The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures

Abstract

The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7-14C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O-beta-D: -glucose (SAG), methylsalicylate 2-O-beta-D: -glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [14C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A1 (a specific inhibitor of the vacuolar H(+)-ATPase) and dissipation of the transtonoplast H(+)-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several beta-glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis-Menten type saturation kinetics with a K(m) and V(max) value of 11 microM and 205 pmol min-1 mg-1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H(+)-antiport-type mechanism.

Proton-Dependent Coniferin Transport, a Common Major Transport Event in Differentiating Xylem Tissue of Woody Plants

“…This result supports that coniferin transport occurs not only in vitro but also in vivo, although this could be reflected by the pH dependency of V-ATPase activity. The K m values of coniferin transport are similar to those of the characterized secondary transporters of various secondary metabolites (Supplemental Table S1; Klein et al, 1996;Frangne et al, 2002;Dean et al, 2005;Marinova et al, 2007). It is noteworthy that the ATPdependent transport activity of aglycone forms of monolignols, such as coniferyl alcohol and sinapyl alcohol, is considerably lower (less than 4% of coniferin transport; Fig.…”

Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance inArabidopsis

“…Analyses of bsmt1 mutants cannot definitively prove this statement because MeSA depletion in these plants seems to negatively affect SA biosynthesis at the transcriptional level ( Figure 7F). In addition to MeSA volatilization, SAG formation and subsequent vacuolar storage is an alternative way to handle an excess of SA (Lee et al, 1995;Dean et al, 2005). MeSA formation might also influence the interplay between SA and JA, which trigger distinct sets of defense responses and thereby often behave in a counteractive manner (Traw et al, 2003;Koornneef et al, 2008).…”

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

“…After SA infiltration into leaves, SGE is formed transiently at early stages, while SAG levels continuously increase over time (Dempsey et al, 2011). During pathogen infection, the vast majority of free SA is converted to SAG (Lee and Raskin, 1998), which is transported to the vacuole for degradation (Dean et al, 2005). In Arabidopsis, UGT74F1, UGT74F2, and UGT75B1 recognize SA as a substrate (Lim et al, 2002).…”