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NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development.

ABSTRACT: Proteinases play a critical role in developmental homeostasis and in response to environ-mental stimuli. Our present research reports that a new cysteine protease, NtCP56, is involved in the development of pollen grains in Nicotiana tabacum L. The NtCP56 gene, which encodes a protein of 361 amino acid residues with a calculated molecular mass of 40 kDa, is strongly expressed in anthers. The recombinant NtCP56 showed a high activity towards casein. Kinetic analysis revealed a K(m) of 2.20 mg ml(-1) and V(max) of 11.07 microg ml(-1) min(-1). The recombinant NtCP56 retained more than 50% of its maximum enzymatic activity from 20 degrees C to 60 degrees C with an optimum Tm range of 30-50 degrees C. The enzyme had a maximum activity at approximately pH 6.5. Suppression of the NtCP56 gene in anti-sense transgenic tobaccos resulted in the sterility of pollen grains. Our data indicated that, as a cysteine protease, NtCP56 might play an important role in pollen development.

SUBMITTER: Zhang XM

PROVIDER: S-EPMC2671612 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development.

Zhang Xiao-mei XM Wang Ying Y Lv Xiao-meng XM Li Hui H Sun Peng P Lu Hai H Li Feng-lan FL

Journal of experimental botany 20090226 6

Proteinases play a critical role in developmental homeostasis and in response to environ-mental stimuli. Our present research reports that a new cysteine protease, NtCP56, is involved in the development of pollen grains in Nicotiana tabacum L. The NtCP56 gene, which encodes a protein of 361 amino acid residues with a calculated molecular mass of 40 kDa, is strongly expressed in anthers. The recombinant NtCP56 showed a high activity towards casein. Kinetic analysis revealed a K(m) of 2.20 mg ml(- ...[more]

PMID: 19246592

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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:Pollen, an extremely reduced bicellular or tricellular male reproductive structure of flowering plants, serves as a model for numerous studies covering wide range of developmental and physiological processes. The pollen development represents a fragile and vital phase of plant ontogenesis and pollen was among the first singular plant tissues thoroughly characterized at the transcriptomic level (Honys and Twell [5]). Arabidopsis pollen developmental transcriptome has been published over a decade ago (Honys and Twell, 2004) and transcriptomes of developing pollen of other species have followed (Rice, Deveshwar et al. [2]; Triticeae, Tran et al. [11]; upland cotton, Ma et al. [8]). However, the transcriptomic data describing the development of tobacco pollen, a bicellular model for cell biology studies, have been missing. Here we provide the transcriptomic data covering three stages (Tupý et al., 1983) of wild type tobacco (Nicotiana tabacum, cv. Samsun) pollen development: uninucleate microspores (UNM, stage 1), early bicellular pollen (eBCP, stage 3) and late bicellular pollen (lBCP, stage 5) as a supplement to the mature pollen (MP), 4 h-pollen tube (PT4), 24 h-pollen tubes (PT24), leaf (LF) and root (RT) transcriptomic data presented in our previous studies (Hafidh et al., 2012a; Hafidh et al., 2012b). We characterized these transcriptomes to refine the knowledge base of male gametophyte-enriched genes as well as genes expressed preferentially at the individual stages of pollen development. Alongside updating the list of tissue-specific genes, we have investigated differentially expressed genes with respect to early expressed genes. Pollen tube growth and competition of pollen tubes in female pistil can be viewed as a race of the fittest. Accordingly, there is an apparent evolutionary trend among higher plants to store significant material reserves and nutrients during pollen maturation. This supply ensures that after pollen germination, the pollen tube utilizes its resource predominantly for its rapid elongation in the female pistil. Previous transcriptomic data from Arabidopsis showed massive expression of genes encoding proteins forming both ribosomal subunits that were accumulated in developing pollen, whereas their expression was not detectable in growing pollen tubes (Honys and Twell, 2004). We observed a similar phenomenon in less advanced bicellular tobacco pollen. Here, we describe in detail how we obtained and analyzed validated microarray dataset deposited in Gene Expression Omnibus (GSE62349).

Project description:In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil.

Dataset Information

NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development.

Publications

NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development.

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