S. Kamatham et al. / Phytochemistry 143 (2017) 160e169
161
and Jones, 1996). The signaling compounds generated at the pri-
mary site of infection elicit increased resistance to secondary
infection in uninfected parts of the plants and this phenomenon is
known as systemic acquired resistance (SAR) (Delaney et al., 1994;
Ross, 1961; Ryals et al., 1994; Ward et al., 1991). It is largely
considered that salicylic acid (SA) is a major contributor to the
development of SAR. The development of SAR is mainly associated
with the coordinate expression of a large number of defense genes
in systemic parts of the plants (Ryals et al., 1995).
Salicylic acid (SA) plays a critical role in SAR induction by acting
as an endogenous signal for the induction of SAR genes (Delaney
et al., 1994; Dempsey and Klessig, 2012). It was also reported that
the translocating, SAR-inducing signal is not SA (Vernooij et al.,
1994). Reciprocal grafts demonstrated that the signal requires the
presence of SA in tissues distant from the infection site to induce
systemic resistance (Vernooij et al., 1994). The intermediates of SA
biosynthesis and the molecular events in plants were detailed by
several researchers (Dempsey et al., 2011; Lee et al., 1995; Ribnicky
et al., 1998; Silverman et al., 1995; Wildermuth et al., 2001). During
pathogen infection, the endogenously accumulated SA is rapidly
chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA), 3-acetonyl-
3-hydroxyoxindole (AHO), glycerol-3-phosphate (G3P) induced SAR
via PR gene expression (Chanda et al., 2011; Gorlach et al., 1996;
Lawton et al., 1996; Li et al., 2008; Nakashita et al., 2002; Yasuda
et al., 2003a, 2003b).
In the present study, we have chemically synthesized and
characterized BzSA derivatives. Pre-treatment of BzSA derivatives
(1e14) activated NPR1 dependent SAR gene expression in systemic
leaves of tobacco plants. Similarly, pre-treatment of SA- deficient
NahG transgenic Arabidopsis plants with BzSA derivatives induced
PR gene expression. Furthermore, pre-treatment of local leaves of
tobacco plants with BzSA derivatives (1e14) prevented the spread
of tobacco mosaic virus (TMV) infection to uninfected systemic
leaves, thus reducing TMV lesion number and diameter more
potently than known SAR inducers such as SA, ASA, and BzSA.
Likewise, pre-treatment of these derivatives reduced leaf necrosis
and curling in case of Arabidopsis NahG plants.
2. Results and discussion
metabolized to its conjugates such as SA-O-b-D-glucoside and
2.1. Chemical synthesis of BzSA derivatives
methylsalicylate (MeSA) and subsequent studies have demon-
strated that MeSA is a critical mobile signal for SAR in plants (Enyedi
et al., 1992; Lee et al., 1995; Park et al., 2007). Recently, it is also
reported that plants regulate SA levels by converting it to 2, 3-
dihydroxybenzoic acid (2, 3-DHBA) to prevent SA over-
accumulation (Zhang et al., 2013). The requirement of SA in SAR
development was demonstrated in transgenic NahG plants that
harbor a bacterial gene encoding salicylate hydroxylase, which
converts SA into inactive catechol (Gaffney et al., 1993). In our pre-
vious study, we reported benzoylsalicylic acid (BzSA) as a new de-
rivative of SA that induces SAR more potently than SA and its
derivative acetyl salicylic acid (ASA) (Kamatham et al., 2016). In-
duction of SAR was blocked when SA methyltransferase that con-
verts SA to MeSA was silenced in primary infected leaves, and
therefore it was concluded that MeSA is a SAR signal in tobacco (Park
et al., 2007).
Recently, several metabolites have been identified as SAR inducing
signals that work either dependent or independent of SA accumula-
tion (Chaturvedi et al., 2012; Dempsey and Klessig, 2012; Gao et al.,
2015; Shah et al., 2014). Dehydroabietinal (DA), an abietane diterpe-
noid, purified fromvascular sap of Arabidopsis thaliana leaves induced
SAR through the accumulation of SA (Chaturvedi et al., 2012). The
non-protein amino acid pipecolic acid (Pip) regulates SAR and basal
immunity to bacterial pathogen infection and biosynthesis of Pip in
systemic tissues contributing to SAR establishment (Ding et al., 2016;
Hartmann et al., 2017). Azelaic acid (AzA) a putative SAR signal, mo-
bilizes Arabidopsis immunity in a concentration-dependent manner
(Jung et al., 2009; Wittek et al., 2014). The other SAR inducers such as
Benzo (1, 2, 3) thiadiazole-7-carbothioic acid S-methyl ester (BTH)
(Gorlach et al.,1996; Lawton et al.,1996) and 2, 6-dichloroisonicotinic
acid (INA) (Metraux et al., 1990; Uknes et al., 1992) induced SAR in-
dependent of SA accumulation. In contrast, probenazole (PBZ) and its
active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT)
induced SAR via SA accumulation (Yoshioka et al., 2001). During SAR,
SA activates NPR1 gene a transcriptional coregulator that activates SA-
dependent defense genes (Wu et al., 2012). The expression of PR
genes leads to the enhancement of SAR in plants (Durrant and Dong,
2004; Ryals et al., 1996). Loss of function studies involving mutations
in NPR1 gene, showed compromised SAR and therefore unable to
develop resistance to pathogen infection (Kinkema et al., 2000; Mou
et al., 2003; Spoel et al., 2009). Previously, it has been reported that SA
and INA derivatives are biologically active and some of them induced
PR-1 expression in tobacco plants (Conrath et al., 1995). In addition,
benzothiadiazole, N-cyanomethyl-2-chloroisonicotinamide (NCI), 3-
In the previous study, we have purified BzSA from the seed coats
of Givotia rottleriformis and reported as an efficient SAR inducer as
compared to SA and ASA (Kamatham et al., 2016). In the present
study, we have synthesized BzSA and its derivatives (1e14) with
different chemical modifications (Fig. 1a and b) according to pre-
viously described method (Cheong et al., 2008). The chemical
synthesis of BzSA derivatives was performed in two steps, in the
first step benzoic acid with different moieties were converted into
corresponding benzoyl chlorides. The commercially available
various substituted precursors such as (3, 4-dimethoxy), (4-
methoxy), (3, 4, 5 e tri methoxy), 4-nicotinyloxy, (4-fluoro), (6-
chloronicotinolyoxy), (4-chloro), (4-bromo), (4-ido), (3-chloro),
(4-trifluoro), (4-nitro), (3-bromo), (thiophenyl-2 carbonyloxy)
benzoyl chlorides were purchased from Avera chemicals, India. In
the second step, the acid chloride was conjugated to SA and the
resultant BzSA derivatives were named according to their moieties
as 2-(4-methoxy) BzSA [1], 2-(4-iodo) BzSA [2], 2- (3-chloro) BzSA
[3], 2-(3, 4-dimethoxy) BzSA [4], 2- (3, 4, 5-trimethoxy) BzSA [5], 2-
(6-chloro nicotinoyloxy) BzSA [6], 2-(4-nitro) BzSA [7], 2-(4-chloro)
BzSA [8], 2-(4-fluoro) BzSA [9], 2-(4-bromo) BzSA [10], 2-(4-
nicotinoyloxy) BzSA [11], 2-(thiophenyl-2-carbonyloxy) BzSA [12],
2-(4-trifluoromethyl) BzSA [13] and 2-(3-bromo) BzSA [14]. The
unreacted substrates were removed by open silica column chro-
matography and the purity of BzSA derivatives (1e14) was verified
using thin layer chromatography (TLC). The chemical structural
analysis of purified BzSA derivatives (1e14) was performed using
IR, 1H and 13C NMR analysis and the molecular mass of each de-
rivative was determined by HRMS (Supplementary Figs. S1eS30).
Recently, we have reported that purified BzSA from the seed coats
of Givotia rottleriformis induced SAR in tobacco and Arabidopsis
(Kamatham et al., 2016). None of the BzSA derivatives (1e14) are
reported as SAR inducers in plants so far.
2.2. BzSA derivatives induced SAR genes expression in tobacco
plants
The establishment of SAR is mainly associated with the accu-
mulation of a large number of defense genes such as NPR1, PR, HR
and MAPK genes upon pathogen infection or by exogenous appli-
cation of chemical elicitors (Chaturvedi et al., 2012; Dempsey and
Klessig, 2012; Kinkema et al., 2000; Mou et al., 2003; Spoel et al.,
2009).
Kamatham, Samuel
