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 : 12-13

2.1. Identification of benzoylsalicylic acid in seed coats of G. rottleriformis

We extracted the total seed coat compounds in methanol (MeOH) and the methanolic crude seed coat extract was fractionated by open silica column chromatography (see Experimental procedure, Section 4.3.1) and the eluted fractions (1–7) were tested for their SAR inducing bioactivities against TMV in tobacco (Supplementary Table S2 ). Among all the fractions, a fraction number 3 was effective in reducing the development of TMV-induced lesion number and diameter. This active fraction showed a group of peaks with different retention times (RT) when resolved by RP-HPLC ( Fig. 1a ). The major compound of the peak from fraction 3 that eluted at RT 22.8 min ( Fig. 1a ) was purified and tested for its purity by analytical HPLC with RT at 17.6 min ( Fig. 1b ). The purified compound was found to be active against TMV and was characterized as benzoylsalicylic acid (CCDC with accession number 90056) using single crystal X-ray diffraction analysis (Supplementary Fig. S2a and b ). Further structural analysis of purified BzSA was carried out using IR and NMR (Supplementary Fig. S3a–c ). The mass of the purified BzSA was determined by GC–MS/MS as 242 Da (Supplementary Fig. S4 ).

Although the seed coats of Givotia are a rich source of BzSA (0.5 mg /gm DW), the other parts like leaves (0.1 mg /gm DW) and bark (0.15 mg /gm DW) also contained this compound (Supplementary Fig. S5a and b ). The higher accumulation of BzSA, a phenolic compound, in seed coats of G. rottleriformis could be due to its importance in seed germination, seedling growth and interaction with soil microbes. It is well known from the literature that phenolics function as signals in plant–microbe interactions ( Raskin, 1992). Emerging evidence implicates the role of SA in seed germination, flowering, thermogenesis, plant growth and development, and tolerance to abiotic stresses such as drought, chilling, heavy metal toxicity, heat and osmotic stress in plants ( Khan et al., 2015; Rivas-San Vicente and Plasencia, 2011).

The chemical structure of BzSA along with SA and ASA are depicted in Fig. 2a–c . The possibility of BzSA biosynthesis depends on the availability of free SA and benzoyl-CoA ( Fig. 2d ). Literature suggests that SA and benzoyl-CoA are present in plants and the biosynthesis of SA in plants takes place via CoA-dependent or independent way, and during this process benzoyl-CoA is formed as intermediate by the oxidation of cinnamic acid ( CA) to benzoic acid ( BA) ( Ribnicky et al., 1998). Previously, it has been reported that the majority of the endogenously synthesized SA are rapidly converted and stored as biologically inactive derivatives via glucosylation and methylation since accumulation of SA has adverse physiological consequences ( Dempsey et al., 2011; Park et al., 2007; Vlot et al., 2009). Recent studies have shown 2,3-dihydroxybenzoic acid (2,3 DHBA) as a hydroxy derivative of SA ( Zhang et al., 2013). However, the benzoylation of SA has not been reported so far. In this study, we report benzoylation of SA for the first time 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. 2. (a–d) Chemical structures of (a) Salicylic acid. (b) Acetylsalicylic acid. (c) Benzoylsalicylic acid. (d) Proposed scheme of BzSA biosynthesis in plants.

Fig. 3. (a–f) RT-qPCR analysis of NPR1 and PR genes in local, systemic and TMV-inoculated systemic leaves of tobacco after different treatments. (a) NPR1 transcript levels in SA, ASA and BzSA treated local leaves. (b) NPR1 transcript levels in systemic leaves. (c) NPR1 transcript levels in systemic leaves after TMV inoculation (d) PR1a transcript levels in local leaves that were treated with SA, ASA and BzSA. (e) PR1a transcript levels in systemic leaves. (f) PR1a transcript levels in systemic leaves after TMV inoculation. (g) PR1b transcript levels in local leaves. (h) PR1b transcript levels in systemic leaves. (i) PR1b transcript levels in systemic leaves 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 (TMV). Treated leaves were local and distal leaves that did not receive any treatment were systemic.Data are expressed as means ± SD of 3 independent experiments. The expression of the genes was normalized with constitutively expressed EF1a elongation factor. The bars with the same letter are not significantly different (p <0.05) by Newman–Keul’s multiple comparison test.

Fig. 4. (a–c) Expression of PR and other defense genes in local and systemic leaves of tobacco after different treatments. (a) Dose dependent expression of PR1a in systemic leaves. (b) Time dependent expression of PR1a in systemic leaves. (c) Expression of defense marker genes in systemic leaves of tobacco plants. Tobacco plants sprayed with water (mock) or 0.1% dimethylsulphoxide (DMSO) or abraded with carborandum (CARB) served as controls. Treated leaves were local and the distal leaves that did not receive any treatment were systemic. Data represents Mean ± SD of 3 independent experiments using graph-pad prism software. EF1a was used as a loading control. The bars with the same letter are not significantly different (p <0.05) by Newman–Keul’s multiple comparisons test. The 18S rRNA was used as an internal loading control.

Fig. 5. (a–d) Effect of BzSA on TMV-induced lesions in local and systemic tobacco leaves. (a) TMV controls showing lesions in local and systemic tobacco leaves. (b) TMV lesions in local (72 h SA treatment) and systemic leaves (24 h and 72 h SA treatment). (c) TMV lesions in local (72 h ASA treatment) and systemic leaves (24 h and 72 h ASA treatment). (d) TMV lesions in local (72 h BzSA treatment) and systemic leaves (24 and 72 h BzSA treatment). Treated leaves were local and the distal leaves that did not receive any treatment were systemic. Carborandum (CARB) treated leaves served as ontrols. Plants inoculated with TMV served as TMV infection controls.