Environmental changes are known to elicit a range of responses in plants, including alterations in gene expression, metabolism, growth and development. One long-studied example is the process of acclimation to low temperature. Yet our understanding of the early steps in sensing a decrease in temperature and the associated signaling processes leading to alterations in the plant cell is incomplete. In recent years research into the roles of sphingolipids in plants has expanded considerably, and a growing number of reports have demonstrated the involvement of distinct sphingolipids, especially ceramides, long-chain bases (LCBs) and phosphorylated long-chain bases (LCBPs), in signal transduction and cell regulation. In this issue of New Phytologist, a study by Dutilleul et al. (pp. 181–191) provides evidence that the LCBP phytosphingosine phosphate (t18:0-P), previously implicated in abscisic acid (ABA) signaling leading to stomatal closure, plays a role in signaling in response to low temperature.
‘This rapidly manifested increase in t18:0-P following a temperature shift suggests it could be an early participant in a signaling pathway, but which of the many potential downstream components might be regulated by t18:0-P?’
As an environmental stress, temperature is somewhat unique in that it directly impacts every compartment/component in a plant cell; and because temperature influences the rate of physical and chemical reactions, essentially every vital process is altered. Since all cellular processes do not exhibit identical temperature coefficients (Q10), a response to a decrease (or increase) in temperature by the plant must occur that includes modulating a vast number of biochemical reactions and transport processes in order to prevent metabolic imbalances. A related process, cold acclimation, also involves changes in response to low (nonfreezing) temperatures that subsequently permit the plant to tolerate freezing temperatures and the associated osmotic and dehydration stresses associated with ice formation. Not surprisingly, numerous changes in gene expression, post-translational modification, and metabolite levels have been reported to accompany the response to low temperature (Ruelland et al., 2009). Induction of such a complex response to low temperature most likely involves multiple signaling networks that coordinate the response (Vergnolle et al., 2005; Ruelland et al., 2009). The actual ‘receptor’ involved in the perception of temperature and the early steps in signaling are not known, but it is thought the signal originates in the cell membrane, and the earliest signals identified appear to be phospholipid-derived molecules (Ruelland et al., 2002). Other components of temperature signaling in plants have yet to be identified, but recent work by Cantrel et al. (2011) uncovered a rapid phosphorylation of sphingolipids.
Complex sphingolipids are significant components of the plasma membrane, tonoplast and endomembrane system of plant cells. Sphingolipid biosynthetic intermediates and products of sphingolipid catabolism known to play signaling roles in animals and fungi are present in plant cells, and their physiological significance in plants is beginning to be understood (Chen et al., 2009). For example, Arabidopsis acd5 mutants deficient in ceramide kinase activity accumulate endogenous ceramide, are more susceptible to pathogen infection, and undergo a precocious apoptotic-like cell death later in development (Liang et al., 2003). The loss of ERH1 (coding for one of the three known inositolphosphorylceramide synthase genes) in Arabidopsis lines carrying the resistance gene RPW8 results in enhanced hypersensitive response (HR)-like cell death that is accompanied by an increase in the level of ceramide (Wang et al., 2008). An Arabidopsis mutant having a defective subunit of serine palmitoyltransferase (that catalyzes LCB synthesis) is resistant to the mycotoxin fumonisin B1 (FB1) and it was proposed that LCBs are involved in controlling programmed cell death (PCD; Shi et al., 2007). More recently, the accumulation of the LCB sphinganine (d18:0) was associated with PCD induced by FB1 (Saucedo-García et al., 2011). Of relevance to the report by Dutilleul et al., they provided evidence indicating that the mitogen-activated protein (MAP) kinase MPK6 was a downstream component of the sphingolipid-mediated PCD pathway.
To date, most evidence regarding LCBP function in plants involves signaling in response to ABA (Worrall et al., 2008). Increases in sphingosine phosphate (d18:1-P) content accompanied drought stress in Commelina communis (Ng et al., 2001). Stomatal closure occurred following incubation of leaf epidermal strips with exogenous d18:1-P, while incubation with an inhibitor of LCB kinase attenuated the stomatal response to added ABA. Subsequent studies in Arabidopsis showed that LCB kinase activity was transiently stimulated by ABA, and inhibition of kinase activity diminished the stomatal response to ABA treatment, as found for C. communis. Exogenous d18:1-P was capable of influencing guard cell behavior in wild-type (WT) protoplasts but not in protoplasts from mutants lacking the G-protein α-subunit, evidence that the G-protein is downstream of exogenously supplied d18:1-P in the ABA signaling pathway (Coursol et al., 2003). Sphingosine and d18:1-P are virtually absent in many plants, including Arabidopsis; however, t18:0-P can influence guard cell behavior in a similar manner to d18:1-P (Coursol et al., 2005). Indeed, Arabidopsis mutants incapable of producing d18:1 or d18:1-P responded to ABA in a manner indistinguishable from WT (Michaelson et al., 2009), supporting the contention that t18:0-P is the likely signal molecule in Arabidopsis.
Dutilleul et al. reveal another signaling role for t18:0-P, as a participant in the early response to a decrease in temperature in Arabidopsis. The transient formation of a phosphosphingolipid in response to a shift in temperature from 22 to 4°C previously reported by Cantrel et al. (2011) was confirmed, and its identity was established to be t18:0-P. Its formation in response to cold was rapid, being detectable within 1 min, and transient, peaking between 3 and 30 min. Formation of t18:0-P was partially inhibited by LCB kinase inhibitors. However, the use of these inhibitors does not allow the identification of a specific LCB kinase in manifesting this response. Arabidopsis has at least three LCB kinases, and any one (or more) could be involved. The investigators examined the ability of different LCB kinase mutants to synthesize t18:0-P following a temperature shift. Intriguingly, only mutants lacking one particular LCB kinase, LCBK2 (At2g46090), failed to exhibit t18:0-P synthesis in response to temperature; the other lcbk mutants did exhibit a temperature-induced increase in t18:0-P similar to WT. They also noted that the lcbk2 lines exhibited constitutively elevated t18:0-P synthesis at 22°C, possibly a consequence of a compensatory activity by the other LCB kinases.
This rapidly manifested increase in t18:0-P following a temperature shift suggests it could be an early participant in a signaling pathway, but which of the many potential downstream components might be regulated by t18:0-P? Dutilleul et al. decided to examine two members of the MAP kinase pathway, MPK4 and MPK6, known to be activated following a temperature shift. While both MAP kinases exhibited increased enzyme activity within 30 min of a temperature shift in WT plantlets, MPK6 activity was not stimulated following a temperature shift to 4°C in the lcbk2 mutants, suggesting that t18:0-P regulates (directly or indirectly) MPK6, but not MPK4. It was also found that the lcbk2 mutants exhibited higher constitutive MPK6 activity than WT at 22°C; however, recall that the lcbk2 mutants exhibited constitutively higher t18:0-P synthesis at 22°C, so, together, these results are consistent with the contention that t18:0-P influences MPK6 activity. To further test the role of t18:0-P in regulating MPK6, LCBs and LCBPs were supplied to cultured cells and subsequently monitored for changes in MPK6 activity. Consistent with the previous results, the activity of MPK6 was stimulated by exogenous t18:0-P, but not by d18:0-P or t18:0.
Further experiments to explore the consequences of impaired t18:0-P formation on cold-regulated genes were also reported, and these results support and expand potential signaling roles for t18:0-P. The expression of the cold-responsive gene NOI3 was impaired in lcbk2 mutants following a temperature shift. Differences in the expression of the DELLA gene RGL3 and HB12, a putative target of DELLA factors, were also observed between WT and lcbk2 mutants. Exogenous t18:0-P and t18:0 were shown to alter expression of RGL3 and HB12, but in reciprocal fashion. While further studies are needed to clarify the respective roles of free and phosphorylated t18:0 in altering the expression of these genes involved in repressing growth while facilitating survival, the results contribute to the growing body of evidence that t18:0-P affects multiple targets involved in responding to biotic and abiotic stresses and regulating growth and development.
Taken together, the report by Dutilleul et al. and that of Saucedo-García et al. (2011) point to MPK6 acting as a downstream transducer for both LCBP and LCB, responding to environmental stress in the presence of elevated t18:0-P but promoting PCD in the presence of elevated d18:0. While the results presented by Dutilleul et al. shed light on the various downstream targets of t18:0-P in signaling, one significant question surrounds the identity of the upstream signal stimulating LCBK2 enzyme activity in response to low temperature. At present, a leading candidate is phosphatidic acid, which has been shown to stimulate the activity of Arabidopsis LCB kinases (Guo et al., 2011) and to be generated rapidly (within 1–2 min) by the enzymatic cleavage of phospholipids in response to a temperature shift (Ruelland et al., 2002).
These studies indicate critical roles for specific sphingolipids in signaling pathways and suggest complex interrelationships between sphingolipids and components of pathways involved in the response to hormones, pathogens and environmental stresses.