Formation and vacuolar localization of salicylic acid glucose conjugates in soybean cell suspension cultures

Abstract

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max[L.] cv Williams 82) cell suspension cultures. [7–14C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–14C]SA was taken up rapidly from the culture media and converted primarily to SA 2‐O‐β‐d‐glucose (SAG). Lower levels of glucosylated 2,5‐dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2‐O‐β‐d‐glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP‐glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 mM SA. The SAGT activity in these cells was 88‐fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.

Salicylic Acid Biosynthesis and Metabolism

“…NASCArray (Craigon et al, 2004) Experiment 414 (P. syringae) and Experiment 123 (Phytophthora infestans)). In tobacco (Lee and Raskin, 1998) and soybean (Dean et al, 2003), where the specific glucose conjugates were examined, SAG was the dominant glucosylated form of SA. Current protocols widely used in the Arabidopsis community, however, may not distinguish SGE and SAG, as both acid hydrolysis (e.g.…”

“…Similar to tobacco (Dean et al, 2005) and soybean (Dean et al, 2003;Dean and Mills, 2004), SAG in Arabidopsis is thought to be produced in the cytosol and then (actively) transported into the vacuole as a non-toxic storage form of SA (Figure 3). Transport of SAG into the vacuole also ensures there is no end-product inhibition of the cytosolic glucosyltransferase, and can thus increase yield.…”

“…Production of MeSAG appears to be mediated by SGT1, as recombinant SGT1 converted MeSA to MeSAG in vitro. Soybean and tobacco cell suspension cultures also have been shown to convert radio-labelled SA to MeSA and/or MeSAG (Dean et al, 2003;2005). The biological role of MeSAG remains unknown; one possible function is to serve as a non-volatile storage form of MeSA (Dean et al, 2003;2005).…”

“…The biological role of MeSAG remains unknown; one possible function is to serve as a non-volatile storage form of MeSA (Dean et al, 2003;2005). In contrast to SAG, which is predominantly in the vacuole, only 50% or less of the MeSAG detected in soybean and tobacco was found in this organelle (Dean et al, 2003;2005).…”

“…[ 14 C]SA was shown to be incorporated into 2,3-DHBA, 2,5-DHBA, or their sugar conjugates in several plant species, with the percent incorporation varying dramatically between the species tested (Ibrahim and Towers, 1959). Later studies showed exogenously applied SA also was effectively converted to 2,5-DHBA glycosides in Arabidopsis (Dean and Delaney, 2008), Glycine max (soybean; Dean et al, 2003) and C. roseus (Shimoda et al, 2002). By contrast, Bartsch et al (2010) found only a small fraction of exogenously supplied [ 2 H]SA was converted into 2,3-DHBA (detected as the pentoside) and another unidentified DHBA-pentoside, possibly that of 2,5-DHBA, in avrRpm1-expressing Arabidopsis transgenics.…”