Nejvíce citovaný článek - PubMed ID 15908605
Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.
- Klíčová slova
- Arabidopsis, Male gametophyte, Microgametogenesis, Proteome, RNA-seq,
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- proteomika MeSH
- pyl genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- reprodukovatelnost výsledků MeSH
- transkriptom MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny huseníčku * MeSH
Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.
- Klíčová slova
- gametophyte, mating system, ploidy, pollen competition, sexual selection,
- MeSH
- Arabis * MeSH
- genomika MeSH
- pohlavní výběr MeSH
- pyl genetika MeSH
- samooplození MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function.
- MeSH
- fenotyp MeSH
- genetická variace MeSH
- genotyp MeSH
- Magnoliopsida genetika růst a vývoj MeSH
- organogeneze rostlin genetika fyziologie MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny metabolismus MeSH
- rozmnožování genetika fyziologie MeSH
- sekvenční analýza RNA MeSH
- stanovení celkové genové exprese * MeSH
- transkripční faktory metabolismus MeSH
- vyšší rostliny genetika růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- rostlinné proteiny MeSH
- transkripční faktory MeSH
Formins are a large, evolutionarily conserved family of actin-nucleating proteins with additional roles in regulating microfilament, microtubule, and membrane dynamics. Angiosperm formins, expressed in both sporophytic and gametophytic tissues, can be divided into two subfamilies, Class I and Class II, each often exhibiting characteristic domain organization. Gametophytically expressed Class I formins have been documented to mediate plasma membrane-based actin assembly in pollen grains and pollen tubes, contributing to proper pollen germination and pollen tube tip growth, and a rice Class II formin, FH5/RMD, has been proposed to act as a positive regulator of pollen tube growth based on mutant phenotype and overexpression data. Here we report functional characterization of the Arabidopsis thaliana pollen-expressed typical Class II formin FH13 (At5g58160). Consistent with published transcriptome data, live-cell imaging in transgenic plants expressing fluorescent protein-tagged FH13 under the control of the FH13 promoter revealed expression in pollen and pollen tubes with non-homogeneous signal distribution in pollen tube cytoplasm, suggesting that this formin functions in the male gametophyte. Surprisingly, fh13 loss of function mutations do not affect plant fertility but result in stimulation of in vitro pollen tube growth, while tagged FH13 overexpression inhibits pollen tube elongation. Pollen tubes of mutants expressing a fluorescent actin marker exhibited possible minor alterations of actin organization. Our results thus indicate that FH13 controls or limits pollen tube growth, or, more generally, that typical Class II formins should be understood as modulators of pollen tube elongation rather than merely components of the molecular apparatus executing tip growth.
- Klíčová slova
- Arabidopsis thaliana, At5g58160, Class II formin, pollen tube, tip growth,
- Publikační typ
- časopisecké články MeSH
Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.
- MeSH
- Arabidopsis cytologie MeSH
- časové faktory MeSH
- centrifugace - gradient hustoty ekonomika metody MeSH
- oxid křemičitý chemie MeSH
- povidon chemie MeSH
- pyl cytologie MeSH
- separace buněk ekonomika metody MeSH
- viabilita buněk MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- oxid křemičitý MeSH
- Percoll MeSH Prohlížeč
- povidon MeSH
In our previous study we applied the Agilent 44K tobacco gene chip to introduce and analyze the tobacco male gametophyte transcriptome in mature pollen and 4h pollen tubes. Here we extended our analysis post-pollen mitosis II (PMII) by including a new data set obtained from more advanced stage of the ongoing progamic phase - pollen tubes cultivated in vitro for 24 h. Pollen mitosis II marks key events in the control of male gametophyte development, the production of two sperm cells. In bicellular species covering cca 70% of angiosperms including Nicotiana tabacum, PMII takes place after pollen germination in growing pollen tube. We showed the stable and even slightly increasing complexity of tobacco male gametophyte transcriptome over long period of progamic phase-24 h of pollen tube growth. We also demonstrated the ongoing transcription activity and specific transcript accumulation in post-PMII pollen tubes cultivated in vitro. In all, we have identified 320 genes (2.2%) that were newly transcribed at least after 4h of pollen tube cultivation in vitro. Further, 699 genes (4.8%) showed over 5-fold increased accumulation after the 24h of cultivation.
Male gametophyte development leading to the formation of a mature pollen grain is precisely controlled at various levels, including transcriptional, post-transcriptional and post-translational, during its whole progression. Transcriptomic studies exploiting genome-wide microarray technologies revealed the uniqueness of pollen transcriptome and the dynamics of early and late successive global gene expression programs. However, the knowledge of transcription regulation is still very limited. In this study, we focused on the identification of pollen-expressed transcription factor (TF) genes involved in the regulation of male gametophyte development. To achieve this, the reverse genetic approach was used. Seventy-four T-DNA insertion lines were screened, representing 49 genes of 21 TF families active in either early or late pollen development. In the screen, ten phenotype categories were distinguished, affecting various structural or functional aspects, including pollen abortion, presence of inclusions, variable pollen grain size, disrupted cell wall structure, cell cycle defects, and male germ unit organization. Thirteen lines were not confirmed to contain the T-DNA insertion. Among 61 confirmed lines, about half (29 lines) showed strong phenotypic changes (i.e., ≥ 25% aberrant pollen) including four lines that produced a remarkably high proportion (70-100%) of disturbed pollen. However, the remaining 32 lines exhibited mild defects or resembled wild-type appearance. There was no significant bias toward any phenotype category among early and late TF genes, nor, interestingly, within individual TF families. Presented results have a potential to serve as a basal information resource for future research on the importance of respective TFs in male gametophyte development.
- MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- DNA bakterií MeSH
- fenotyp MeSH
- multigenová rodina MeSH
- pyl růst a vývoj MeSH
- rostlinné geny MeSH
- transkripční faktory metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- T-DNA MeSH Prohlížeč
- transkripční faktory MeSH
Activated cortical domains (ACDs) are regions of the plant cell cortex performing localized membrane turnover, delimited by concerted action of the cortical cytoskeleton and endomembrane compartments. Arabidopsis thaliana rhizodermis consists of two cell types differing by a single ACD (trichoblasts, carrying tip-growing root hairs, and hairless atrichoblasts), providing a model for the study of ACD determination. We compiled a set of genes specifically upregulated in root hairs from published transcriptome data, and compared it with a "virtual Arabidopsis root hair proteome", i.e. a list of computationally identified homologs of proteins from the published soybean root hair proteome. Both data sets were enriched in genes and proteins associated with root hairs in functional studies, but there was little overlap between the transcriptome and the proteome: the former captured gene products specific to root hairs, while the latter selected those abundant in root hairs but not necessarily specific to them. Decisive steps in ACD specification may be performed by signaling proteins of high expression specifity and low abundance. Nevertheless, 73 genes specifically transcribed in Arabidopsis trichoblasts or root hairs encode homologs of abundant root hair proteins from soybean. Most of them encode "housekeeping" proteins required for rapid tip growth. However, among the "candidates" is also a generative actin isoform, ACT11. Preliminary characterization of an act11 mutant allele indeed suggests a hitherto unexpected role for this gene in root and root hair development.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- fenotyp MeSH
- kořeny rostlin genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku metabolismus MeSH
- pyl metabolismus MeSH
- regulace genové exprese u rostlin fyziologie MeSH
- stanovení celkové genové exprese * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny huseníčku MeSH
Sexual plant reproduction depends on the production and differentiation of functional gametes by the haploid gametophyte generation. Currently, we have a limited understanding of the regulatory mechanisms that have evolved to specify the gametophytic developmental programs. To unravel such mechanisms, it is necessary to identify transcription factors (TF) that are part of such haploid regulatory networks. Here we focus on bZIP TFs that have critical roles in plants, animals and other kingdoms. We report the functional characterization of Arabidopsis thaliana AtbZIP34 that is expressed in both gametophytic and surrounding sporophytic tissues during flower development. T-DNA insertion mutants in AtbZIP34 show pollen morphological defects that result in reduced pollen germination efficiency and slower pollen tube growth both in vitro and in vivo. Light and fluorescence microscopy revealed misshapen and misplaced nuclei with large lipid inclusions in the cytoplasm of atbzip34 pollen. Scanning and transmission electron microscopy revealed defects in exine shape and micropatterning and a reduced endomembrane system. Several lines of evidence, including the AtbZIP34 expression pattern and the phenotypic defects observed, suggest a complex role in male reproductive development that involves a sporophytic role in exine patterning, and a sporophytic and/or gametophytic mode of action of AtbZIP34 in several metabolic pathways, namely regulation of lipid metabolism and/or cellular transport.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- buněčná stěna metabolismus ultrastruktura MeSH
- fluorescenční mikroskopie MeSH
- geneticky modifikované rostliny MeSH
- květy genetika růst a vývoj metabolismus MeSH
- metabolické sítě a dráhy genetika fyziologie MeSH
- mikroskopie elektronová rastrovací MeSH
- mutace MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny huseníčku genetika fyziologie MeSH
- pyl genetika metabolismus ultrastruktura MeSH
- pylová láčka genetika růst a vývoj metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- stanovení celkové genové exprese MeSH
- testy genetické komplementace MeSH
- trans-aktivátory genetika fyziologie MeSH
- transmisní elektronová mikroskopie MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bZIP34 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- trans-aktivátory MeSH
