JXB Advance Access published online on November 27, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm269
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns
1INRA-UMR DAP, 2 place Viala, 34060 Montpellier Cedex 01, France
2INRA/SupAgro-UMR DIA-PC, 2 place Viala, 34060 Montpellier Cedex 01, France
3CNB, Genética Molecular de Plantas, C/Darwin 3 Cantoblanco, 28049 Madrid, Spain
4CSIRO Plant Industry, PO Box 350, Glen Osmond, SA 5064, Australia
* To whom correspondence should be addressed. E-mail: laurent.torregrosa{at}supagro.inra.fr
Received 6 July 2007; Revised 3 October 2007 Accepted 8 October 2007
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Abstract |
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Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower model.
Key words: Flower development, grapevine, MADS-box, reproductive organs, somatic variants
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Genes involved in flower development have been extensively studied in the Angiosperms using Arabidopsis and Antirrhinum as genetic models, leading to the identification of genes and gene networks controlling these processes (Theißen, 2001; Ausín et al., 2005). Among them, the MADS-box gene family has been shown to play a central role in the determination of flower meristem and flower organ identity. The current genetic model explaining the specification of the flower organ identifies five genetic functions (A-B-C-D-E) mainly specified by MADS-box genes with specific roles in the initiation and development of flower organs in the different flower whorls, from sepals to carpels (Ferrario et al., 2004). While spectacular progress has been made in the understanding of the molecular regulation of flower development in model herbaceous plants, an increasing number of reports show differences in the regulation and the function of homeotic genes in other plant species (Ferrario et al., 2004).
We are interested in the regulation of reproductive development in grapevine (Vitis vinifera L.), a woody perennial vine with a pattern of organ formation and development distinct from those previously described for other plants (Mullins et al., 1992; Boss et al., 2003; Carmona et al., 2007). Grapevine flowers, with four whorls from sepals to carpels, belong to the regular flower type of Eudicot plants and have been well described at the anatomical and morphological level (Pratt, 1971; Hardie et al., 1996). However, the underlying molecular genetic mechanisms regulating flower development are poorly understood. Some putative orthologues of genes controlling flower initiation and development in Arabidopsis have been isolated and characterized (Boss et al., 2001, 2002; Carmona et al., 2002, 2007; Calonje et al., 2004; Boss et al., 2006; Sreekantan et al., 2006), and their expression analyses have suggested some functional differences in this species compared with model plants (Boss and Thomas, 2002; Calonje et al., 2004; Sreekantan et al., 2006). Still, expression of some of these genes in Arabidopsis transgenic plants displayed phenotypes similar to those produced by overexpression of the endogenous Arabidopsis genes (Boss et al., 2006; Carmona et al., 2007). However, their role in grapevine flower development remains unknown since genetic transformation is still a lengthy and tedious process in this species (Bouquet et al., 2003).
The use of natural genetic variation and induced artificial mutations can be very informative in establishing gene function in the absence of genetic transformation (Emmanuel and Levy, 2002; Jack, 2004; Koornneef et al., 2004). This genetic variation can be used in forward and reverse genetic approaches to support causal relationships between gene sequences and phenotypes. Natural genetic variation is starting to be exploited in the identification of genomic regions responsible for specific quality traits in grape (Cabezas et al., 2003; Doligez et al., 2006) and could also be used in both forward and reverse approaches. However, phenotypic variations for early development of reproductive organs have not been described in grapevine and although induced mutagenesis could be an alternative approach to generate phenotypic variation for this trait, mutagenesis studies are scarce in grapevine (Kuksova et al., 1997) and have not been reported to induce flowers with altered formation patterns. One interesting alternative to be considered in grapevine and other woody perennial species is somatic genetic variation, which can be maintained through vegetative propagation. In grapevine, somatic genetic variation has been successfully used so far in three cases to identify genes involved in gibberellic acid signalling (Boss and Thomas, 2002), anthocyanin production (Kobayashi et al., 2004; Walker et al., 2006), and berry early morphogenesis (Fernandez et al., 2006).
The goal in this work was to characterize grapevine somatic variants altered in flower development that could serve as genetic tools to identify the roles of genes involved in this developmental process. The phenotypic description of six somatic variants is presented here, as well as the corresponding RNA expression analysis of three MADS-box genes, previously reported to be related to the formation of the innermost floral whorls throughout flower development (Boss et al., 2001, 2002). The results allowed the development of working hypotheses on the role of genes that could be altered in each case.
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Materials and methods |
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Plant material
The collection of grapevines at the INRA Domaine de Vassal (Marseillan, France, http://www.montpellier.inra.fr/vassal) was examined and found to include six accessions with developmentally aberrant flowers, with or without cluster architecture modifications. These were studied in comparison with the original variety grown under the same field conditions. Abnormal flowers were found in clonal mutants derived from the international cultivars Pinot and Gamay, and from the Southern Europe traditional cultivars Bouchalès, Carignan (known as Mazuelo in its country of origin Spain), Mourvèdre, and Morrastel (similarly known as Monastrel and Graciano, respectively). Early descriptions led to the introduction of these variants in the collection as double-flowered (DF) Gamay, Morrastel, and Pinot, ramose bunch (RB) Carignan, female Bouchalès, and female Mourvèdre. The genetic identity of cultivars and somatic variants was confirmed through genotyping the plants with 20 unlinked microsatellite loci; VVMD5, VVMD7 (Bowers et al., 1996), VVMD21, VVMD24, VVMD25, VVMD27, VVMD28, VVMD32 (Bowers et al., 1999), VVS2 (Thomas and Scott, 1993), VVIB01, VVIH54, VVIN16, VVIN73, VVIN73, VVIP31, VVIP60, VVIQ52, VVIV37, VVIV67 (Merdinoglu et al., 2005), VMC1b4 (unpublished), and VMC4f3 (Di Gaspero et al., 2000). PCR conditions were as described in Lacombe et al. (2003).
Microscopy analyses
Inflorescence samples were taken at anthesis from the original clones and simultaneously from the corresponding somatic variant of at least two of the five representatives established in the field. Alternatively, sampling used greenhouse-grown fertile cuttings according to Mullins et al. (1966). Samples were preserved in a 0.2 M phosphate buffer (pH 7.2), with 10% paraformaldehyde and 2.5% glutaraldehyde for a minimum of 48 h at 4 °C, and stored at the same temperature in 70% ethanol until further processing. For scanning electronic microscopy, after dehydration through a graded ethanol series, explants were critically-point dried with CO2, sputtered with platinum, and observed with a JEOL JSM 6300F microscope.
Histological observations were performed on flowers embedded in Technovit Historesin® after dehydration as described above and following the manufacturer's specifications. For improved sample embedding, an extra step was introduced after ethanol dehydration using three successive (4, 24, and 24 h) 1-butanol baths before impregnation with resin. Semi-thin sections (3 µm) were stained with periodic acid–Schiff to reveal polysaccharide-rich structures and further stained with Naphthol Blue Black to reveal protein-containing bodies.
RNA extraction for gene expression studies
Sampling was done for several stages of inflorescence development defined as follows: stage 1, inflorescence primordium still enclosed within the bud; stage 2, inflorescence clearly separated from the vegetative axis; stage 3, first flower buds visible in the top of the inflorescence; stage 4, initiation of new inflorescence primordia finished, all flowers well separated; stage 5, all flowers formed on fully expanded pedicels; stages 5+, cap drop starting. Flowers and inflorescences were randomly sampled for each clone from the five representatives cultivated in the field.
Total RNA was extracted from 1–2 g of inflorescence tissue as described by Tesnière and Vayda (1991). To get sufficient material, stage 1 and 2 samples were combined before extraction processing. A 100 µg aliquot of crude RNA was further purified using the Qiagen RNeasy cleanup mini-protocol including a DNase treatment, resulting in 10–30 µg of clean RNA. Quality and quantity were evaluated by both electrophoresis and spectrofluorometry.
Real-time PCR analysis
Real-time PCR analyses were performed as described in Fernandez et al. (2006). Using Primer3 software, specific primers (Tm=58–60 °C) were designed (Rozen and Skaletsky, 2000) to amplify 100–200 bp in the 3' untranslated region of the VvMADS1 (GenBank ID: AF265562
[GenBank]
), VvMADS2 (GenBank ID: AF373601
[GenBank]
), VvMADS3 (GenBank ID: AF373602
[GenBank]
), and ubiquitin extension protein VvUBI (GenBank ID: CF406001) genes (see Table 1). The expression levels of the MADS-box genes were determined for three technical replicates and corrected using the corresponding expression level of ubiquitin, VvUBI.
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Origin of the somatic variants
The somatic variants analysed were collected from a wide range of geographical origins and over a long period of time. DF Gamay was introduced from the Ravaz collection (France) in 1949, DF Pinot from the Champagne area in 1951, and DF Morrastel from a French grower in 1956. RB Carignan was introduced from Perpignan Research Station (France) in 1955. Female Bouchalès and female Mourvèdre were introduced more recently from SEPPIC Montauban (France) in 1973 and from the Magaratsh collection (Crimea) via the Oberlin Institute (Colmar, France) in 1979, respectively. The somatic variants, established as five plants for each accession (as were their putative original clones), were recorded as displaying stable phenotypes after repeated vegetative propagation and over successive reproductive cycles. The genetic identity of cultivars and somatic variants was confirmed by genotyping 20 independent microsatellite loci. All the somatic variants displayed genotypes identical to those of the plants from which they were derived (data not shown).
Grapevine flower development
The reproductive development of V. vinifera has been extensively described (Srinivasan and Mullins, 1981). While most of the Vitis species are dioecious, in almost all V. vinifera cultivars, the flower assumes a classical hermaphrodite floral organization with a whorl of five fused sepals followed by a five-petal whorl, five stamens, and the gynaecia being composed of an ovary with two carpels each containing two ovules (Fig. 1A–D).
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Flower development in somatic variants
The vegetative development of the somatic variants was similar to that of the corresponding original plants in each case. No differences in inflorescence number or position were observed between the cultivars and the respective variants. Based on initial morphological examinations, the variants were classified in three major phenotypic groups: (i) reiteration of reproductive meristems (RRM; Carignan variant); (ii) reiteration of perianth whorls (Gamay, Morrastel, and Pinot variants), and (iii) abnormal development of the reproductive whorl (Bouchalès and Mourvèdre variants). Scanning electronic microscopy and histological observations further confirmed this preliminary phenotypic classification and led to the suggestion of specific denominations better suited to the actual flower defects extensively explained below. The Carignan variant was described by the name RRM and all the variants showing reiteration of perianth whorls were described as ‘multiple perianth whorls’ (MPW). Somatic variants displaying abnormal development of the reproductive whorls and initially known as female variants were more specifically described as ‘unfused carpels’ (UFC) for the Bouchalès and ‘carpel-less’ (CLS) for the Mourvèdre variants. Flower development within each one of these phenotypic groups is described below.
Reiterated reproductive meristems (RRM)—Carignan variant
This variant was characterized by an alteration of early inflorescence architecture. After the early stages of inflorescence development, the differentiation of flower meristems (stages 1–2) was impaired and primordia reiterated the development of inflorescence ramifications (Fig. 2A). Histological observations showed reiteration of adjacent bract-floral meristems in tandem, with at least two floral meristems being formed (Fig. 2B). These formations collapsed a few weeks after their initiation. This reiteration was probably the origin of the high number of inflorescence ramifications, hence the ‘ramose bunch’ (RB) description (Fig. 2C, E). This reiteration process caused a delay of 
30 d in the cluster development of the variant with respect to its corresponding cultivar (Fig. 2D). By the end of cluster development some flowers could reach full regular development, although it was frequently observed that the basic floral organization was altered with some reiteration of petals or formation of petaloid anther filaments.
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In general, most regular flowers eventually differentiated at the most distal inflorescence positions that corresponded to the ones developing later. Regular mature flowers were fertile and some berries developed normally up to the ripening stage, although with the delay accumulated in the previous developmental phases. At ripening, mutant vines were therefore observed to bear long, very large bunches with a high number of ramifications and berries (Fig. 2F).
Multiple perianth whorls (MPW)—Gamay, Morrastel, and Pinot variants
Inflorescence and flower primordia initiation in these variants followed the regular pattern observed in normal plants (data not shown). However, in these variants, after the flower primordia differentiated (stages 2–3), the organ primordia generated by the flower meristem reiteratively differentiated as whorl 1 (double, short sepals could sometimes be observed in Pinot) and especially whorl 2 organs. Furthermore, the number of whorls produced was not limited to four, as the flower meristem continued to generate additional whorls of perianth organs (Fig. 3A, E, I, H). As a result of this altered developmental pattern, these somatic variants generated flower structures consisting of multiple reiterations of sepal and petal whorls lacking reproductive organ whorls (Fig. 3B and C, F and G, J and K). In addition to these common features, anther-like structures were occasionally found fused to the upper part of inner petals of MPW Pinot (Fig. 3C, D) and some ovule-like formations appeared in the innermost organs of MPW Gamay (Fig. 3L). Moreover, the MPW Gamay inflorescence exhibited additional specific features such as lack of secondary rachis and very short main rachis and pedicels. Sometimes, MPW Gamay flowers displayed the formation of a secondary pedicel separating reiterated flowers in the position of whorls 3 and 4 (Fig. 3K). Somatic variants reached their most advanced inflorescence and flower development at the anthesis time of the corresponding wild-type plants. After this stage, somatic variant inflorescences necrosed within a few weeks without berry formation.
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Abnormal reproductive whorl development variants
UFC Bouchalès
Inflorescence and flower primordia developed normally in this mutant. However, later flower development was altered as some flowers displayed a double sepal or petal whorl (Fig. 4C), while all of them showed anomalies in the development of stamens and carpels. Stamens were normal in number and position but filaments were shorter than in the wild type or sometimes absent with anthers fused to ovaries. The abnormal anther structure did not allow a normal closure of the petal cap (calyptra). Furthermore, ovary development was also compromised and most ovaries exhibited only partially fused carpels (Fig. 4C). The most conspicuous phenotypic modification in this variant was premature flower opening (Fig. 4A, B) as a result of abnormal stamen and carpel development. The premature opening of flowers prevented complete development and decreased flower set. Still, after open pollination, some ovaries were able to develop into normal, seeded berries but, at ripening, bunches were mainly composed of small seedless berries as sometimes observed for female grapevine genotypes with stamens or pollen not fully functional (Fig. 4D).
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CLS Mourvèdre
This somatic variant displayed larger flower buds than its wild-type plant counterpart (Fig. 5A). Microscopic analysis did not reveal alterations in the initiation of inflorescence and flower primordia, which initiated in regular positions and developed normally in the first and second whorls. However, primordia in whorls 3 and 4 did not differentiate regular reproductive organs but only carpelloid structures (Fig. 5B, D). Regular carpels with normal ovaries could not be observed, but ovule-like structures were often found fused to carpelloid structures in the fourth central whorl. These ovule-like structures displayed some normal ovule features such as recognizable integuments (Fig. 5C). Anthers were absent or displayed a completely abnormal shape, although microspores could be observed (Fig. 5E, F). Developed flowers were not functional and necrosed a few weeks after full development of the inflorescence. This variant was completely sterile and did not develop fruit, even under open pollination, indicating that the carpelloid structures were not functional.
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A synoptic view of the main morphological alterations observed in the six somatic variants described herein is presented in Fig. 6.
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Expression of VvMADS- box genes during flower development of somatic variants
VvMADS1, VvMADS2, and VvMADS3 were initially identified as the putative orthologues of AG/SHP, SEP1/2, and AGL13, respectively (Boss et al., 2001, 2002). In Arabidopsis, SEPALLATA genes are involved in the specification of petal, stamen, and carpel identity (Pelaz et al., 2000), AGAMOUS is required to establish the identity of reproductive organs (Drews et al., 1991), while SHATTERPROOF seems to be more involved in carpel development (Savidge et al., 1995). Finally, the role of AGL13 is not yet clear, although based on its expression pattern it could be involved in ovule formation. Expression of the corresponding grapevine orthologous genes was analysed by real-time PCR in the somatic variants to obtain information on the molecular events associated with the described flower developmental phenotypes. The results are shown in Fig. 7.
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The correspondence between inflorescence development stages used for RNA extraction sampling and floral organ development was: stage 1, formation of first flower meristems and initiation of outermost whorl primordia; stage 2, more advanced flower primordia initiating sepals; stage 3, most flowers developing petals and stamens, and initiating ovary development; stage 4, sepals fully developed in most flowers, petals and stamens finishing development, and ovaries developing; stage 5, petals and stamens formed and ovary development completing in most flowers; stages 5+, most flowers with dehiscing anthers. Stages 1 and 2 were analysed together as the corresponding inflorescence samples were combined.
Expression profiles and levels of VvMADS1, VvMADS2, and VvMADS3 were very similar in the six original cultivars. VvMADS1 and VvMADS2 showed a clear increase in their expression from inflorescence initiation to the mature flower stage (Fig. 7). On the other hand, the expression profile of VvMADS3 showed a maximum at developmental stages 4–5 (Fig. 7).
The RRM somatic variant of Carignan was characterized by an overexpression of all VvMADS-box genes at stage 3 of flower development as well as at stage 5 for VvMADS1. All the MPW variants, independently of their origin, were characterized by a strong reduction in VvMADS1 expression throughout flower development. They also shared coincident expression of VvMADS2, which was expressed at levels similar to the corresponding original cultivars during the initial stages of flower development but showed a significant reduction during late stages (4–5). Finally, these three different somatic variants displayed slightly different phenotypes regarding the expression of VvMADS3. Expression of this gene did not seem affected in the MPW Morrastel somatic variant, with respect to its wild-type cultivar. However, VvMADS3 was overexpressed in the initial flower developmental stages of MPW Gamay and throughout flower development in the MPW Pinot variant (Fig. 7).
Expression patterns of VvMADS-box genes in the two somatic variants altered in the development of reproductive organs, i.e. CLS Mourvèdre and UFC Bouchalès, differed significantly, as did the morphology of their flowers. CLS Mourvèdre displayed reduced expression of VvMADS1 expression during early flower developmental stages. In contrast to this VvMADS1 expression profile, VvMADS2 and VvMADS3 appeared to be overexpressed in earlier stages (1–2 and 3 for VvMADS2 and 3–4 for VvMADS3). In the case of VvMADS2, there was a strong reduction in its expression in the later stages of 5 and 5+. For UFC Bouchalès, the expression of all three VvMADS-box genes was found to be very close to that of the corresponding wild type.
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Discussion |
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It is significant that out of the 200 variants contained in the 4500 V. vinifera accessions of the Vassal repository, the largest grapevine collection of Vitaceae germplasm, only six variants displayed altered flower morphology (This et al., 2006). This could indicate that little attention so far has been paid to flower development and/or that such perturbations are rare events in Vitis. Indeed, while many reports describe somatic variation for leaf morphology (Durquety and Houbart, 1982), berry form, or colour (Pires et al., 2003; Kobayashi et al., 2004; Fernandez et al., 2006), few authors have reported Vitis variants with abnormal flower morphology. Previous reports have mentioned the existence of multiple whorled flowers (Oprea, 1965; Branas, 1974), but lacked detailed descriptions. Caporali et al. (2003) described flowers with abnormal development of anthers in wild, hermaphrodite V. vinifera Additionally, the Mourvedre and Bouchalès variants have been only briefly reported recently (Sreekantan et al., 2006).
The genetic basis of somatic variants
The utility of somatic grapevine variants in the study of gene function depends largely on the understanding of the mechanisms involved in the generation of somatic variation. In the highly probable case that the described phenotypes are the result of somatic mutations, several considerations are required when interpreting the variant phenotypes. First, the somatic mutation takes place in a single cell belonging to a specific cell layer (L1 and L2 layers are distinct in grapevine shoot apical meristems; see Thompson and Olmo, 1963; Franks et al., 2002; Fernandez et al., 2006). For the mutant phenotype to be observed, the mutant cell has to ‘colonize’ the corresponding cell layer in at least one shoot apical meristem and derived organs. However, unless the mutant cell layer colonizes the other cell layer and this situation is again stabilized, the somatic variant will be a chimera. This in fact has been the case in most grapevine somatic variants described previously (Boss and Thomas, 2002; Franks et al., 2002; Ageorges et al., 2006; Fernandez et al., 2006). This situation means that the observed phenotype results from chimerism and cannot be directly associated with the phenotype of solid mutant plants. Secondly, somatic mutations will only take place in one copy of the target gene. Unless the mutation takes place in the functional allele of a heterozygous locus, an unlikely situation, the resulting phenotypes are more likely to be due to the occurrence of gain-of-function mutations causing an observable change. This has been the case in the two somatic variants that have so far been characterized at the molecular level in grapevine [GAI1 by Boss and Thomas (2002) and the berry colour MYBA1 gene by Kobayashi et al. (2004)]. Thus, it is highly probable that the phenotypes observed in the described variants are the result of gain-of-function mutations in a chimeric state, something that should be considered when attempting to understand the phenotypes under current genetic models of flower development. Thirdly, it is not known which molecular mechanisms are responsible for the generation of these mutant phenotypes and so far the available information is scarce. At least for berry colour, retrotransposable elements through homologous long terminal repeat (LTR) recombination or illegitimate recombination are responsible for a large proportion of the somatic variation observed for the trait (Lijavetzky et al., 2006). If these mechanisms were responsible for some of the flower phenotypes described, one would expect some phenotypes to appear repeatedly in specific cultivars as well as in genetically related cultivars. The MPW phenotype, found in Pinot and Gamay, a derived hybrid variety, would be in agreement with this possibility. Slight phenotypic differences between both variants could be attributed to different genetic backgrounds. The similarity of phenotypes observed even in non-closely related cultivars such as Morrastel and Pinot could also be explained by the presence of those mutable loci in the genome.
Mutant phenotypes and altered gene functions in grapevine flower development
The somatic variants presented here are affected in the specification of identity, of either flower meristems or organ primordia. However, no earlier events regulating flower initiation appear to be affected. The RRM variant exhibited the earliest phenotypic alteration, apparently causing a delay in flower meristem specification. This alteration resulted in clusters with exacerbated ramifications and delayed development. In Arabidopsis, a similar phenotype is caused by lack-of-function mutations in genes required for flower meristem specification such as AP1 (Bowman et al., 1993), LFY (Parcy et al., 2002), FUL (Ferrandiz et al., 2000), or more extremely in the double mutant ap1 cal (Kempin et al., 1995). Similar to what was observed in the Carignan variant, in the Arabidopsis mutants the phenotype of the basal flowers is more strongly affected than that of the uppermost flowers that are almost normal and fertile. In fact, even strong cauliflower (ap1 cal) phenotypes are eventually able to elongate their inflorescence and produce regular, fertile flowers (Kempin et al., 1995). This suggests that both in the Arabidopsis mutants and in the Carignan variant the genes required for floral organ formation are fully functional. The RRM phenotype could also be explained as a result of overexpression or ectopic expression of TFL-like genes in the inflorescence and flower meristems, as has been shown in transgenic Arabidopsis using either Arabidopsis TFL genes or genes from other species, including grapevine (Boss et al., 2006).
Grapevine MPW variants are able to develop perianth organs requiring A and B functions, but are impaired in the specification of anther and carpel identity. This phenotype is consistent with a loss-of-function mutation in class C genes such as AGAMOUS (Coen and Meyerowitz, 1991) which is also required to prevent reiteration of whorl formation (Yanofsky et al., 1990). In agreement with this possibility, the expression of the VvMADS1 gene, a putative AG/SHP homologue (Boss et al., 2001), was strongly reduced in the three variants during all stages of flower development.
The CLS Mourvèdre variant did not exhibit clear homeotic transformations of the ovary into other floral organs as commonly observed in Arabidopsis. Instead, this mutant developed normal sepals and petals, but exhibited alterations in the innermost whorls with the development of carpelloid structures. These whorls displayed ovules together with abnormal, stamen-like tissues. The fact that alterations were restricted to the two inner whorls suggests malfunction in class C or downstream genes. Interestingly, VvMADS1 was found to be repressed in this mutant during early flower development, but later expression was found to be similar to the wild type, and ovule-like structures could develop.
The UFC Bouchalès displayed less severe flower morphology defects and similar expression patterns for VvMADS1, 2, and 3 compared with the wild type. Sreekantan et al. (2006) recently showed a delayed overexpression of VvMADS9 (PI homologue) in the UFC variant compared with the wild type. Interestingly, a PI temporal deregulation was also shown to be associated with some alterations of ovary shape in the fleshless grapevine mutant (Fernandez et al., 2006).
The grapevine genome sequence was recently completed (Jaillon et al., 2007), facilitating the identification and cloning of candidate gene sequences. Grapevine somatic variants exhibiting unusual patterns of flower or berry development represent unique material to enable the investigation of the regulation of reproductive development in grapevine (Fernandez et al., 2006, 2007). However, because of the origin of these phenotypes, the dissociation of L1 and L2 cell layers, through either sexual propagation (as gametes arise from the L2 cell layer) in the fertile mutants or somatic embryogenesis (Franks et al., 2002), will be required for subsequent genetic analysis. This report is the first comprehensive description of grapevine somatic variants showing altered early development of reproductive organs. Further investigations, based on our hypotheses, should eventually result in the identification of the gene(s) responsible for the mutant phenotypes and lead to a better understanding of their function in grapevine flower development.
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Acknowledgements |
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Scanning electron microscopy and light microscopy observations were performed, respectively, at the Montpellier UMII Joint Electronic Microscopy Laboratory and PHIV Montpellier Rio Imaging facilities (http://www.mri.cnrs.fr). The authors thank D Varès, Dr J-M Boursiquot, and Domaine INRA de Vassal staff for the access to somatic variants, passport data, and useful comments. Thanks are extended to G Lopez for plant care and to C Brachet for RNA extractions.
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Abbreviations |
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CLS, carpel-less; DF, double-flowered; MPW, multiple perianth whorls; RB, ramose bunch; RRM, reiterated reproductive meristems; UFC, unfused carpels.
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References |
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Ageorges A, Fernandez L, Vialet S, Merdinoglu D, Terrier N, Romieu C. Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries. Plant Science (2006) 170:372–383.[CrossRef][Web of Science]
Ausin I, Alonso-Blanco C, Martinez-Zapater JM. Environmental regulation of flowering. International Journal of Developmental Biology (2005) 49:689–705.[CrossRef][Web of Science][Medline]
Boss PK, Buckeridge EJ, Poole A, Thomas MR. New insights into grapevine flowering. Functional Plant Biology (2003) 30:593–606.[CrossRef][Web of Science]
Boss PK, Sensi E, Hua C, Davies C, Thomas MR. Cloning and characterisation of grapevine (Vitis vinifera L.) MADS-box genes expressed during inflorescence and berry development. Plant Science (2002) 162:887–895.[CrossRef][Web of Science]
Boss PK, Sreekantan L, Thomas MR. A grapevine TFL1 homologue can delay flowering and alter floral development when over-expressed in heterologous species. Functional Plant Biology (2006) 33:31–41.[CrossRef][Web of Science]
Boss PK, Thomas MR. Association of dwarfism and floral induction with a grape ‘green revolution’ mutation. Nature (2002) 416:847–850.[CrossRef][Medline]
Boss PK, Vivier M, Matsumoto S, Dry IB, Thomas MR. A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development. Plant Molecular Biology (2001) 45:541–553.[CrossRef][Web of Science][Medline]
Bouquet A, Torregrosa L, Chatelet P. Grapevine genetic engineering: tool for genome analysis or plant breeding method? Which future for transgenic vines? AgBiotechNet (2003) 5:1–10.
Bowers J, Boursiquot JM, This P, Chu K, Johansson H, Meredith C. Historical genetics: the parentage of Chardonnay, Gamay and other wine grapes of Northeastern France. Science (1999) 285:1562–1565.
Bowers JE, Dangl GS, Vignani R, Meredith CP. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome (1996) 39:628–633.[Medline]
Bowman JL, Alvarez J, Weigel D, Meyerowitz EM, Smyth DR. Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes. Development (1993) 119:721–743.
Branas J. Viticulture (1974) Montpellier, France: Dehan Imp.
Calonje M, Cubas P, Martínez-Zapater JM, Carmona MJ. Floral meristem identity genes are expressed during tendril development in grapevine. Plant Physiology (2004) 135:1491–1501.
Cabezas JA, Cervera MT, Arroyo-Garcia R, Ibanez J, Rodriguez-Torres I, Borrego J, Cabello F, Martinez-Zapater JM. Garnacha and Garnacha Tintorera: genetic relationships and the origin of teinturier varieties cultivated in Spain. American Journal of Enology and Viticulture (2003) 54:237–245.
Caporali E, Spada A, Marziani G, Failla O, Scienza A. The arrest of development of abortive reproductive organs in the unisexual flower of Vitis vinifera ssp silvestris. Sexual Plant Reproduction (2003) 15:291–300.[Web of Science]
Carmona MJ, Calonje M, Martinez-Zapater JM. The FT/TFL1 gene family in grapevine. Plant Molecular Biology (2007) 63:637–650.[CrossRef][Web of Science][Medline]
Carmona MJ, Cubas P, Martínez-Zapater JM. VFL, the grapevine FLORICAULA/LEAFY ortholog, is expressed in meristematic regions independently of their fate. Plant Physiology (2002) 130:68–77.
Coen ES, Meyerowitz EM. The war of the whorls: genetic interactions controlling flower development. Nature (1991) 353:31–37.[CrossRef][Medline]
Di Gaspero G, Peterlunger E, Testolin R, Edwards J, Cipriani G. Conservation of microsatellite loci within the genus Vitis. Theoretical and Applied Genetics (2000) 101:301–308.[CrossRef][Web of Science]
Doligez A, Audiot E, Baumes R, This P. QTLs for muscat flavor and monoterpenic odorant content in grapevine (Vitis vinifera L.). Molecular Breeding (2006) 18:109–125.[CrossRef][Web of Science]
Drews GN, Bowman JL, Meyerowitz EM. Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell (1991) 65:991–1002.[CrossRef][Web of Science][Medline]
Durquety PM, Houbart JP. Two Tannat sports: ‘meunier’ and ‘ bullé’. Progrès en Agricole Viticole (1982) 99:83–87.
Emmanuel E, Levy AA. Tomato mutants as tools for functional genomics. Current Opinion in Plant Biology (2002) 5:112–117.[CrossRef][Web of Science][Medline]
Fernandez L, Romieu C, Moing A, Bouquet A, Maucourt M, Thomas MR, Torregrosa L. The grapevine fleshless berry mutation: a unique genotype to investigate differences between fleshy and non-fleshy fruit. Plant Physiology (2006) 140:537–547.
Fernandez F, Torregrosa L, Terrier N, Sreekantan L, Grimplet J, Davies C, Thomas MR, Romieu C, Ageorges A. Identification of genes associated with flesh morphogenesis in grapevine (V. vinifera L.) berry. Plant Molecular Biology (2007) 63:307–323.[CrossRef][Web of Science][Medline]
Ferrandiz C, Liljegren SJ, Yanofsky MF. Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science (2000) 289:436–438.
Ferrario S, Imminck RGH, Angenent GC. Conservation and diversity in flower land. Current Opinion in Plant Biology (2004) 7:84–91.[CrossRef][Web of Science][Medline]
Franks T, Botta R, Thomas MR. Chimerism in grapevines: implications for cultivar identity, ancestry and genetic improvement. Theoretical and Applied Genetics (2002) 104:192–199.[CrossRef][Web of Science][Medline]
Hardie WJ, O'Brien TP, Jaudzems VG. Morphology, anatomy and development of the pericarp after anthesis in grape, Vitis vinifera L. Australian Journal of Grape and Wine Research (1996) 2:97–142.[CrossRef]
Jack T. Molecular and genetic mechanism of floral control. The Plant Cell (2004) 16:1–17.
Jaillon O, Aury JO, Noel B, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature (2007) 449:463–467.[CrossRef][Medline]
Kempin SA, Savidge B, Yanofsky MF. Molecular basis of the cauliflower phenotype in. Arabidopsis. Science (1995) 267:522–525.
Kobayashi S, Goto-Yamamoto N, Hirochika H. Retrotransposon-induced mutations in grape skin color. Science (2004) 304:982.
Koornneef M, Alonso-Blanco C, Vreugdenhill D. Naturally occurring genetic variation in Arabidopsis thaliana. Annual Review of Plant Biology (2004) 55:141–172.[CrossRef][Medline]
Kuksova V, Piven N, Gleba Y. Somaclonal variation and in vitro induced mutagenesis in grapevine. Plant Cell, Tissue and Organ Culture (1997) 49:17–27.[CrossRef][Web of Science]
Lacombe T, Laucou V, Di Vecchi M, et al. Contribution à la caractérisation et à la protection in situ des populations de Vitis vinifera L. ssp. silvestris (Gmelin) Hegi, en France. Quatrième colloque national du BRG, La Châtre 14–16 Octobre 2002. Les Actes du BRG (2003) 4:381–404.
Lijavetzky D, Ruiz-Garcia L, Cabezas JA, De Andres MT, Bravo G, Ibanez A, Carreno J, Cabello F, Ibanez J, Martinez-Zapater JM. Molecular genetics of berry colour variation in table grape. Molecular Genetics and Genomics (2006) 276:427–435.[CrossRef][Web of Science][Medline]
Merdinoglu D, Butterlin G, Bevilacqua L, Chiquet V, Adam-Blondon A-F, Decroocq S. Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex. Molecular Breeding (2005) 15:349–366.[CrossRef][Web of Science]
Mullins MG, Bouquet A, Williams LE. Biology of grapevine (1992) Cambridge: Cambridge University Press.
Oprea DD. Lucr
ri pratice di viticultur (1965) Bucarest: Didactica 
i Pedagogica.
Parcy F, Bomblies K, Weigel D. Interaction of LEAFY, AGAMOUS and TERMINAL FLOWER1 in maintaining floral meristem identity in Arabidopsis. Development (2002) 129:2519–2527.[Web of Science][Medline]
Pelaz S, Ditta G, Baumann E, Wisman E, Yanofsky M. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature (2000) 405:200–203.[CrossRef][Medline]
Pires EJP, Sawazaki HE, Terra MM, Bothelho RV, Conagim A, Nogueira NAM. Redimeire: a natural mutation of cv. Italia in Brazil. Vitis (2003) 42:55–56.[Web of Science]
Pratt C. Reproductive anatomy in cultivated grapes: a review. American Journal of Enology and Viticulture (1971) 22:92–101.
Rozen S, Skaletsky HJ. Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics methods and protocols: methods in molecular biology—Krawetz S, Misener S, eds. (2000) Totowa, NJ: Humana Press. 365–386.
Savidge B, Rounsley SD, Yanofsky MF. Temporal relationship between the transcription of two Arabidopsis MADS-box genes and the floral organ identity genes. The Plant Cell (1995) 7:721–733.[Abstract]
Sreekantan L, Torregrosa L, Fernandez L, Thomas MR. VvMADS9, a class B MADS-box gene involved in grapevine flowering, shows different expression patterns in mutants with abnormal petal and stamen structures. Functional Plant Biology (2006) 33:877–886.[CrossRef][Web of Science]
Srinivasan C, Mullins MG. Physiology of flowering in the grapevine: a review. American Journal of Enology and Viticulture (1981) 32:47–53.
Tesniere C, Vayda MEV. Method for the isolation of high quality RNA from grape berry tissues without contaminating tannins or carbohydrates. Plant Molecular Biology Reporter (1991) 9:242–251.[CrossRef]
Theißen G. Development of floral organ identity: stories from the MADS house. Current Opinion in Plant Biology (2001) 4:75–85.[CrossRef][Web of Science][Medline]
This P, Lacombe T, Thomas MR. Historical origins and genetic diversity of wine grapes. Trends in Genetics (2006) 22:511–519.[CrossRef][Web of Science][Medline]
Thomas MR, Scott NS. Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theoretical and Applied Genetics (1993) 86:985–990.[Web of Science]
Thompson MM, Olmo HP. Cyto-histological studies of cytochimeric and tetraploid grapes. American Journal of Botany (1963) 50:901–906.[CrossRef][Web of Science]
Walker AR, Lee E, Robinson S. Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus. Plant Molecular Biology (2006) 62:623–635.[CrossRef][Web of Science][Medline]
Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA, Meyerowitz EM. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature (1990) 5:35–39.
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