However although LCBs are known to induce PCD

However, although LCBs are known to induce PCD in plant cells, the signaling pathway leading to this PCD and its actors remains largely uncharacterized. It has been shown that the mitogen-activated protein kinase 6 (MPK6) is rapidly activated when Arabidopsis seedlings are exposed to FB1 or LCBs, such as PHS or DHS [7]. Moreover, using tobacco BY-2 cells, we showed that DHS is able to trigger PCD in a calcium (Ca2+)-dependent manner mainly through nuclear Ca2+ increases [10]. More recently, we highlighted a new sphingolipid-induced Ca2+-dependent mechanism involving the protein kinase CPK3 and 14-3-3 proteins in the context of LCB-induced cell death in Arabidopsis [11]. As plant proteases, and more particularly Papain Like Cysteine Proteases (PLCPs) are key players in plant immunity [12,13] we investigated their role in LCB-mediated PCD. PLCPs belong to MEROPS protease family C1 A of clan CA, and are characterized by the occurrence in their structure of the papain-like fold that displays two lobes delineating a substrate-binding groove which contains the catalytic site Cys-His-Asn [14,15]. In plants, this specific class of protease has been sub-divided into 9 subfamilies according to phylogenetic analysis and conserved structural features. The number of PLCPs is between 20–40 MRT67307 HCl per plant genome, with 31 genes found in Arabidopsis [15]. PLCPs are involved at various stage of plant development and two of them RD21 A and AALP are the main active in senescing leaves [16]. More interestingly, they also participate in plant immunity and thus rd21 null mutants are more susceptible to the necrotrophic fungal pathogen Botrytis cinerea whereas they do not display a particular phenotype in response to P. syringae or the oomycete pathogen Hyaloperonospora arabidopsidis [17]. In the present study, we combined activity-based protein profiling, biochemical and genetic approaches to identify the PLCPs involved in the sphingolipid pathway leading to plant PCD. By using Arabidopsis cells and leaves, we demonstrate that this sub-class of plant proteases is activated in response to PHS or FB1, respectively and we identify RD21 (responsive-to-desiccation-21) as one of the main PLCPs activated in response to sphingolipid treatment. Using a reverse genetic approach with two null mutants of RD21,we show that this protease acts as a negative regulator of FB1-induced cell death in Arabidopsis.
Material and methods
Results
Discussion In plants, programmed cell death plays a fundamental role in development and defense responses. As an example, during necrotrophic pathogen-plant interactions, host death is induced enabling pathogen development [28]. Interestingly, the necrotrophic fungus Fusarium moniliforme interferes with plant sphingolipid metabolism by secreting a mycotoxin called fumonisin B1 (FB1). This molecule, which is a structural analog of LCBs, inhibits the ceramide synthase and induces a concomitant increase in free intracellular levels of PHS and DHS, metabolites known to elicit a subsequent PCD in plants [6,7]. However, the LCB-mediated signaling pathway leading to PCD remains largely unknown so far. Nevertheless, previous studies have shown that different second messengers including intracellular Ca2+ ions and reactive oxygen species (ROS) or proteins such as MPK6, 14-3-3 s and the calcium-dependent protein kinase CPK3 are involved in this process [7,10,11]. In normal conditions, dimeric 14-3-3 proteins interact with CPK3 [11]. In response to PHS, a cytosolic Ca2+ transient is induced and leads to CPK3 activation. Ca2+-activated CPK3 phosphorylates 14-3-3 s on a specific serine residue, which is located at the 14-3-3 dimer interface [11]. Recent studies have shown that this phosphorylation event leads to plant 14-3-3 monomerization [29,30], that could disrupt the 14-3-3/CPK3 complex, leading to the release of CPK3 [11].