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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: 3.94). 06/2009; 9:52. DOI: 10.1186/1471-2229-9-52
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ABSTRACT 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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    • "( see below ) and evidence has also been found for partic - ipation of the Arabidopsis serpins , AtSRP2 ( At2g14540 ) and AtSRP3 ( At1g64030 ) , in growth responses to plant exposure to the DNA - alkylating agent methyl methane - sulfonate ( Ahn et al . 2009 ) ."
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    ABSTRACT: Protease inhibitors of the serpin family are ubiquitous in the plant kingdom but relatively little is known about their biological functions in comparison with their counterparts in animals. X-ray crystal structures have provided crucial insights into animal serpin functions. The recently solved structure of AtSerpin1 from Arabidopsis thaliana, which has the highly conserved reactive center P2-P1' Leu-Arg-Xaa (Xaa = small residue), displays both conserved and plant-specific serpin features. Sequence homology suggests that AtSerpin1 belongs to serpin Clade B, composed of intracellular mammalian serpins, which is consistent with the lack of strong evidence for secretion of serpins from plant cells. The major in vivo target protease for AtSerpin1 is the papain-like cysteine RD21 protease, a match reminiscent of the inhibition of cathepsins K, L and S by the Clade-B mammalian serpin, SCCA-1 (SERPINB3). The function of AtSerpin1 and other serpins that contain P2-P1' Leu-Arg-Xaa (the 'LR' serpins) in plants remains unknown. However, based on its homology and interactive partners, AtSerpin1 and perhaps other serpins are likely to be involved in regulating programmed cell death or associated processes such as senescence. Abundant accumulation of serpins in seeds and their presence in phloem sap suggest additional functions in plant defense by irreversible inhibition of digestive proteases from pests or pathogens. Here we review the most recent findings in plant serpin biology, focusing on advances in describing the structure and inhibitory specificity of the LR serpins.
    Physiologia Plantarum 11/2011; 145(1):95-102. DOI:10.1111/j.1399-3054.2011.01540.x
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    • "Interestingly , a recent paper postulated that AtSerpin-1 is unlikely to interact with AtMCP2f to yield a stable complex in vivo: instead this protease inhibitor mainly interacts with RD21, a cysteine protease localized in the apoplast (Lampl et al., 2010). Eleven serpin genes are annotated in the Arabidopsis genome, and two serpin genes [AtSRP2 (At2g14540) and AtSRP3 (At1g64030)] were recently shown to be required for normal growth sensitivity to DNA damaging stress (Ahn et al., 2009). Analysis of GFP fusions with SRP2 or SRP3 suggests that AtSRP2 and AtSRP3 localize to the nucleus and cytosol, respectively. "
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    ABSTRACT: Cysteine proteases such as caspases play important roles in programmed cell death (PCD) of metazoans. Plant metacaspases (MCPs), a family of cysteine proteases structurally related to caspases, have been hypothesized to be ancestors of metazoan caspases, despite their different substrate specificity. Arabidopsis thaliana contains six type II MCP genes (AtMCP2a-f). Whether and how these individual members are involved in controlling PCD in plants remains largely unknown. Here we investigated the function and regulation of AtMCP2d, the predominant and constitutively expressed member of type II MCPs, in stress-inducible PCD. Two AtMCP2d mutants (mcp2d-1 and mcp2d-3) exhibited reduced sensitivity to PCD-inducing mycotoxin fumonisin B1 as well as oxidative stress inducers, whereas AtMCP2d over-expressors were more sensitive to these agents, and exhibited accelerated cell-death progression. We found that AtMCP2d exclusively localizes to the cytosol, and its accumulation and self-processing patterns were age-dependent in leaves. Importantly, active proteolytic processing of AtMCP2d proteins dependent on its catalytic activity was observed in mature leaves during mycotoxin-induced cell death. We also found that mcp2d-1 leaves exhibited reduced cell death in response to Pseudomonas syringae carrying avirulent gene avrRpt2, and that self-processing of AtMCP2d was also detected in wild-type leaves in response to this pathogen. Furthermore, increases in processed AtMCP2d proteins were found to correlate with conditional cell-death induction in two lesion-mimic mutants (cpr22 and ssi4) that exhibit spontaneous cell-death phenotypes. Taken together, our data strongly suggest that AtMCP2d plays a positive regulatory role in biotic and abiotic stress-induced PCD.
    The Plant Journal 03/2011; 66(6):969-82. DOI:10.1111/j.1365-313X.2011.04554.x
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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.
    Methods in enzymology 01/2011; 499:347-66. DOI:10.1016/B978-0-12-386471-0.00017-1
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