing inflorescence cDNA. These were then used to probe a cDNA library made from a similar stage of inflorescence development from which full-length clones were obtained (VvMADS2
Conclusion
The economic value of a grapevine depends on its production of flowers and the development of the pistils into berries following pollination. To understand these processes we are characterising MADS-box genes from grapevine as this family of genes has been demonstrated to play key roles in flower and fruit development in Arabidopsis and other species. We compared the grapevine cDNAs with many of the MADS-box genes identified in Arabidopsis. Each of the grapevine MADS-box genes aligned closely
Acknowledgements
This work was supported in part by grants from the Grape and Wine Research and Development Corporation, the Dried Fruits Research and Development Council and the Cooperative Research Centre for Viticulture. The authors wish to thank Di and John Harvey for allowing us to sample material from their vineyard, Don MacKenzie and Melissa Pickering for excellent technical assistance and Stuart McClure for help with the scanning electron microscope. E. Sensi was supported by a grant from the University
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The nucleotide sequences of the VvMADS2, VvMADS3, VvMADS4 and VvMADS5 cDNAs described in this paper can be obtained from GenBank™ under the accession numbers AF373601, AF373602, AF373603 and AF373604.
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Present address: John Innes Centre, Norwich Research Park, Norwich, Norfolk NR4 7UH, UK.