Givotia rottleriformis, Griff.

Kamatham, Samuel, Neela, Kishore Babu, Pasupulati, Anil Kumar, Pallu, Reddanna, Singh, Surya Satyanarayana & Gudipalli, Padmaja, 2016, Benzoylsalicylic acid isolated from seed coats of Givotia rottleriformis induces systemic acquired resistance in tobacco and Arabidopsis, Phytochemistry 126, pp. 11-22 : 13

2.2. Detection of SA precursors in seed coats of G. rottleriformis

The other compounds of fraction 3 were identified as BA that eluted at RT 14.7 min, salicylic acid (SA) at RT 15.3 min, benzaldehyde (BD) at RT 19.4 min, and CA at RT 21.4 min ( Fig. 1 and Supplementary Fig. S6a–d ). The purified BA and SA were also analysed by IR and NMR (Supplementary Figs. S7a and b and S8a–c). We also detected methyl salicylate (MeSA) and methyl benzoate (MeBA) in active fraction 3 (Supplementary Fig. S9). The conjugates of the SA and BA have already been reported previously (Chong et al., 2001; Lee et al., 1995). The biosynthesis of SA and its role in disease resistance has been established in various plants ( Meuwly et al., 1995; Ribnicky et al., 1998; Silverman et al., 1995; Vogt, 2010; Wildermuth et al., 2001). The precursors of SA pathway were identified as CA, BD, benzoyl-CoA and BA ( Boatright et al., 2004; Gao et al., 2015; Ribnicky et al., 1998). Though the biosynthesis of SA is well known in plants ( Ribnicky et al., 1998; Seyfferth and Tsuda, 2014; Wildermuth et al., 2001), identification of SA along with its precursors and its new derivative BzSA in the present study adds new information with respect to metabolism of SA in plants.

Figures

Fig. 1. (a and b). RP-HPLC chromatogram of active fraction-3 showing salicylic acid precursors and BzSA. (a) Preparative HPLC chromatogram showing benzoic acid (RT at 14.5 min), salicylic acid (RT at 15.3 min), benzaldehyde (RT at 19.4 min), cinnamic acid (RT at 22.5 min) and benzoylsalicylic acid (RT at 22.8 min). (b) Analytical HPLC chromatogram of purified BzSA. The single peak eluted at RT 17.6 min is purified BzSA.

Fig. 6. (a–d) Development of SAR in Arabidopsis NahG plants after different treatments followed by TMV inoculation. (a) AtPR1 and AtPR5 expression in systemic leaves of WT plants. (b) AtPR1 and AtPR5 expression in systemic leaves of NahG plants. (c) TMV-induced leaf necrosis in SA, ASA and BzSA treated local and systemic leaves of WT (Upper panel) and SA deficient NahG Arabidopsis plants after treatment with SA, ASA and BzSA followed by TMV inoculation (Lower panel). (d) Western-blot analysis of TMV coat protein (CP) levels in SA, ASA and BzSA treated systemic leaves of NahG plants after TMV inoculation. Tobacco plants sprayed with water (mock) or 0.1% dimethylsulphoxide (DMSO) or abraded with carborandum (CARB) served as controls. Plants inoculated with TMV served as infection controls.Treated leaves were local and the distal leaves that did not receive any treatment were systemic. Actin8 was used as a loading control.