Dataset Information

Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes.

ABSTRACT: Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2. Here, we show that fluorescence-tagged tobacco NtPLC3 displays a subapical plasma membrane distribution which frames that of fluorescence-tagged PI4P 5-kinases, suggesting that NtPLC3 may modulate PtdIns(4,5)P2-mediated processes in pollen tubes. The expression of a dominant negative NtPLC3 variant resulted in pollen tube tip swelling, consistent with a delimiting effect on PtdIns(4,5)P2 production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P2 function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P2 antagonizes the effects of PtdIns(4,5)P2 overproduction in vivo. When analyzed by spinning disc microscopy, fluorescence-tagged NtPLC3 displayed discontinuous membrane distribution omitting punctate areas of the membrane, suggesting that NtPLC3 is involved in the spatial restriction of plasma membrane domains also at the nanodomain scale. Together, the data indicate that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P2 in the subapical plasma membrane of pollen tubes.

SUBMITTER: Stenzel I

PROVIDER: S-EPMC7238183 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes.

Stenzel Irene I Ischebeck Till T Vu-Becker Linh Hai LH Riechmann Mara M Krishnamoorthy Praveen P Fratini Marta M Heilmann Ingo I

Plants (Basel, Switzerland) 20200403 4

Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2. Here, we show that fluorescence-tagged tobacc ...[more]

PMID: 32260253

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Project description:BackgroundPollen 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).ResultsDe 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.ConclusionsA 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.

Project description:BACKGROUND: Arabinogalactan proteins (AGPs) are cell wall proteoglycans that have been shown to be important for pollen development. An Arabidopsis double null mutant for two pollen-specific AGPs (agp6 agp11) showed reduced pollen tube growth and compromised response to germination cues in vivo. A microarray experiment was performed on agp6 agp11 pollen tubes to search for genetic interactions in the context of pollen tube growth. A yeast two-hybrid experiment for AGP6 and AGP11 was also designed. RESULTS: The lack of two specific AGPs induced a meaningful shift in the gene expression profile. In fact, a high number of genes showed altered expression levels, strengthening the case that AGP6 and AGP11 are involved in complex phenomena. The expression levels of calcium- and signaling-related genes were found to be altered, supporting the known roles of the respective proteins in pollen tube growth. Although the precise nature of the proposed interactions needs further investigation, the putative involvement of AGPs in signaling cascades through calmodulin and protein degradation via ubiquitin was indicated. The expression of stress-, as well as signaling- related, genes was also changed; a correlation that may result from the recognized similarities between signaling pathways in both defense and pollen tube growth.The results of yeast two-hybrid experiments lent further support to these signaling pathways and revealed putative AGP6 and AGP11 interactors implicated in recycling of cell membrane components via endocytosis, through clathrin-mediated endosomes and multivesicular bodies. CONCLUSIONS: The data presented suggest the involvement of AGP6 and AGP11 in multiple signaling pathways, in particular those involved in developmental processes such as endocytosis-mediated plasma membrane remodeling during Arabidopsis pollen development. This highlights the importance of endosomal trafficking pathways which are rapidly emerging as fundamental regulators of the wall physiology.

Dataset Information

Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes.

Publications

Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes.

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