Isolation and Characterization of cDNAs Expressed in the Early Stages of Flavonol-Induced Pollen Germination in Petunia (original) (raw)
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Plant Physiology, 2003
Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division. ; fax 351-21-4407970.
Genes & Genetic Systems, 2010
After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for upregulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.
Transcriptome profiling of tobacco (Nicotiana tabacum) pollen and pollen tubes
BMC Genomics
Background: Pollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6). Results: De novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization. Conclusions: A major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.
BMC Genomics, 2010
Background: Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to quickly germinate, produce a polar-growth pollen tube derived from the vegetative cell, and deliver two sperm cells into the embryo sac for double fertilization. The gene expression profiles of developing and germinated pollen have been characterised by use of the eudicot model plant Arabidopsis. Rice, one of the most important cereal crops, has been used as an excellent monocot model. A comprehensive analysis of transcriptome profiles of developing and germinated pollen in rice is important to understand the conserved and diverse mechanism underlying pollen development and germination in eudicots and monocots. Results: We used Affymetrix GeneChip ® Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stageenriched transcripts displayed a "U-type" change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/ 26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each. Conclusions: Our results demonstrated that rice pollen expressed a set of reduced but specific transcripts in comparison with vegetative tissues, and the number of stage-enriched transcripts displayed a "U-type" change during pollen development, with the lowest at the bicellular pollen stage. These features are conserved in rice and Arabidopsis. The shift in gene expression program at the bicellular pollen stage may be important to the transition from earlier cell division to later pollen maturity. Pollen at maturity presynthesized transcripts needed for germination and early pollen tube growth. The transcription regulation associated with pollen development would have divergence between the two species. Our results also provide novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination.
Unveiling the gene-expression profile of pollen
Genome biology, 2003
Four recent papers have characterized the transcription profile of pollen grains, showing striking differences between gene expression in pollen and other plant tissues. These studies increase the number of known pollen-expressed genes by as much as 50-fold and have identified many novel genes that are potentially pollen-specific.
Transcript profiling on developing Petunia hybrida floral organs
Sexual Plant Reproduction, 2003
The cDNA-AFLP transcript profiling technique was used to analyse gene expression during flower development in Petunia hybrida. Reproductive and vegetative floral organs were sampled at five developmental stages and gene expression profiles were compared. This allowed us to assemble an inventory of genes expressed mainly in anthers during microspore development and in ovaries during macrosporogenesis. About 6,000 transcript tags were generated, 354 of which showed a modulated and/or organ-specific expression pattern. Stamen-specific transcripts exhibiting an upregulation in gene expression were well represented in our screening. Ovary-specific transcripts were less frequently observed and often displayed a constant level of gene expression. Of 194 fragments characterised further by sequencing, 35% showed homology with known genes in a database search. They belong to a wide range of gene classes, such as proteases, transcription factors and genes involved in metabolism, cell cycle and disease resistance. The usefulness of cDNA-AFLP transcript profiling as a tool to unravel complex developmental processes at the molecular level is discussed.
Analysis of expressed sequence tags from Petunia flowers
Plant Science, 2007
Petunia cDNA libraries were prepared from whole flower parts (including buds), pollen, and pollen tubes to generate expressed sequence tags (ESTs). A total of 7001 random clones were subjected to unidirectional sequencing, resulting in identification of 611 groups of related sequences and 2410 singletons. Highly conserved 1098 ESTs were functionally assigned. ESTs encoding proteins involved in the calcium-dependent signal pathway and in cell wall metabolism such as pectin degradation and modification were frequently found in the pollen and pollen tube libraries. The 2976 cDNA clones from the bud/flower cDNA libraries were used for the construction of microarrays. The 112 functionally annotated genes were up-regulated in the buds just before opening, including the genes for anthocyanin pigmentation and protein degradation. These ESTs and microarrays will serve the analysis of floral traits of petunias. #
Journal of the American Society for Horticultural Science, 1999
Mature pollen from Petunia hybrida contains significant levels of 1-aminocyclopropane-1-carboxylic acid (ACC), and this ACC is thought to play a role in pollination-induced ethylene by the pistil. We investigated the developmental accumulation of ACC in anthers and pollen. The level of ACC in anthers was very low until the day before anthesis, at which time it increased 100-fold. A 1.1-kb partial ACC synthase cDNA clone (pPHACS2) was amplified from total RNA isolated from mature anthers by reverse transcriptase, followed by polymerase chain reaction using oligonucleotide primers synthesized to conserved amino acid sequences in ACC synthases. The expression of pPHACS2 mRNA during anther development was correlated with the accumulation of ACC and was localized to the pollen grain. The pPHACS2 cDNA was used to identify the PH-ACS2 gene from a library of genomic DNA fragments from Petunia hybrida. PH-ACS2 encoded an ACC synthase transcript of four exons interrupted by three introns. The...
Functional genomics of pollen tube–pistil interactions in Arabidopsis
Biochemical Society Transactions, 2010
The pollen tube represents an attractive model system for functional genomic analysis of the cell–cell interactions that mediate guided cellular growth. The pollen tube extends through pistil tissues and responds to guidance cues that direct the tube towards an ovule, where it releases sperm for fertilization. Pollen is readily isolated from anthers, where it is produced, and can be induced to produce a tube in vitro. Interestingly, pollen tube growth is significantly enhanced in pistils, and pollen tubes are rendered competent to respond to guidance cues after growth in a pistil. This potentiation of the pollen tube by the pistil suggested that pollen tubes alter their gene-expression programme in response to their environment. Recently, the transcriptomes of pollen tubes grown in vitro or through pistil tissues were determined. Significant changes in the transcriptome were found to accompany growth in vitro and through the pistil tissues. Reverse genetic analysis of pollen-tube-in...
Pollen development, a genetic and transcriptomic view
Pollen Tube: Cellular and Molecular Perspective, 2006
The haploid gametophyte generation occupies a short but vital phase in the life cycle of flowering plants. The male gametophyte consists of just two or three cells when shed from the anthers as pollen grains. It is this functional specialization that is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen development offers an excellent model system to study many fundamentally important biological processes such as polarity, cell fate determination, cell cycle regulation, cell signaling and mechanisms of gene regulation.