Stages Of Anther Development Research Articles

Relationship between flower characteristics and androsporogenesis in wild species and interspecific hybrids of Passiflora

This study was carried out aiming to characterize the different stages of androsporogenesis (meiotic) and androgametogenesis (post-meiotic), and analyze the relationship between the lengths of the flower bud and the anther in relation to the development stages of gametophytes in wild species of Passiflora of ornamental potential and interspecific hybrids F1, to generate subsidies for researches with anther culture and genetic improvement programs. Due to overlaps between the lengths of the buds and anthers and the stages of meiosis I and II, the analyses have allowed only the differentiation of five different classes: prophase I, meiocytes with cells in metaphase I to telophase II, tetrad, androspores, and androphyte I. The use of the anther length was more indicated for differentiation between the androsporogenesis and androgametogenesis than bud length. The occurrence of symmetric binucleate androphyte was considered an anomaly in view of its low occurrence (lower than 7.5 %). There was no association between the different locations of anther sections and developmental stages of androgametogenesis, with overlaps between these variables.

Transcriptomic analysis of differentially expressed genes during anther development in genetic male sterile and wild type cotton by digital gene-expression profiling.

BackgroundCotton (Gossypium hirsutum) anther development involves a diverse range of gene interactions between sporophytic and gametophytic tissues. However, only a small number of genes are known to be specifically involved in this developmental process and the molecular mechanism of the genetic male sterility (GMS) is still poorly understand. To fully explore the global gene expression during cotton anther development and identify genes related to male sterility, a digital gene expression (DGE) analysis was adopted.ResultsSix DGE libraries were constructed from the cotton anthers of the wild type (WT) and GMS mutant (in the WT background) in three stages of anther development, resulting in 21,503 to 37,352 genes detected in WT and GMS mutant anthers. Compared with the fertile isogenic WT, 9,595 (30% of the expressed genes), 10,407 (25%), and 3,139 (10%) genes were differentially expressed at the meiosis, tetrad, and uninucleate microspore stages of GMS mutant anthers, respectively. Using both DGE experiments and real-time quantitative RT-PCR, the expression of many key genes required for anther development were suppressed in the meiosis stage and the uninucleate microspore stage in anthers of the mutant, but these genes were activated in the tetrad stage of anthers in the mutant. These genes were associated predominantly with hormone synthesis, sucrose and starch metabolism, the pentose phosphate pathway, glycolysis, flavonoid metabolism, and histone protein synthesis. In addition, several genes that participate in DNA methylation, cell wall loosening, programmed cell death, and reactive oxygen species generation/scavenging were activated during the three anther developmental stages in the mutant.ConclusionsCompared to the same anther developmental stage of the WT, many key genes involved in various aspects of anther development show a reverse gene expression pattern in the GMS mutant, which indicates that diverse gene regulation pathways are involved in the GMS mutant anther development. These findings provide the first insights into the mechanism that leads to genetic male sterility in cotton and contributes to a better understanding of the regulatory network involved in anther development in cotton.

Expression Profile in Rice Panicle: Insights into Heat Response Mechanism at Reproductive Stage

Rice at reproductive stage is more sensitive to environmental changes, and little is known about the mechanism of heat response in rice panicle. Here, using rice microarray, we provided a time course gene expression profile of rice panicle at anther developmental stage 8 after 40°C treatment for 0 min, 20 min, 60 min, 2 h, 4 h, and 8 h. The identified differentially expressed genes were mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response. The predominant transcription factor gene families responsive to heat stress were Hsf, NAC, AP2/ERF, WRKY, MYB, and C2H2. KMC analysis discovered the time-dependent gene expression pattern under heat stress. The motif co-occurrence analysis on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3 in response to heat stress. The regulation model central to ROS combined with transcriptome and ROS quantification data in rice panicle indicated the great importance to maintain ROS balance and the existence of wide cross-talk in heat response. The present study increased our understanding of the heat response in rice panicle and provided good candidate genes for crop improvement.

The SlFRK4 promoter is active only during late stages of pollen and anther development

Carbohydrates are essential for male gametophyte development. However, our understanding of the mechanism by which the sugar supply is controlled in the stamen is still in its infancy. We previously reported on the stamen-specific expression of the tomato (Solanum lycopersicum) sugar metabolic gene, fructokinase 4 (SlFRK4). Here, we present the cloning and the characterization of the SlFRK4 promoter and show its differential activation during anther development. We also show that the tissue-specific expression of SlFRK4 promoter is maintained in Arabidopsis thaliana. By histochemical analyses of the GUS reporter gene and DTA toxin driven by the SlFRK4 promoter, we show that the SlFRK4 promoter is gradually activated in pollen grains throughout the later stages of anther development and upon pollen germination. In addition, we analyzed the expression profile of SlFRK4 and other sugar metabolic genes and found that SlFRK4 and the invertase LIN7 are co-expressed in mature and germinated pollen. These findings point to the existence of a specialized mechanism in which carbohydrates are provided to the male gametophyte during the later stages of its development and suggest a valuable tool for manipulating the development of male gametophytes in crop species.

Expression analyses of Brachiaria brizantha genes encoding ribosomal proteins BbrizRPS8, BbrizRPS15a, and BbrizRPL41 during development of ovaries and anthers

Brachiaria brizantha is a forage grass of the Poaceae family. Introduced from Africa, it is largely used for beef cattle production in Brazil. Brachiaria reproduces sexually or asexually by apomixis, and development of biotechnological tools for gene transfer is being researched to support the breeding programs. The molecular bases of reproduction have not yet been fully elucidated; it is known that gametophyte formation and main reproductive events occur inside the anthers and ovaries. There is therefore much interest in identifying genes expressed in these organs and their corresponding upstream regulatory sequences. In this work we characterized three cDNA from ovaries of B. brizantha plants (CL 09, CL10, and CL21) which show similarity in databases with genes encoding ribosomal proteins S8, S15a, and L41 and were named BbrizRPS8, BbrizRPS15a, and BbrizRPL41, respectively. These clones show higher expression in ovaries, anthers and roots, mitotically active tissues, when compared to leaves of B. brizantha. Localization of transcripts of BbrizRPS8, BbrizRPS15a, and BbrizRPL41 was investigated in the reproductive organs, ovaries, and anthers, from the beginning of development up to maturity. Their activity was higher in early stages of anther development, while expression was detected in all developmental stages in the ovaries, except for BbrizS15a, which was detected only in synergids of apomictic plants.

The MYB80 Transcription Factor Is Required for Pollen Development and the Regulation of Tapetal Programmed Cell Death inArabidopsis thaliana

Arabidopsis thaliana MYB80 (formerly MYB103) is expressed in the tapetum and microspores between anther developmental stages 6 and 10. MYB80 encodes a MYB transcription factor that is essential for tapetal and pollen development. Using microarray analysis of anther mRNA, we identified 404 genes differentially expressed in the myb80 mutant. Employing the glucocorticoid receptor system, the expression of 79 genes was changed when MYB80 function was restored in the myb80 mutant following induction by dexamethasone. Thirty-two genes were analyzed using chromatin immunoprecipitation, and three were identified as direct targets of MYB80. The genes encode a glyoxal oxidase (GLOX1), a pectin methylesterase (VANGUARD1), and an A1 aspartic protease (UNDEAD). All three genes are expressed in the tapetum and microspores. Electrophoretic mobility shift assays confirmed that MYB80 binds to all three target promoters, with the preferential binding site containing the CCAACC motif. TUNEL assays showed that when UNDEAD expression was silenced using small interfering RNA, premature tapetal and pollen programmed cell death occurred, resembling the myb80 mutant phenotype. UNDEAD possesses a mitochondrial targeting signal and may hydrolyze an apoptosis-inducing protein(s) in mitochondria. The timing of tapetal programmed cell death is critical for pollen development, and the MYB80/UNDEAD system may regulate that timing.

Analysis of anther transcriptomes to identify genes contributing to meiosis and male gametophyte development in rice.

BackgroundIn flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development.ResultsTranscriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far.ConclusionsNot only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation.

N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N2O) gas to obtain 2n male gametes. N2O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N2O treatment restores fertility in sterile hybrids. To establish optimal N2O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N2O gas at different anther developmental stages from fertile and sterile hybrid lilies. N2O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N2O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC1 plants. Thus N2O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.

Cytochemical and ultrastructural observations of anthers and pollen grains in Lathyrus undulatus Boiss

Cytochemical and ultrastructural observations of anthers and pollen grains inLathyrus undulatusBoissInLathyrus undulatusBoiss. (Fabaceae), the young microspore stage of anther development was characterized by the enlarged secretory tapetal cells, which presented an intense reaction with regard to protein, insoluble polysaccharides and lipids. At bicellular pollen stage, the middle layer and the tapetum degenerated. After degradation of the tapetum, epidermis and single row U-shaped endothecium existed in the mature anther wall, and pollen grains remained in the locus. Young microspores had a spherical and centrally located nucleus with one or two nucleoli, many spherical lipid bodies and starchy plastids. A mature pollen grain contains insoluble polysaccharides, proteins, lipids and calcium. The mature pollen had the following morphological characteristics: 3-zonocolporate, prolate, tectate (imperforate) type of exine and perforate type of structure. The intine formed an important constituent portion of the wall, and consisted two sublayers: an outer intine (exintine) and an inner intine (endintine). The well-defined exine was made up of lipoidal substances and protein, but the intine composed of insoluble polysaccharides and protein. The bicellular state of the pollen grains persisted to anthesis.

DROUGHT STRESS: Role of Carbohydrate Metabolism in Drought‐Induced Male Sterility in Rice Anthers*

AbstractRice plants exposed to three consecutive days of water stress (−0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT‐PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers.

A flavonol synthase gene GtFLS defines anther-specific flavonol accumulation in gentian

Flavonoids are important for male gametophyte development. Here we report on the flavonoid components and their biosynthetic regulation in gentian anthers. Among flavonoids, flavonols, including kaempferol, quercetin and isorhamnetin derivatives, accumulated abundantly in gentian anthers. However, flavones and anthocyanins, which are the main flavonoids accumulating in petals, were not detected. Northern blot analysis of nine flavonoid biosynthetic genes showed that the ‘early’ flavonoid biosynthetic genes were expressed in both anthers and petals, and that flavonol synthase (FLS) transcripts were restricted to anthers. In contrast, flavone synthase II (FNSII) and ‘late’ flavonoid biosynthetic genes were expressed specifically in gentian petals. To confirm anther-specific expression of FLS, the 5′-upstream region of FLS (GtFLSpro) was cloned by inverse PCR and fused to the uidA (GUS) reporter gene. Tobacco, Arabidopsis and gentian plants, transformed with the GtFLSproGUS construct, exhibited anther-specific GUS expression. Expression was observed in the tapetum and in pollen at late stages of anther development in transgenic plants. These results revealed that flavonol accumulation in gentian anthers was regulated by the spatial expression of GtFLS. Our results also suggest that anther-specific regulation of FLS is conserved among higher plants and the GtFLS promoter is useful for induction of specific gene expression in anthers.

Water Deficit Reduced Fertility of Young Microspores Resulting in a Decline of Viable Mature Pollen and Grain Set in Rice

AbstractPollen formation in rice (Oryza sativa L.) is highly vulnerable to environmental stresses such as heat, chilling and drought. In rice plants exposed to drought during male reproductive development, the most obvious damage often observed is a decline in the number of engorged pollen and grain set. This has been well characterized in rice under chilling and to a lesser extent under drought stress. Moreover, detailed literature on the immediate effects of drought on developing young microspores in rice is still limited. Here, we report findings from experiments on rice plants exposed to water deficit for three consecutive days during early stages of anther development. When the osmotic potential of the growing medium was equal to or less than −0.5 MPa, as induced by polyethylene glycol, the leaf water potential was significantly lowered and grain set was reduced. A strong correlation between grain set and viable young microspores (P < 0.001, r2 = 0.8223) indicates that water deficit immediately reduced fertility of rice plants at the time of exposure. This result suggests a new underlying mechanism of water deficit‐induced pollen abortion in rice.

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase (PG) gene previously isolated from the flower bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). This gene was found to be expressed specifically in tapetum and pollen after the tetrad stage of anther development. Antisense RNA technology was used to study the function of BcMF2 in Chinese cabbage. Scanning and transmission electron microscopy revealed that there were deformities in the transgenic mature pollen grains such as abnormal location of germinal furrows. In addition, the homogeneous pectic exintine layer facing the exterior seemed to be overdeveloped and predominantly occupied the intine, thus reversing the normal proportional distribution of the internal endintine layer and the external exintine layer. Since it is a continuation of the intine layer, the pollen tube wall could not grow normally. This resulted in the formation of a balloon-like swelling structure in the pollen tube tip in nearly 80% of the transgenic pollen grains. Premature degradation of tapetum was also found in these transgenic plants, which displayed decreased expression of the BcMF2 gene. BcMF2 might therefore encode a new PG with an important role in pollen wall development, possibly via regulation of pectin's dynamic metabolism.

Inhibition of chalcone synthase Expression in Anthers of Raphanus sativus with Ogura Male Sterile Cytoplasm

Expression of the mitochondrial gene orf138 causes Ogura cytoplasmic male sterility (CMS) in Raphanus sativus, but little is known about the mechanism by which CMS takes place. A preliminary microarray experiment revealed that several nuclear genes concerned with flavonoid biosynthesis were inhibited in the male-sterile phenotype. In particular, a gene for one of the key enzymes for flavonoid biosynthesis, chalcone synthase (CHS), was strongly inhibited. A few reports have suggested that the inhibition of CHS causes nuclear-dependent male sterile expression; however, there do not appear to be any reports elucidating the effect of CHS on CMS expression. In this study, the expression patterns of the early genes in the flavonoid biosynthesis pathway, including CHS, were investigated in normal and male-sterile lines. In order to determine the aberrant stage for CMS expression, the characteristics of male-sterile anthers are observed using light and transmission electron microscopy for several stages of flower buds. The expression of CHS and the other flavonoid biosynthetic genes in the anthers were compared between normal and male-sterile types using real time RT-PCR. Among the flavonoid biosynthetic genes analysed, the expression of CHS was strongly inhibited in the later stages of anther development in sterility cytoplasm; accumulation of putative naringenin derivatives was also inhibited. These results show that flavonoids play an important role in the development of functional pollen, not only in nuclear-dependent male sterility, but also in CMS.

Gene trap strategy, an effective tool for identification of novel genes expressed in anther tissues in Arabidopsis thaliana

Analysis of GUS expression patterns was previously carried out for 20,000 gene trap lines of Arabidopsis thaliana generated by the Kazusa DNA Research Institute. This analysis indicated that 51 lines showed GUS expression within stamens. In this paper, we further focused on 42 of these lines in order to examine the detailed expression patterns at different anther developmental stages. Seven lines showed GUS expression exclusively in the tapetum during the early stage of anther development, thirty lines in pollen grains at the tricellular pollen stage and five lines in filaments at the tricellular pollen stage. We identified four trapped-genes by sequencing the adjacent fragments to T-DNA insertions. RT-PCR analysis confirmed that the mRNA expression patterns in these four genes were consistent with the expression pattern observed by GUS activity. Our results demonstrated that the gene trap system is an efficient strategy for identifying stamen specific genes in Arabidopsis.

Co-suppression of Si401, a maize pollen specific Zm401 homologous gene, results in aberrant anther development in foxtail millet

We previously reported that the maize pollen-specific gene Zm401 functions in anther development. In this study, a Zm401’s ortholog Si401 was amplified and cloned from foxtail millet (Setaria italica). Zm401 and Si401 are highly conserved with 99% of their coding sequences identical to each other. Southern blot and RT-PCR analysis reveal that Si401 has only one copy in the genome and is expressed exclusively in the panicle. Co-suppression of Si401 in foxtail millet results in multiple abnormalities during the late stages of anther development, including premature degeneration of the tapetum, pre-deposition of fibrous bands in endothecium cells, and aborted pollen grains. Our results demonstrate that Si401 plays an essential role in anther development in foxtail millet and might be useful for generation of male-sterile plants in cereal crops.

Characterization and functional analysis of a novel PCP gene BcMF5 from Chinese cabbage ( Brassica campestris L. ssp. chinensis Makino)

Brassica campestris Male Fertile 5 (BcMF5), a novel member of the pollen coat protein class A (PCP-A) gene family, was identified from Brassica campestris L. ssp. chinensis Makino (Chinese cabbage-pak-choi). Temporal and spatial expression analysis showed that BcMF5 is a late-expressed PCP gene related to the process of determining pollen fertility. Functional analysis by hairpin RNA (hpRNA)-mediated RNA interference also showed that the expression of BcMF5 is inhibited, which resulted in the low germination ability of the pollen and also in an abnormality of the pollen exemplified by a collapsed germination furrow. This demonstrates that the expression of BcMF5 is closely related to the tapetum. Further, the expression profile of the BcMF5 promoter in Arabidopsis was also analyzed. This analysis indicated that the BcMF5 promoter began expression in the early stage of anther development and drove high levels of glucuronidase (GUS) expression in anthers, pollen, and the pollen tube in the late stage of pollen development, but did not drive any expression in petals, sepals, or pistils. Together with the functional analysis, the hypothesis that BcMF5 may have a sporophytic or gametophytic expression pattern is presented.

The pea PsEND1 promoter drives the expression of GUS in transgenic wheat at the binucleate microspore stage and during pollen tube development

Many plant genetic engineering applications require spatial expression of genes which in turn depends upon the availability of specific promoters. In cereals, genetic modification of flowering and grain setting to influence yield and grain quality is of significant interest. PsEND1 is a pea promoter that displays expression in the epidermis, connective tissue, endothecium and middle layers during different stages of anther development. No homeologous sequence of this promoter or its coding sequence has been found in cereals. This present work aimed at the characterization of the pea PsEND1 promoter driving the expression of the gusA gene in transgenic wheat. Nine transgenic lines were produced by particle bombardment and analyzed for the expression of the gusA gene throughout development by histochemical GUS staining and by RT-PCR in vegetative and reproductive tissues and organs. Expression of the gusA gene was first detected during pollen development, in microspores at binucleate stage. Activity of the gusA gene was also found in mature pollen, after anthesis. Following pollen grain germination, expression of the gusA gene was seen from an early stage of pollen tube formation until advanced stages, approaching the ovary. No further expression of the gusA gene was detected after fertilization, nor during seed development. The results reported here show that the PsEND1 promoter is functional in wheat and its patterns of expression may be of interest for the application of genetic modification in wheat breeding.

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, -609 to +3 bp and -377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both -609 to +3 bp and -377 to +3 bp fragments of BcMF5 promoter were capable of driving beta-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan(R) plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.

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

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