- Research article
- Open Access
Flower development and sex specification in wild grapevine
- Miguel Jesus Nunes Ramos†1,
- João Lucas Coito†1,
- Helena Gomes Silva2,
- Jorge Cunha3, 4,
- Maria Manuela Ribeiro Costa2 and
- Margarida Rocheta1Email author
© Ramos et al.; licensee BioMed Central. 2014
- Received: 31 March 2014
- Accepted: 26 November 2014
- Published: 12 December 2014
Wild plants of Vitis closely related to the cultivated grapevine (V. v. vinifera) are believed to have been first domesticated 10,000 years BC around the Caspian Sea. V. v. vinifera is hermaphrodite whereas V. v. sylvestris is a dioecious species. Male flowers show a reduced pistil without style or stigma and female flowers present reflexed stamens with infertile pollen. V. vinifera produce perfect flowers with all functional structures. The mechanism for flower sex determination and specification in grapevine is still unknown.
To understand which genes are involved during the establishment of male, female and complete flowers, we analysed and compared the transcription profiles of four developmental stages of the three genders. We showed that sex determination is a late event during flower development and that the expression of genes from the ABCDE model is not directly correlated with the establishment of sexual dimorphism. We propose a temporal comprehensive model in which two mutations in two linked genes could be players in sex determination and indirectly establish the Vitis domestication process. Additionally, we also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role involved in sex differentiation. Also, the detection of differentially transcribed regions that extended existing gene models (intergenic regions) between sexes suggests that they may account for some of the variation between the subspecies.
There is no evidence of differences of expression levels in genes from the ABCDE model that could explain the shift from hermaphroditism to dioecy. We propose that sex specification occurs after floral organ identity has been established and therefore, sex determination genes might be having an effect downstream of the ABCDE model genes.
For the first time a full transcriptomic analysis was performed in different flower developmental stages in the same individual. Our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across developmental stages and genders that will contribute for future work in sex determination in seed plants.
- Vitis sylvestris
- Intronic RNA
The grape family (Vitaceae) has been widely recognized for its economic importance as the source of wine, table grapes and raisins. The family consists of 14 genera and ~ 900 species . It has been suggested that this family appeared 58.5 ± 5.0 million years ago in North America . In the Vitaceae family two subspecies still co-exist in Eurasia and in North Africa: the cultivated one, V. vinifera subsp. vinifera (to simplify in this work will be referred as V. v. vinifera) and the wild form V. vinifera subsp. sylvestris (simply referred as V. v. sylvestris). The cultivation of V. v. vinifera seems to have occurred between the seventh and the fourth millennia BC, in a geographical area between the Black Sea and Iran [3, 4] and it seems to be linked to the discovery of wine, making this the Vitis species with major agronomic and economic importance .
The wild grapevine plants are dioecious, in contrast with practically all cultivated varieties that are hermaphroditic and self-fruitful. This shift in sexual system from dioecy to self-pollination, i.e. hermaphroditism, was fundamental for grapevine productivity. V. v. sylvestris male flowers produce erect stamens and fertile pollen and have a reduced pistil with no style or stigma. On the contrary, female flowers have a well developed pistil but present reflexed stamens and produce infertile pollen incapable of pollination [6–8]. Therefore, in V. v. sylvestris, flowers are hermaphrodite at early stages of development and become unisexual due to the arrest of the reproductive organs .
The induction of flowering is provided by key genes that act as switches that signal the transition from vegetative to reproductive organ development. The genes involved in this switch and during flower development have been extensively studied in Arabidopsis thaliana. The induction of floral meristem identity is largely achieved by LEAFY (LFY) and APETALA1 (AP1) . Other genes, such as UNSUAL FLOWER ORGANS (UFO), WUSCHEL (WUS) and SEPALATA3 (SEP3) can act as co-factors for LFY in the activation of genes that specify flower organ identity (the ABCDE model genes) [11, 12]. According to the model, APETALA2 (AP2) and AP1 belong to A function, that are responsible for sepal development in the first floral whorl. APETALA3 (AP3) and PISTILLATA (PI) are B function genes and together with A function genes specify petals in the second whorl and with AGAMOUS (AG), a C function gene, specify stamens in the third whorl. C function alone specifies carpels in the fourth whorl. SEP1-3 (E function) interacts with A, B and C function genes to correctly establish the identities in the four floral whorls. SEEDSTICK (STK) and SHATERPROOF 1-2 (SHP1-2) are D function, and with the E class genes specify ovule identity. Interaction between these genes provides the signalling for flower organ development: sepals, petals, stamens, carpels and ovules [13, 14]. When LFY is activated it promotes, with UFO, the expression of AP3 and PI [15, 16]. LFY, through the activation of AP1, can promote the expression of SEP3 . LFY/SEP3 will then activate AP3 and PI . SEP3 acts as a cofactor of AP1 for the activation of AP3 and PI. Once activated, these two genes can auto-regulate themselves [19–21]. LFY along with WUS, also promotes the expression of AG [22, 23], which can also positively auto-regulate itself . AP2 is expressed in the entire floral meristem but is repressed by miRNA172 in the third and fourth whorls [25–27]. These interactions promote a temporal delay in the activation of the floral homeotic genes. This delay might be by essential to ensure that differentiation of floral organs occurs before the termination of the floral meristem .
During the development of unisexual flowers, a particular genetic control involved in the arrest of reproductive organs becomes operative . This stage differs between species, spanning the developmental spectrum from the appearance of reproductive organ primordia to the formation of fully developed but non-functional organs. Probably, in dioecious species, the point of divergence from hermaphroditic to unisexual developmental pathway is controlled by sex determining genes. Regarding Vitis sex evolution, a model was proposed  that suggests that two closely linked genes were responsible for the establishment of a dioecious population. In this model of digenic linked inheritance, Sp is the allele responsible for perfect pollen development and sp the allele that inhibits pollen development; So is the allele that inhibits ovule development and so the allele responsible for perfect ovule development. Very little is known about the nature of the genes controlling sexual determination and the mechanism in dioecious species that triggers the developmental arrest of male or female organs. The aim of this work was to identify in the wild grapevine differentially expressed genes during early flower development and, as a consequence, potentially important in sex determination. In order to assess differences between developmental stages and between genders, we sequenced the female, male and hermaphrodite flower transcriptome using Pinot Noir as the reference genome and employed global gene expression analysis. This allowed a better understanding of the expression levels of the ABCDE genes as a whole, as well as to determine their performance as putative players in sex determination. We also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role related to sex differentiation. Additionally, we detected transcribed regions that are not annotated in the reference genome, and disparities in those regions between wild and domesticated Vitis, suggests that they may play a role in the differences observed in the flower. In conclusion, our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across flower developmental stages and genders.
Sexual differentiation of wild grapevine is an interesting problem in developmental biology. The history of Vitis evolution may be starts with hermaphroditic ancestors that switched to a dioecious population that underwent a domestication process [30, 31].
Wild grapevine morphology and flower bud development
In summary, in V. v. sylvestris, the establishment of male and female characteristic flower morphology is quite a late event during flower development. Flowers of both sexes are bisexual at inception and become unisexual by cessation of organ development that in male plants consists on the suppression of the style and the stigma and in female flowers in the abnormal morphology of stamens that become reflexed with non-functional pollen.
RNA-Seq analysis of three flower sex types in grapevine
RNA-Seq reads summary and number of expressed genes
Putative exons and “dark matter”?
A different number of putative exons in different stages of flower development may indicate that different protein isoforms with possibly different functions may be required during flower development. Apart from the genes referred above, it is very important to consider genes with RPKM value lower than 1 (that were considered as not being expressed), but for which putative exons were found. As the RPKM takes into consideration the number of reads on exon-annotated regions, but ignore reads on introns, the RPKM value is not correctly calculated. These kind of genes represent a situation where no reads, or very few ones, match with the annotated exons and a large amount of transcripts matched with intronic regions (Figure 7B). The presence of these putative exons transcribed but not annotated indicates that the Vitis genome needs to be revised. Another situation comes from transcripts outside the boundaries of known genes, that could revealed the presence of a “dark matter” as it has been referred for noncoding regions that produce an important set of transcripts in mammalian genomes . We have excluded regions flanking genes up to a distance of 10 kb, as reads in these regions are more frequent and possibly correlated with known genes (Figure 7C). It has been thought that the reads found in intronic or intergenic regions are just pre-mRNAs en route to splicing or spliced-out introns en route to degradation. Nevertheless, we observed that the signal from intronic RNA was not evenly spread among genes: different amounts of intronic RNA were found along genes whose expression is associated with sex or flower development. The same situation occurs with intergenic transcripts. Contrary to the general idea, our data reinforce the opinion from other studies [39, 40] that this class of RNAs should not be ignored.
All predicted genes were assigned to functional categories and annotation using 12x V1 of the reference genome and we have manually inspected the genes mentioned in this work to confirm their sequence.
A second RT-qPCR was performed with samples collected one year latter from other V. v. sylvestris plants, in order to compare results with the previous data in terms of consistency and possible variations that may occur, for example, due to climatic or genotype changes (Figure 8). The expression of VvPi and VvAP3, both B function genes, was similar to the previous year. However, VvAP1 an A function gene responsible to make sepals in the first whorl and together with B function genes petals in the second whorl, show lower expression mainly in the B stage in male and female plants. On the other hand, VvTFL displays a decline of expression in stages D and G whereas VvLFY and VvAP1 show an increase in transcription abundance. These results are in accordance with the role of TFL as a meristem identity gene that also prevents expression of AP1 and LFY in shoot apical meristem . An exception was found in the male plants where the expression of VvTFL drops almost continuously along development while in the first year a decrease was observed only in the two latter stages. The regulation of VvTFL, in the second year, could be a response related to the growth of the organs for the same agronomic developmental stage influenced by climate (Figure 8).
The world of sex specification
According to the ABCDE model, A-class function in Arabidopsis is required for perianth identity and, AP2, in particular, leads to repression of C-class function . The lowest expression of VvAG in female and hermaphrodite stage B is possible due to VvAP2 action that is higher expressed at initial stages of flower development. The role of VvAG in determining the carpel seems to happen later than this developmental stage, as we can see an increased in expression during development until stage H. Also of interest at the early stage B is the expression of genes responsible for sepal and petal development, normally through the assembly of the protein complexes AP1/AP1/SEP/SEP and AP1/SEP/AP3/PI that specify sepal and petal identity, respectively  (Figure 10) confirming what was observed by Calonje et al.  at in situ hybridizations experiments, where VvAP1 was largely expressed in the inflorescence branch meristems. RNA-Seq data reveled that between D and G stage VvLFY expression increases as well as VvAP1, which suggests a transition to flower development through induction of VvAP1 expression in regions of the shoot apical meristem that give rise to flower primordia . However, these changes are accompanied by an increase in VvAP2, VvPI and VvSEP3 in the three sexes and an increase of VvAP3 and VvSEP2 in male and hermaphrodite plants and a decrease of these genes in female flowers (Figure 10). These changes might corroborate a delay in the formation of sepal and petal and the initiation of stamens formation . On these initial stages of floral development, it is interesting to point out that the expression levels of these genes, in female and hermaphrodite plants, are very similar, whereas male plants seem to have slightly different expression levels of the ABCDE model genes. This might provide a hint of how sexual differentiation affects flower development in Vitis plants. In situ hybridization experiments performed by Yao et al. , show that VvLFY expression reached its maximum in the floral meristem primordia. At later stages of flower development (G and H), where the floral meristem with all organ primordia are formed, we observe a decrease of VvLFY expression and an increase of VvAG [10, 51], which is being highly expressed (Figure 10). In these stages, we also observe an increase expression of all the genes of the ABCDE model in all genders. The exception is VvAP2 in the males plants which show a decrease in expression between stage G and H, this may be due as normal female organs development is compromised in male flowers whose development requires VvAP2 expression, as has been shown for the VvAP2 homologue gene in Arabidopsis [52, 53]. Another exception was a slight decrease of VvAP1 in female flowers that somehow may be related to sepal and petal formation at these stages.
A hint that sexual determination does not occur during flower organ onset is the expression of D function genes, VvSTK and VvSHP1-2. These genes show low expression in B and D stages, as expected, since they are required for ovule formation (function D) [54, 55], but their expression does not vary between the sexes in the later stages (Figure 10). This corroborates with the fact that male flowers develop ovules just like female and hermaphrodite plants but, for an yet unknown reason, male flowers do not form some of the most complex flower structures: the style and the stigma on a fully functional carpel. Whatever genes are promoting flower sex specification, they might be having an effect downstream of the ABCDE model genes. Another possibility is that although the expression level of the ABCDE genes is similar in the analysed stages of the three different flower types, their spatial pattern of expression might be slightly different. So, these results could be complemented in the future by in situ hybridization experiments to provide a complete spatial gene expression analysis of the three types of Vitis flower meristems.
Putative genes involved in sex specification
On the other hand, genes only expressed in male flowers can lead to pistil abortion by inhibiting stigma development. Our data show that genes already described as being involved in different carpel structures development did not show significant expression changes until stage H, particularly in male flowers where style and stigma are aborted (Additional file 3). However, there are quite a few number of genes that are exclusively expressed in the female flower, in particular in the later stage H. These genes may be the putative key players in sex determination because the sexual dimorphism in Vitis occurs in a late stage of flower development. We noticed this type of expression in the gene annotated as Nodulin MtN3 and in members of the Lipoxygenase (LO) gene family. The members of these gene families seem to be involved in sexual organ differentiation in different species. In Arabidopsis thaliana, Ruptured Pollen Grain1 (RPG1), a member of the MtN3 gene family, plays an important role in exine pattern determination and in the cellular integrity of microspores  and in rice, the homologue gene of Medicago truncatula Nodulin MtN3 (Xa1) when suppressed originate plants with small anthers and reduced fertility due to the production of mostly abortive pollen . In Pea (Pisum sativum L.), the natural pattern of flower and fruit development is associated with a lipoxygenase gene repression, and the carpel senescence pathway is associated with high levels of lipoxygenase gene expression . As VvNodulin MtN3 and VvLOs in Vitis are not expressed in hermaphrodite flowers, these genes may not be important for a perfect flower organ development so the exclusive expression in female flower could be associated with pollen abortion.
We also observed genes with no expression in any stage of male flower development that are highly expressed during female and hermaphrodite flower development. A gene encoding a Pentatricopeptide repeat-containing protein is not expressed in male flowers, which could mean that this protein is essential for carpel development. In male sterile Petunia , Kosena radish  and Oryza sativa , pentatricopeptide repeat-containing genes can restore fertility showing that this gene could be essential for the perfect development of sexual floral organs. On the other hand, genes with no expression during female flower development, which are highly expressed in male and hermaphrodite flowers, can play an important role in pollen viability. In Arabidopsis, YUCCA flavin monooxygenase mutants appeared to have a floral indeterminacy problem , suggesting that some members of the flavin monooxygenase family seem to be essential for the development of perfect flowers. In V. v. sylvestris we found a member of flavin-containing monooxygenase gene family that is not expressed in the female flowers. This fact suggests that there might be a flavin monooxygenase that might also contribute to the reproductive organ dimorphism in this species. Flower development can be regulated by plant hormones , as it happens in curcubit species . In Vitis, the 1-aminocyclopropane-1-carboxylate oxidase 1 (ACS1) homologue is differentially expressed, with a higher expression in stage D and H of female flowers when compared with the same stages in male and hermaphrodite flower. This enzyme is involved in the ethylene biosynthesis pathway . In gynoecious cucumber plants, that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds, whereas in monoecious cucumber, the two types of flower buds (male and female) can be distinguished on the basis of their levels of CSACS2 gene expression , being less abundant in the male flowers. This correlation between high levels of CSACS2 gene expression and femininity may also occur in V. v. sylvestris. In our transcriptomic data, Alpha-expansin 3 gene involved in auxin signaling in Arabidopsis  and CTR1-like protein kinase gene involved in ethylene-mediated signaling pathway , have higher expression levels in stage H of female flower when compared with the same stage in the male and hermaphrodite flowers. In grapevine, endogenous application of cytokinin, 6-benezylamino-9-(2-tetrahydropyranyl)-purine can induce hermaphroditic flowers in staminate plants . Therefore, sex reversion by hormone application may indicate that genes involved in hormone signaling could be important players in the development of the androecium or gynoecium.
With this analysis, we conclude that there are genes involved in the reproductive development and sex specification in other species that are also differentially expressed between grapevine male, female and hermaphrodite flower and may be good candidates for conferring sex identity to these flowers.
Inheritance and evolution of flower sex in grapevine
In the present study, we analysed RNA-Seq results from buds belonging to four developmental stages from three Vitis genders: female and male ancestor (V. v. sylvestris) and a hermaphrodite cultivar (V. v. vinifera). Our primary analyses confirm that Vitis flowers start their development as hermaphrodites until they reach developmental stage G to H, when male plants became distinguishable by the presence of a defective pistil and the female by reflexed stamens. RNA-Seq also provided the evidence that on developmental stage G, female and hermaphrodite flowers share a large number of expressed genes, whereas the number of active genes on male buds abruptly diminishes, suggesting that, the these genes are involved in the development of a fully functional carpel in female and hermaphrodite flowers.
Data also revealed a higher number of intronic matched reads that are good candidate for new exons or it could be what some authors call “dark matter”. The evidence that this “dark matter” exists came from the existence of match reads in intergenic regions that change across development and sexes. These results encourage an extensive revision on the V. v. vinifera databases and reinforce the opinion that this class of RNAs should not be ignored or automatically assigned to the category of annotated pre-mRNAs. Our data suggests that these regions may be related to flower development and/or sex determination. We also show that the ABCDE model genes expression levels are not related to flower sex specification, but other genes, including some classified as “unknown” function, may have a role in sex differentiation. We consider the model where females are homozygotes for so and sp genes, and hermaphrodite are heterozygous to sp (so so/Sp sp) to be the most probable. Alongside, a comprehensive evolutionary model is proposed based on the knowledge available. Maybe the domestication happened in an ancestral hermaphrodite or alternatively, a male reversion originated a new hermaphrodite form that is used, nowadays, in grape industry.
Sampling and biological material manipulation
Flowering buds from V. v. sylvestris female and male plants and also from the hermaphrodite V. v. vinifera cv. Touriga Nacional were collected in Dois Portos (Lisbon district, Portugal; 75 – 100m), where a Vitis collection was established. Floral buds were collected every week during the month of April of 2012 (temperature interval during sampling month: 7.4 – 24.3°C; total precipitation during sampling month: 59.3 mm) and frozen in liquid nitrogen.
The harvesting of floral buds was made from a single plant of each gender to perform RNA-Seq. Developmental stages B, D, G and H (according to Baggiolini ) were collected for each gender performing a total of twelve samples (Additional file 1). Additionally, similar developmental stages were collect from the same plants in the following year, as well as from six other male and eleven female plants in the collection (temperature interval during sampling month: 8.1 – 27.0°C; total precipitation during sampling month: 71.8 mm). Buds were classified according to their developmental stage and treated separately. About 5 frozen buds per stage/gender were ground into a fine powder in liquid nitrogen and total RNA was extracted and purified with the Spectrum™Total RNA kit (Sigma-Aldrich, Inc, Spain) and Qiagen RNAeasy Mini Spin Columns (Qiagen, Valencia, CA, USA), respectively, according to the manufacturer’s instructions. The concentration and purity of total RNA were determined using a Synergy HT Nanodrop system (Biotek, Germany), with the software Gen5™ (Biotek, Germany). The quality of the RNA was confirmed on a denaturing gel electrophoresis. For each sample, 25 μg of total RNA were utilized for transcriptome sequencing (2012 samples).
Transcriptome sequencing and quality analysis of sequence reads
cDNA library construction and transcriptome sequencing were performed by BaseClear, B.V, Netherlands. The cDNA libraries were constructed following the procedures outlined in the Illumina platform. Sequence reads were generated using the Illumina Casava pipeline version 1.8.2. Two Illumina runs were performed on each sample that functioned as technical replicas, to evaluate data consistency. Prior to the assembly and mapping, filters were applied to remove low quality reads resulting an average of 30 million of single reads (Table 1) 50 bp in length equivalent to 1.5 Gb of total sequence data for each sample. Initial quality assessment was based on data passing the Illumina Chastity filtering. The second quality assessment was based on the remaining reads using the FASTQC tool version 0.10.0. The quality of the FASTQ sequences was enhanced by trimming off low-quality bases using CLC Genomics Workbench version 5.5.1. The quality-filtered sequence reads were used for further analysis with the CLC Genomics Workbench. First, an alignment against the V. v. vinifera reference genome that is a near-homozygous and non-cultivated accession, PN40024 , (http://genomes.cribi.unipd.it/DATA/GFF/V1.tar.gz) and calculation of the expression values was performed, and then, a comparison of expression values and a statistical analysis was made. The selected expression measurement was the Reads per Kilobase of exon model per Million mapped reads (RPKM) with the aim of normalizing for the difference in number of mapped reads between samples, as well as the transcript length . RPKM is calculated by dividing the total number of exon reads by the number of mapped reads (in Millions) times the exon length (in kilobases). The statistical analysis to assess the significance of expression differences was performed using Kal’s Z-test. The Phred quality scores ranged from 37.3 to 38.2, which meant that the accuracy of the base call was higher than 99.9%. Quality control was performed to examine the consistency of the experiment and the variability between samples and groups. In brief, the overall distribution of the RPKM expression values is compared between samples and groups through a box plot representation that provide an effective summary of this large amount of data (Additional file 4).
Software used to process Vitistranscriptomes
IGV (http://www.broadinstitute.org/igv/) was used for manually inspect the reads assembled to each gene. MATLAB® (2012b, The MathWorks, Inc., Natick, USA) was used to retrieve gene transcript sequences from *.bam files.
RNA-Seq validation and reproducibility through RT-qPCR
cDNA was obtained with the RETROscript® kit (Ambion, Life Technologies, Spain), following the manufacturer’s protocol. RNA from the samples that were sequenced, as well as RNA from other plants collected at the same time and in the following year were retrotranscribed. cDNA concentration was measured in a microplate reader Synergy HT (Biotek, Germany), using the software Gen5™ (Biotek, Germany) for nucleic acid quantification.
Primers used on RNA-Seq validation
5’ – GAACAAGATCAATCGCCAAG – 3’
5’ – ACAGCTTTCCTTTAGTGGAG – 3’
5’ – GAATTTGATGCAAGGGACAG – 3’
5’ – TAAAGGTCAAATCAGAGCCC – 3’
5’ – GAGAGGCAGAGGGAGCATCC – 3’
5’ – GCTTGCTCCCGCCTTCTTCGC – 3’
5’ – GTTGGTAGAGTGATTGG – 3’
5’ – GAAATGCCAAGGCCAAATAT – 3’
5’ – GGAGAATGATAGCATGCAGA – 3’
5’ – TTTCCACCTCTCTCACATTG – 3’
RT-qPCR runs were perform, for each gene, with a series of decimal dilutions of a precisely calculated number of plasmid copies (3,000 pg to 3 pg) to create a calibration ruler. Cqs (threshold cycles) obtained from RNA samples was matched against the calibration ruler to estimate the copy number of each transcript on samples. The absolute copy number was calculated using the following formula: Copy number = C × NA/M; where Copy number, number of molecules/μL contained in the purified cDNA; C, concentration of the purified cDNA (g/μL); M, the molecular weight of the cDNA gene fragment; NA, Avogadro’s number = 6.023 × 1023 molecules/mole. The coefficient of correlation (r) between RNA-Seq samples and RT-qPCR were also calculated.
cDNA sequence cloning
To verify the sequence of the five selected genes putatively involved in flower development (VvTFL1, VvLFY, VvAP1, Vv AP3, VvPI) we cloned and sequenced the corresponding open reading frame of wild grapevine, as well as V. v. vinifera (Table 2). Amplification reactions were performed in 25 μL composed by 1 μg of cDNA; PCR buffer (20 mM Tris HCl [pH 8.4], 50 mM KCl; 1.5 mM of MgCl2); 0.2 mM of dNTPs; 0.4 μM of each primer; 5 U of DNA taq polymerase and autoclaved MiliQ water. The applied program had 4 min for initial denaturation at 94°C; 30 cycles of 45 s at 94°C for denaturation, 45 s at 55°C for annealing and 90 s at 72°C for extension; and a final extension at 72°C for 4 minutes. PCR products were purified with Zymoclean Gel DNA Recovery (Zymo Research Corp., Orange, CA, USA) and cloned with TOPO TA Cloning Kit (Invitrogen™, Carlsbad, Calif.) following the manufacturer’s protocols. Plasmids were extracted with Wizard Plus SV Minipreps DNA Purification (Promega, Leiden, The Netherlands). Plasmid concentration was measured in a microplate reader Synergy HT (Biotek, Germany), using the software Gen5™ (Biotek, Germany) for nucleic acid quantification.
RNA-Seq data analyzed in this study have been submitted to the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under the number GSE56844.
This work was supported by the funded project PTDC/AGR-GPL/119298/2010 from Fundação para a Ciência e Tecnologia and MRocheta, JLCoito and JCunha by the fellowships FRH/BPD/64905/2009, SFRH/BD/85824/2012 and SFRH/BPD/74895/2010, respectively. We are also grateful to Eng. Eiras-Dias, curator from Portuguese Ampelographic Collection (property of Instituto Nacional de Investigação Agrária e Veterinária, Dois Portos), for the collaboration in this work allowing the access to the Vitis collection.
- Wen J: Vitaceae. The families and genera of vascular plants. Edited by: Kubitzki K. 2007, Berlin: Springer Berlin Heidelberg, 466-478. Volume 9Google Scholar
- Chen I, Manchester SR: Seed morphology of modern and fossil Ampelocissus (Vitaceae) and implications for phytogeography. Am J Bot. 2007, 94: 1534-1553. 10.3732/ajb.94.9.1534.PubMedView ArticleGoogle Scholar
- McGovern PE, Glusker DL, Lawrence LJMMV: Neolithic resinated wine. Nature. 1996, 381: 480-481. 10.1038/381480a0.View ArticleGoogle Scholar
- Zohary D, Hopf M: Domestication of plants in the Old World. 2000, New York: Oxford University Press, 3Google Scholar
- William DA, Su YH, Smith MR, Lu M, Baldwin DA, Wagner D: Genomic identification of direct target genes of LEAFY. Proc Natl Acad Sci U S A. 2004, 101 (6): 1775-1780. 10.1073/pnas.0307842100.PubMed CentralPubMedView ArticleGoogle Scholar
- Kimura PH, Okamoto GKKH: Flower types, pollen morphology and germination, fertilization, and berry set in Vitis coignetiae Pulliat. Am J Enol Vitic. 1997, 48: 323-327.Google Scholar
- Caporali E, Spada A, Marziani G, Failla Q, Scienza A: The arrest of development of abortive reproductive organs in the unisexual flower of Vitis vinifera ssp. silvestris. Sex Plant Reprod. 2003, 15: 291-300.Google Scholar
- Gallardo A, Ocete R, López MA, Lara M, Rivera D: Assessment of pollen dimorphism in populations of Vitis vinifera L. subsp. sylvestris (Gmelin). Hegi in Spain Vitis. 2009, 48 (2): 59-62.Google Scholar
- Diggle PK, Stilio VSD, Gschwend AR, Golenberg EM, Moore RC, Russell JRW, Sinclair JP: Multiple developmental processes underlie sex differentiation in angiosperms. Trends Genet. 2011, 27 (9): 368-376. 10.1016/j.tig.2011.05.003.PubMedView ArticleGoogle Scholar
- Liljegren SJ, Gustafson-Brown C, Pinyopich A, Ditta GS, Yanofsky MF: Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate. Plant Cell. 1999, 11: 1007-1018. 10.1105/tpc.11.6.1007.PubMed CentralPubMedView ArticleGoogle Scholar
- Rijpkema AS, Vandenbussche M, Koes R, Heijmans K, Gerats T: Variations on a theme: Changes in the floral ABCs in angiosperms. Semin Cell Dev Biol. 2010, 21: 100-107. 10.1016/j.semcdb.2009.11.002.PubMedView ArticleGoogle Scholar
- Murai K: Homeotic genes and the ABCDE model for floral organ formation in wheat. Plants. 2013, 2: 379-395. 10.3390/plants2030379.View ArticleGoogle Scholar
- Krizek BA, Fletcher JC: Molecular mechanisms of flower development: an armchair guide. Nat Rev Genet. 2005, 6 (9): 688-698. 10.1038/nrg1675.PubMedView ArticleGoogle Scholar
- Immink RG, Kaufmann K, Angenent GC: The 'ABC' of MADS domain protein behaviour and interactions. Semin Cell Dev Biol. 2010, 21 (1): 87-93. 10.1016/j.semcdb.2009.10.004.PubMedView ArticleGoogle Scholar
- Levin JZ, Meyerowitz EM: UFO: an Arabidopsis gene involved in both floral meristem and floral organ development. Plant Cell. 1995, 7: 529-548. 10.1105/tpc.7.5.529.PubMed CentralPubMedView ArticleGoogle Scholar
- Wilkinson MD, Haughn GW: UNUSUAL FLORAL ORGANS controls meristem identity and organ primordia fate in Arabidopsis. Plant Cell. 1995, 7 (9): 1485-1499. 10.1105/tpc.7.9.1485.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu C, Xi W, Shen L, Tan C, Yu H: Regulation of floral patterning by flowering time genes. Dev Cell. 2009, 16 (5): 711-722. 10.1016/j.devcel.2009.03.011.PubMedView ArticleGoogle Scholar
- Wagner D, Sablowski RW, Meyerowitz EM: Transcriptional activation of APETALA1 by LEAFY. Science. 1999, 285 (5427): 582-584. 10.1126/science.285.5427.582.PubMedView ArticleGoogle Scholar
- Hill TA, Day CD, Zondlo SC, Thackeray AG, Irish VF: Discrete spatial and temporal cis-acting elements regulate transcription of the Arabidopsis floral homeotic gene APETALA3. Development. 1998, 125 (9): 1711-1721.PubMedGoogle Scholar
- Tilly JJ, Allen DW, Jack T: The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects. Development. 1998, 125 (9): 1647-1657.PubMedGoogle Scholar
- Honma T, Goto K: The Arabidopsis floral homeotic gene PISTILLATA is regulated by discrete cis-elements responsive to induction and maintenance signals. Development. 2000, 127 (10): 2021-2030.PubMedGoogle Scholar
- Lenhard M, Bohnert A, Jurgens G, Laux T: Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS. Cell. 2001, 105 (6): 805-814. 10.1016/S0092-8674(01)00390-7.PubMedView ArticleGoogle Scholar
- Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F, Simon R: A molecular link between stem cell regulation and floral patterning in Arabidopsis. Cell. 2001, 105: 793-803. 10.1016/S0092-8674(01)00384-1.PubMedView ArticleGoogle Scholar
- Gomez-Mena C, de Folter S, Costa MM, Angenent GC, Sablowski R: Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis. Development. 2005, 132 (3): 429-438. 10.1242/dev.01600.PubMedView ArticleGoogle Scholar
- Aukerman MJ, Sakai H: Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell. 2003, 15 (11): 2730-2741. 10.1105/tpc.016238.PubMed CentralPubMedView ArticleGoogle Scholar
- Chen X: A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science. 2004, 303: 2022-2025. 10.1126/science.1088060.PubMedView ArticleGoogle Scholar
- Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D: Specific effects of microRNAs on the plant transcriptome. Dev Cell. 2005, 8 (4): 517-527. 10.1016/j.devcel.2005.01.018.PubMedView ArticleGoogle Scholar
- Wagner D: Flower morphogenesis: timing is key. Dev Cell. 2009, 16 (5): 621-622. 10.1016/j.devcel.2009.05.005.PubMedView ArticleGoogle Scholar
- Kater MM, Franken J, Carney KJ, Colombo L, Angenent GC: Sex determination in the monoecious species cucumber is confined to specific floral whorls. Plant Cell Environ. 2001, 13: 481-493. 10.1105/tpc.13.3.481.View ArticleGoogle Scholar
- Oberle GD: A genetic study of variations in floral morphology and function in cultivated forms of Vitis. N Y AgrExpt Sta Tech Bul. 1938, 250: 3-32.Google Scholar
- Charlesworth D: Plant sex determination and sex chromosomes. Heredity. 2002, 88: 94-101. 10.1038/sj.hdy.6800016.PubMedView ArticleGoogle Scholar
- Cunha J, Baleiras-Couto M, Cunha JP, Banza J, Soveral A, Carneiro LC, Eiras-Dias JE: Characterization of Portuguese populations of Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi. Genet Resour Crop Evol. 2007, 54: 981-988. 10.1007/s10722-006-9189-y.View ArticleGoogle Scholar
- Baggiolini M: Les stades repères dans le developpement annuel de la vigne et leur utilisation pratique. Rev Romande Agric Vitic Arbor. 1952, 8: 4-6.Google Scholar
- Morin RD, O'Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu AL, Zhao Y, McDonald H, Zeng T, Hirst M, Eaves CJ, Marra MA: Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res. 2008, 18 (4): 610-621. 10.1101/gr.7179508.PubMed CentralPubMedView ArticleGoogle Scholar
- Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008, 5: 621-628. 10.1038/nmeth.1226.PubMedView ArticleGoogle Scholar
- Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyère C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Fabbro CD, Alaux M, Gaspero GD, Dumas V, et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007, 449 (7161): 463-467. 10.1038/nature06148.PubMedView ArticleGoogle Scholar
- Grimplet J, Van Hemert J, Carbonell-Bejerano P, Diaz-Riquelme J, Dickerson J, Fennell A, Pezzotti M, Martinez-Zapater JM: Comparative analysis of grapevine whole-genome gene predictions, functional annotation, categorization and integration of the predicted gene sequences. BMC Res Notes. 2012, 5: 213-10.1186/1756-0500-5-213.PubMed CentralPubMedView ArticleGoogle Scholar
- Johnson JM, Edwards S, Shoemaker D, Schadt EE: Dark matter in the genome: evidence of widespread transcription detected by microarray tiling experiments. Trends Genet. 2005, 21 (2): 93-102. 10.1016/j.tig.2004.12.009.PubMedView ArticleGoogle Scholar
- Bakel HV, Nislow C, Blencowe BJ, Hughes TR: Most “dark matter” transcripts are associated with known genes. PLoS Biol. 2010, 8: e1000371-10.1371/journal.pbio.1000371.PubMed CentralPubMedView ArticleGoogle Scholar
- Bakel HV, Nislow C, Blencowe BJ, Hughes TR: Response to “The reality of pervasive transcription”. PLoS Biol. 2011, 9: e1001102-10.1371/journal.pbio.1001102.PubMed CentralView ArticleGoogle Scholar
- Carmona MJ, Calonje M, Martinez-Zapater JM: The FT/TFL1 gene family in grapevine. Plant Mol Biol. 2007, 63 (5): 637-650. 10.1007/s11103-006-9113-z.PubMedView ArticleGoogle Scholar
- Carmona MJ, Cubas P, Martinez-Zapater JM: VFL, the grapevine FLORICAULA/LEAFY ortholog, is expressed in meristematic regions independently of their fate. Plant Physiol. 2002, 130 (1): 68-77. 10.1104/pp.002428.PubMed CentralPubMedView ArticleGoogle Scholar
- Calonje M, Cubas P, Martinez-Zapater JM, Carmona MJ: Floral meristem identity genes are expressed during tendril development in grapevine. Plant Physiol. 2004, 135 (3): 1491-1501. 10.1104/pp.104.040832.PubMed CentralPubMedView ArticleGoogle Scholar
- Poupin MJ, Federici F, Medina C, Matus JT, Timmermann T, Arce-Johnson P: 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. Gene. 2007, 404: 10-24. 10.1016/j.gene.2007.08.005.PubMedView ArticleGoogle Scholar
- Marioni JCMC, Mane SM, Stephens M, Gilad Y: RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 2008, 18: 1509-1517. 10.1101/gr.079558.108.PubMed CentralPubMedView ArticleGoogle Scholar
- Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Baren MJV, Salzberg SL, Wold BJ, Pachter L: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010, 28 (5): 511-515. 10.1038/nbt.1621.PubMed CentralPubMedView ArticleGoogle Scholar
- Ratcliffe OJ, Bradley DJ, Coen ES: Separation of shoot and floral identity in Arabidopsis. Development. 1999, 126 (6): 1109-1120.PubMedGoogle Scholar
- Wollmann H, Mica E, Todesco M, Long JA, Weigel D: On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development. Development. 2010, 137: 3633-3642. 10.1242/dev.036673.PubMed CentralPubMedView ArticleGoogle Scholar
- Theissen G, Saedler H: Floral quartets. Nature. 2001, 409 (6819): 469-471. 10.1038/35054172.PubMedView ArticleGoogle Scholar
- Yao A, Yang Y, Liao K, Zhang L, Hu J: The expression of VFL and VvTFL1 genes in relation to the effects of gibberellins in different organs of “Xiangfei” grapevine. Afr J Biotechnol. 2010, 9: 2748-2755.Google Scholar
- Weigel D, Meyerowitz EM: Activation of floral homeotic genes in Arabidopsis. Science. 1993, 261 (5129): 1723-1726. 10.1126/science.261.5129.1723.PubMedView ArticleGoogle Scholar
- Leonkloosterziel KM, Keijzer CJ, Koornneef M: A Seed Shape Mutant of Arabidopsis That Is Affected in Integument Development. Plant Cell. 1994, 6 (3): 385-392. 10.1105/tpc.6.3.385.View ArticleGoogle Scholar
- Modrusan Z, Reiser L, Feldmann KA, Fischer RL, Haughn GW: Homeotic Transformation of Ovules into Carpel-Like Structures in Arabidopsis. Plant Cell. 1994, 6 (3): 333-349. 10.1105/tpc.6.3.333.PubMed CentralPubMedView ArticleGoogle Scholar
- Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF: Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature. 2003, 424 (6944): 85-88. 10.1038/nature01741.PubMedView ArticleGoogle Scholar
- Brambilla V, Battaglia R, Colombo M, Masiero S, Bencivenga S, Kater MM, Colombo L: Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis. Plant Cell. 2007, 19: 2544-2556. 10.1105/tpc.107.051797.PubMed CentralPubMedView ArticleGoogle Scholar
- Aarts MG, Hodge R, Kalantidis K, Florack D, Wilson ZA, Mulligan BJ, Stiekema WJ, Scott R, Pereira A: The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J. 1997, 12 (3): 615-623. 10.1046/j.1365-313X.1997.d01-8.x.PubMedView ArticleGoogle Scholar
- Ito T, Nagata N, Yoshiba Y, Ohme-Takagi M, Ma H, Shinozaki K: Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development. Plant Cell. 2007, 19 (11): 3549-3562. 10.1105/tpc.107.054536.PubMed CentralPubMedView ArticleGoogle Scholar
- Yang C, Vizcay-Barrena G, Conner K, Wilson ZA: MALE STERILITY1 is required for tapetal development and pollen wall biosynthesis. Plant Cell. 2007, 19 (11): 3530-3548. 10.1105/tpc.107.054981.PubMed CentralPubMedView ArticleGoogle Scholar
- Dobritsa AA, Nishikawa S, Preuss D, Urbanczyk-Wochniak E, Sumner LW, Hammond A, Carlson AL, Swanson RJ: LAP3, a novel plant protein required for pollen development, is essential for proper exine formation. Sex Plant Reprod. 2009, 22 (3): 167-177. 10.1007/s00497-009-0101-8.PubMedView ArticleGoogle Scholar
- Guan YF, Huang XY, Zhu J, Gao JF, Zhang HX, Yang ZN: RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis. Plant Physiol. 2008, 147 (2): 852-863. 10.1104/pp.108.118026.PubMed CentralPubMedView ArticleGoogle Scholar
- Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, Bennetzen JL, Zhang Q, Wang S: Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 2006, 20 (10): 1250-1255. 10.1101/gad.1416306.PubMed CentralPubMedView ArticleGoogle Scholar
- Rodriguez-Concepcion M, Beltran JP: Repression of the pea lipoxygenase gene loxg is associated with carpel development. Plant Mol Biol. 1995, 27 (5): 887-899. 10.1007/BF00037017.PubMedView ArticleGoogle Scholar
- Bentolila S, Alfonso AA, Hanson MR: A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants. Proc Natl Acad Sci U S A. 2002, 99 (16): 10887-10892. 10.1073/pnas.102301599.PubMed CentralPubMedView ArticleGoogle Scholar
- Koizuka N, Imai R, Fujimoto H, Hayakawa T, Kimura Y, Kohno-Murase J, Sakai T, Kawasaki S, Imamura J: Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. 2003, 34 (4): 407-415. 10.1046/j.1365-313X.2003.01735.x.PubMedView ArticleGoogle Scholar
- Kazama T, Toriyama K: A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice. FEBS Lett. 2003, 544 (1–3): 99-102.PubMedView ArticleGoogle Scholar
- Cheng Y, Dai X, Zhao Y: Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev. 2006, 20 (13): 1790-1799. 10.1101/gad.1415106.PubMed CentralPubMedView ArticleGoogle Scholar
- Chandler JW: The hormonal regulation of flower development. J Plant Growth Regul. 2011, 30: 242-254. 10.1007/s00344-010-9180-x.View ArticleGoogle Scholar
- Tanurdzic M, Banks JA: Sex-determining mechanisms in land plants. Plant Cell. 2004, 16 (Suppl): S61-71.PubMed CentralPubMedView ArticleGoogle Scholar
- Chae HS, Kieber JJ: Eto Brute? Role of ACS turnover in regulating ethylene biosynthesis. Trends Plant Sci. 2005, 10 (6): 291-296. 10.1016/j.tplants.2005.04.006.PubMedView ArticleGoogle Scholar
- Saito S, Fujii N, Miyazawa Y, Yamasaki S, Matsuura S, Mizusawa H, Fujita Y, Takahashi H: Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants. J Exp Bot. 2007, 58 (11): 2897-2907. 10.1093/jxb/erm141.PubMedView ArticleGoogle Scholar
- Kwon C, Neu C, Pajonk S, Yun HS, Lipka U, Humphry M, Bau S, Straus M, Kwaaitaal M, Rampelt H, El Kasmi F, Jürgens G, Parker J, Panstruga R, Lipka V, Schulze-Lefert P: Co-option of a default secretory pathway for plant immune responses. Nature. 2008, 451 (7180): 835-840. 10.1038/nature06545.PubMedView ArticleGoogle Scholar
- Merchante C, Alonso JM, Stepanova AN: Ethylene signaling: simple ligand, complex regulation. Curr Opin Plant Biol. 2013, 16 (5): 554-560. 10.1016/j.pbi.2013.08.001.PubMedView ArticleGoogle Scholar
- Negi SS, Olmo HP: Sex conversion in a male Vitis vinifera L. by a kinin. Science and Sports. 1966, 152: 1624-1625.Google Scholar
- Negi SS, Olmo HP: Studies on sex conversion in male Vitis vinifera L. (sylvestris). Vitis. 1970, 9: 89-96.Google Scholar
- Barrett SCH, Hough J: Sexual dimorphism in flowering plants. J Exp Bot. 2013, 64 (1): 67-82. 10.1093/jxb/ers308.PubMedView ArticleGoogle Scholar
- Lioyd DG: The maintenance of gynodioecy and androdioecy in angiosperms. Genetica. 1975, 45: 325-339. 10.1007/BF01508307.View ArticleGoogle Scholar
- Wolf DE, Satkoski JA, White K, Rieseberg LH: Sex determination in the androdioecious plant Datisca glomerata and its dioecious sister species D. cannabina. Genetics. 2001, 159 (3): 1243-1257.PubMed CentralPubMedGoogle Scholar
- Pannell J: Mixed genetic and environmental sex determination in an androdioecious population of Mercurialis annua. Heredity. 1997, 78 (Pt 1): 50-56.PubMedView ArticleGoogle Scholar
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