Sphingolipid signaling in plants

While relatively few reports have focused on bioactive sphingolipids in plants, several studies have implicated sphingolipids in signaling and regulation. Free LCBs apparently have diverse effects on plants, although in many of the published studies the effects cannot be unambiguously attributed to

LCBs since the levels of related molecules, including LCBP and Cer, were not monitored. Studies of plant-pathogen interactions have implicated disruption of sphingolipid synthesis in programmed cell death (PCD) as part of the hypersensitive response associated with plant defense. The fungal toxins fumonisin B1 and AAL-toxin are LCB analogs that inhibit sphinganine acyltransferase activity, resulting in an accumulation in plant tissues of sph-inganine and 4-hydroxysphinganine (Abbas etal., 1994; Wright etal.,2003) and their phosphorylated intermediates (Lynch et al., unpublished), and promote plant necrosis (Tanaka et al., 1993) and PCD in tissues and protoplasts (Wang et al., 1996; Asai et al., 2000). The response to these toxins appears complex, involving the participation of the ethylene, jasmonate, and salicylate signaling pathways (Asai et al., 2000), but microarray results demonstrated that AAL toxin does not significantly alter the expression of any of the included sphingolipid genes (Gechev et al, 2004). The tomato Asc1 gene, involved in ce-ramide synthesis and conferring AAL-toxin resistance, permits sphingolipid synthesis and prevents long-chain base accumulation in tissues exposed to toxin (Brandwagt et al., 2000, 2002; Spassieva et al., 2002). In sensitive tomato plants, AAL-toxin prevents ceramide (and complex sphingolipid) synthesis, resulting in the accumulation of LCB and death. But if long-chain base synthesis (serine palmitoyltransferase) is simultaneously inhibited, LCB accumulation is prevented and the toxic effects of AAL-toxin are at least partially blocked (Spassieva et al., 2002). As well, overexpression of Asc1 in sensitive plants confers resistance to infection (Brandwagt et al., 2002). These results suggest that LCB may play important regulatory/signaling roles in plant defense, and elevated levels in plant tissues promote cell/tissue death. It must be reiterated, however, that in the above studies LCBP levels were not determined, thus the pathologies cannot be attributed unequivocally to elevated LCBs per se. Indeed, evidence from mutant lines of A. thaliana suggests that the fumonisin-induced accumulation of LCBPs (possibly, specific species of LCBP) is associated with death. That is, mutant lines lacking the sole LCBP lyase exhibit hypersensitivity to fumonisin B1 and a greater accumulation of LCBP in comparison to treated wild type tissues, while mutant lines lacking any one of the three identified LCB kinases are resistant to the toxin (Lynch et al., unpublished).

Additional evidence implicating LCBs in signaling and regulation was provided by a knockout in A. thaliana of the ACD11 gene that results in increased PCD and defense (Brodersen et al., 2002). It was demonstrated that the gene product encodes a putative sphingosine LCB transfer protein. Further studies are needed to better define the specific function of this protein and its sphingolipid LCB specificity in order to understand its role in programmed cell death and defense pathways. Nevertheless, if actual disruption of LCB transfer is responsible for the mutant phenotype, it suggests that the cellular localization of specific sphingolipids may be significant in promoting cell death and other sphingolipid-influenced processes.

A role for Cer in mediating the effects of AAL-toxin exposure was suggested by the partial rescue of AAL-toxin-treated tomato leaves by exogenous ceramide application (Brandwagt et al., 2000). While this does not seem consistent with the proposed role for LCBs (or LCBPs) described above, it was suggested that the relative levels (or ratio) of LCB and Cer may constitute a switch, triggering PCD (Spassieva et al., 2002). Such a "rheostat model" has been proposed to operate in animal cells for LCBP and Cer (Spiegel and Milstien, 2003; Taha et al, 2006). While this merits testing in plants as well, it should include an assessment of the contribution of LCBPs to the rheostat. As well, testing of the rheostat hypothesis should also take into account the levels of specific species of LCB, LCBP, and Cer, in light of recent studies indicating that accumulation of C24 ceramide species induces cell cycle arrest in MCF-7 breast cancer cells while C16 ceramide accumulation is associated with apoptosis (Marchesini et al., 2004).

As with mammalian systems, Cer has been implicated in mediating cell death in plants (Liang et al., 2003; Townley et al., 2005). A. thaliana acd5 mutants deficient in Cer kinase activity are more sensitive to added Cer, accumulate endogenous Cer (kinase substrate), are more susceptible to pathogen infection, and undergo apoptotic-like cell death late in development. These results point to a role for Cer in promoting PCD in plants and demonstrate a role for the ACD5 encoded Cer kinase activity in modulating Cer levels in the plant cell. However, it is unclear whether the sole role of Cer kinase is to convert and sequester Cer, or whether Cer-1-phosphate also functions in signaling/cell regulation as demonstrated in mammalian cells where it activates phospholipase A2 (Pettus et al., 2004). As well, open questions pertaining to the fate of Cer-1-phosphate remain: Is there a specific phosphatase capable of dephosphorylating Cer-1-phosphate as demonstrated in brain (Shinghal et al., 1993), or is Cer-1-phosphate used in some as yet uncharacterized pathway? Consistent with the above results, Townley and colleagues demonstrated that exogenous short-chain Cer induces PCD in A. thaliana suspension cultures (Townley et al., 2005). Treatment with Cer was accompanied by the generation of a calcium transient and an increase in ROS. Inhibition of the calcium transient was found to prevent cell death, whereas inhibition of ROS had no effect on cell survival. These observations suggest that calcium signaling, but not generation of ROS, is involved in ceramide-induced PCD.

A role for S1P in guard cell signaling and stomatal closure has been investigated (Ng et al, 2001; Ng and Hetherington, 2001; Coursol et al, 2003). S1P was identified in lipid extracts from C. communis and increases in S1P content accompanied drought stress (Ng et al., 2001). Stomatal closure occurred following incubation of leaf epidermal strips with exogenous S1P whereas incubation with sphinganine-1-phosphate did not have such an effect, suggesting the significance of the A4 double bond in signaling, although phytosphingosine-1-phosphate can also function in signaling stomatal closure (Coursol et al., 2005). It was found that S1P influences calcium mobilization in guard cells and incubation with an inhibitor of sphingosine LCB kinase attenuates the stomatal response to added abscisic acid (ABA) (Ng et al., 2001; Ng and Het-herington, 2001).

This role for S1P in the ABA signaling pathway leading to reduction of guard cell turgor was further investigated in A. thaliana (Coursol et al., 2003), where it was found that LCB kinase activity is transiently stimulated by ABA, and inhibition of kinase activity (using inhibitors of the mammalian kinase) diminishes the stomatal response to ABA treatment, as found for C. communis. Exogenous S1P is capable of influencing guard cell behavior (both inhibition of stomatal opening and promotion of closure) via inhibition of K+ influx and stimulation of anion efflux in wild type plant protoplasts but not in protoplasts from knockout plants lacking the heterotrimeric G-protein a-subunit, providing evidence that the G-protein is downstream of S1P in the ABA signaling pathway (Coursol et al., 2003) (see Chapter 2 on heterotrimeric G proteins).

Sphingosine and S1P are virtually absent in plants, so while exogenous S1P may act as a signal molecule, it is doubtful that it is the endogenous signal. However, phytosphingosine-1-phosphate can influence guard cell behavior similar to S1P (Coursol et al, 2005). Since phytosphingosine is relatively abundant in plant tissues (it and sphinganine are the prevalent free LCBs) and it serves as a substrate for the three plant LCB kinases (Coursol et al., 2005; Imai and Nishiura, 2005; Tsegaye et al., unpublished), it is likely that phytosphingosine-1-phosphate, rather than S1P, is the LCBP species involved in a guard cell signaling pathway.

Cumulatively, the above studies support the contention that LCBPs can influence stomatal behavior, but the nature of the interaction between LCBP and the G-protein as well as the identification of the components of the pathway leading from ABA to changes in stomatal aperture need to be investigated further, especially given the recent identification of the ABA receptor as a G-protein-linked receptor (Liu et al, 2007). For example, the location of the putative LCBP receptor merits investigation: that exogenous LCBPs can elicit stomatal closure suggests that LCBP must be either transported into the cell to reach an intracellular target, or act through a receptor/binding protein on the cell surface. If a surface receptor for LCBP exists, then internally generated LCBP (in response to ABA) would need to be exported. In mammals, S1P has been reported to act as both a ligand for certain G-protein-coupled receptors at the plasma membrane and as an intracellular second messenger (Payne et al., 2002; Spiegel and Milstien, 2003). The identification of a plant LCBP receptor, including its location and specificity (and affinity) for LCBP, would improve our understanding of this signaling pathway and its contribution to stomatal behavior in plants.

Although S1P is thought to stimulate proliferation in mammalian cells (Spiegel and Milstien, 2003), and a role for LCBP in "re-entering" the cell cycle following heat stress-induced arrest in yeast has been suggested (Jenkins and Hannun, 2001), such a role for LCBP in plants has not been demonstrated. While the responses to fumonisin described above suggest that LCBP hyperaccumulation is lethal, a basic understanding of the effects of physiological concentrations of LCBP is lacking. The report of G protein involvement in cell proliferation in A. thaliana (Ullah et al., 2001) and the evidence from guard cell studies (above) suggesting that the G-protein a-subunit is downstream of LCBP in signaling stomatal closure leads to the suggestion that LCBPs may influence plant cell proliferation (Chalfant and Spiegel, 2005).

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