B<sub>1</sub>-Phytoprostanes Trigger Plant Defense and Detoxification Responses<sup><a href="#fn1" rid="fn1" class=" fn">1</a>,</sup><sup><a href="#fn2" rid="fn2" class=" fn">[w]</a></sup>
Abstract
Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B1-phytoprostanes (PPB1) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB1-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB1 regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB1 considerably prevented cell death caused by severe CuSO4 poisoning. Several Arabidopsis genes induced by PPB1, such as those coding for adenylylsulfate reductase, tryptophan synthase β-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB1 treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB1. In three different plant species, PPB1-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB1-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB1 represent stress signals that improve plants capacity to cope better with a variety of stresses.
Phytoprostanes belong to a novel family of plant effectors that are formed nonenzymatically by a free radical catalyzed biochemical mechanism from α-linolenic acid. Via an identical nonenzymatic mechanism, isoprostanes (isomers of prostaglandins) are formed in animals from arachidonic acid. Nomenclature used to name different phytoprostane classes conforms with the general isoprostane/prostaglandin terminology (Thoma et al., 2004). Due to the nonenzymatic formation of phytoprostanes, two racemic regioisomers (type I and II) of each class can be generated. One pathway leads to the formation of B1-phytoprostanes (PPB1; Fig. 1) that are chemically stable end products of lipid peroxidation. In animals, isoprostanes are not only extremely reliable markers of oxidative stress but also display potent (prostaglandin-) receptor-mediated biological activities in the nanomolar concentration range (Cracowski et al., 2002). Therefore, it is postulated that isoprostanes represent mediators of oxidant injury in animals.
PPB1. Free radical catalyzed oxidation of α-linolenate yields two racemic PPB1 regioisomers (type I and type II).
In plants, several classes of phytoprostanes are constitutively present, and, notably, their levels increase in a variety of conditions associated with enhanced free radical generation (Thoma et al., 2003). Previously, it has been shown that PPB1 formation, together with the synthesis of a series of other phytoprostane classes, can be triggered in vivo by fungal pathogens (Botrytis cinerea) or peroxides (Thoma et al., 2003). Interestingly, endogenous accumulation of PPB1 or F1-phytoprostanes after peroxide, heavy metal challenge, or wounding is a transient process, suggesting that phytoprostanes, similar to isoprostanes in animals, can be rapidly generated and metabolized in vivo (Imbusch and Mueller, 2000a; Thoma et al., 2003). In this study, we show that not only endogenous but also exogenously administered PPB1 are rapidly metabolized by tobacco (Nicotiana tabacum) cell cultures, suggesting that PPB1 are continuously synthesized de novo as a consequence of free radical formation during aerobic metabolism. So far, little is known about the spectrum of biological activities displayed by phytoprostanes. Previously, it has been shown that cyclopentenone phytoprostanes with the deoxy-J1-prostaglandin, A1-prostaglandin (PPA1), and PPB1 ring system activate mitogen-activated protein kinase activity in tomato (Lycopersicon esculentum) cell suspension cultures. In addition, in tomato cell cultures, a gene involved in primary metabolism, extracellular invertase, was induced by PPB1 but not by PPA1 (Thoma et al., 2003). There is also evidence that several classes of phytoprostanes, including PPB1, trigger secondary metabolism in taxonomically distant plant species (Thoma et al., 2003, 2004; Iqbal et al., 2005). PPB1 are structurally related to jasmonates and in fact, biological effects on invertase and secondary metabolism appear to be qualitatively similar to jasmonic acid (JA) or methyl jasmonate (MeJA). However, it has also been shown that biological activities of JA/MeJA and PPB1 differ in various bioassay systems (Thoma et al., 2003).
It is to be expected that even different isomers of one class of phytoprostanes exhibit a different qualitative and quantitative profile of biological activities similar to isoprostane isomers in mammals (Cracowski et al., 2002; Cracowski, 2003, 2004; Janssen, 2004). For studying the biological properties of isoprostanes, chemically pure isoprostane isomers are required that usually can be provided only by total synthesis. Recently, synthetic strategies have been developed for deoxy-J1-, E1-, and F1-phytoprostanes. Due to the large number of possible isomers (32 of each class), only a few isomers are currently available (Rodriguez and Spur, 2003; El Fangour et al., 2004; Iqbal et al., 2005) and under investigation. However, selected isomers may not be representative for any one of the classes.
Although PPB1 are not the most abundant phytoprostanes in vivo, the possibility to isolate large quantities of all theoretically possible PPB1 isomers from linolenate autoxidation mixtures (as demonstrated in this work) allows us to study the biological properties of one class of phytoprostanes. We have prepared methyl esters of all four isomers of PPB1 and compared their effect on secondary metabolism with MeJA in three different plant species (Eschscholzia californica Cham., Crotalaria cobalticola Duvign. & Plancke, tobacco cv Xanthi L.). Moreover, we also probed the spectrum of biological activity of PPB1 using an Arabidopsis (Arabidopsis thaliana) L. Heyn., ecotype Columbia (Col-0) DNA array approach that indicates that PPB1 might induce enzymes that protect cells from the consequences of oxidative stress. To this end, cell death of PPB1 primed tobacco cells in response to severe heavy metal stress was investigated.
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We thank Barbara Dierich and Beate Hilbert for excellent technical assistance.
Notes
This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (grant no. SFB 567 to S.B., G.B., and M.J.M.) and by the Fonds der Chemischen Industrie.
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