Serpin genes AtSRP2 and AtSRP3 are required for normal growth sensitivity to a DNA alkylating agent in Arabidopsis

Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia.
BMC Plant Biology (Impact Factor: 3.81). 06/2009; 9(1):52. DOI: 10.1186/1471-2229-9-52
Source: PubMed


The complex responses of plants to DNA damage are incompletely understood and the role of members of the serpin protein family has not been investigated. Serpins are functionally diverse but structurally conserved proteins found in all three domains of life. In animals, most serpins have regulatory functions through potent, irreversible inhibition of specific serine or cysteine proteinases via a unique suicide-substrate mechanism. Plant serpins are also potent proteinase inhibitors, but their physiological roles are largely unknown.
Six Arabidopsis genes encoding full-length serpins were differentially expressed in developing seedlings and mature tissues. Basal levels of AtSRP2 (At2g14540) and AtSRP3 (At1g64030) transcripts were highest in reproductive tissues. AtSRP2 was induced 5-fold and AtSRP3 100-fold after exposure of seedlings to low concentrations of methyl methanesulfonate (MMS), a model alkylating reagent that causes DNA damage. Homozygous T-DNA insertion mutants atsrp2 and atsrp3 exhibited no differential growth when mutant and wild-type plants were left untreated or exposed to gamma-radiation or ultraviolet light. In contrast, atsrp2 and atsrp3 plants exhibited greater root length, leaf number and overall size than wild-type plants when exposed to MMS. Neither of the two serpins was required for meiosis. GFP-AtSRP2 was localized to the nucleus, whereas GFP-AtSRP3 was cytosolic, suggesting that they target different proteinases. Induction of cell cycle- and DNA damage-related genes AtBRCA1, AtBARD1, AtRAD51, AtCYCB1;1 and AtCYCD1;1, but not AtATM, was reduced relative to wild-type in atsrp2 and atsrp3 mutants exposed to MMS.
Expression of specific serpin genes (AtSRP2 and AtSRP3 in Arabidopsis) is required for normal responses of plants following exposure to alkylating genotoxins such as MMS.

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    • "However, functions of merely a few serpins are known. Two Arabidopsis serpins, AtSRP2 and AtSRP3 play a significant role in maintaining normal growth in the presence of DNA alkylating agents (Ahn et al. 2009). AtSerpin1 is the best-characterized plant serpin till date. "
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    ABSTRACT: Serine protease inhibitors (serpins) and their cognate proteases control diverse physiological processes, including program cell death and immune response, in insects and mammals. Arabidopsis AtSerpin1 inhibits pro-cell-death proteases and protects cells from excessive cell death induced by pathogens. The rice genome contains at least 14 serpin coding genes, but a biological role is not known for any of them. We show here that expression of rice serpin1 (OsSRP-LRS; the closest homolog of AtSerpin1 in rice) is induced by necrotrophic fungal pathogen Rhizoctonia solani inoculation, UV and salt treatment. The transgenic RNAi lines having reduced expression of OsSRP-LRS are morphologically unaffected under the stress-free condition, but show exaggerated cell death upon pathogen, UV or salt treatment. Our results suggest that OsSRP-LRS negatively regulates stress-induced cell death in rice.
    Journal of Plant Biology 10/2015; 58(5):327-332. DOI:10.1007/s12374-015-0283-6 · 1.21 Impact Factor
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    • "More recently, the major in vivo target proteinase for AtSerpin1 was identified as the papain-like cysteine proteinase RESPONSIVE TO DESICCATION-21 (RD21) [7], an enzyme that also has transpeptidase activity [38]. Two other Arabidopsis serpins, AtSRP2 (ArathZ2; At2g14540) and AtSRP3 (ArathZ1; At1g64030), are associated with plant responses to alkylating DNA damage [39]. Serpins found at high concentrations in seeds (up to 4% total protein in wheat grain [34]) are assumed to provide direct defence against exogenous proteinases from insects and other organisms that attack the endosperm and other seed tissues [21,22,34]. "
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    ABSTRACT: Most members of the serpin family of proteins are potent, irreversible inhibitors of specific serine or cysteine proteinases. Inhibitory serpins are distinguished from members of other families of proteinase inhibitors by their metastable structure and unique suicide-substrate mechanism. Animal serpins exert control over a remarkable diversity of physiological processes including blood coagulation, fibrinolysis, innate immunity and aspects of development. Relatively little is known about the complement of serpin genes in plant genomes and the biological functions of plant serpins. A structurally refined amino-acid sequence alignment of the 14 full-length serpins encoded in the genome of the japonica rice Oryza sativa cv. Nipponbare (a monocot) showed a diversity of reactive-centre sequences (which largely determine inhibitory specificity) and a low degree of identity with those of serpins in Arabidopsis (a eudicot). A new convenient and functionally informative nomenclature for plant serpins in which the reactive-centre sequence is incorporated into the serpin name was developed and applied to the rice serpins. A phylogenetic analysis of the rice serpins provided evidence for two main clades and a number of relatively recent gene duplications. Transcriptional analysis showed vastly different levels of basal expression among eight selected rice serpin genes in callus tissue, during seedling development, among vegetative tissues of mature plants and throughout seed development. The gene OsSRP-LRS (Os03g41419), encoding a putative orthologue of Arabidopsis AtSerpin1 (At1g47710), was expressed ubiquitously and at high levels. The second most highly expressed serpin gene was OsSRP-PLP (Os11g11500), encoding a non-inhibitory serpin with a surprisingly well-conserved reactive-centre loop (RCL) sequence among putative orthologues in other grass species. The diversity of reactive-centre sequences among the putatively inhibitory serpins of rice point to a range of target proteases with different proteolytic specificities. Large differences in basal expression levels of the eight selected rice serpin genes during development further suggest a range of functions in regulation and in plant defence for the corresponding proteins.
    BMC Genomics 09/2012; 13(1):449. DOI:10.1186/1471-2164-13-449 · 3.99 Impact Factor
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    • "Protoplast preparation was from 2-week-old Arabidopsis seedlings. A reduced version of this figure was published previously (Ahn et al., 2009). "
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    ABSTRACT: Serpins appear to be ubiquitous in the Plant Kingdom and have several unique properties when compared to the substantial number of other families of protease inhibitors in plants. Serpins in plants are likely to have functions distinct from those of animal serpins, partly because plants and animals developed multicellularity independently and partly because most animal serpins are involved in animal-specific processes, such as blood coagulation and the activation of complement. To encourage and facilitate the discovery of plant serpin functions, here we provide a set of protocols for detection of serpins in plant extracts, localization of serpins in plant tissues and cells, purification of serpins from a range of organs from monocot and eudicot plants, production and purification of recombinant plant serpins, and analysis of plant-protease interactions including identification of in vivo target proteases.
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