Discussion
Resistance inducers have considerable potential in integrated pest
management, but remain rarely used due to concerns including
phytotoxicity and limited efficacy (Walters & Fountaine 2009). Our work
identifies piperonylic acid (PA) as an effective, broad-spectrum and
non-phytotoxic novel resistance inducer. PA treatment significantly
reduces the susceptibility of several crop plants to a panel of pests
and pathogens that includes organisms with different lifestyles
(necrotrophs, (hemi)biotrophs and herbivores), degrees of specialization
(generalists and specialists), and host tissues (roots and shoots).
PA is a well-known inhibitor of C4H, the second enzyme of the
phenylpropanoid pathway (PPP) (Schalk et al. 1998). Prolonged
inhibition of this pathway by growing plants on medium containing PA is
detrimental to plant growth and development (Naseer et al. 2012;
Van de Wouwer et al. 2016), whereas we show that pulsed PA
application has no such effect. The absence of growth defects upon pulse
treatment with PA is likely explained by rapid inactivation of PA
through conjugation, which we have observed here in tomato and which had
previously been reported in A. thaliana (Steenackers et
al. 2016).
Foliar PA treatment does not affect growth, but does trigger several IR
hallmarks. PA-treated plants experience a transient, local ROS burst
which begins within one hour and peaks several hours later, a response
consistent with our understanding of ROS as an early messenger in plant
immunity (Nanda, Andrio, Marino, Pauly & Dunand 2010; Barna, Fodor,
Harrach, Pogány & Király 2012; Segal & Wilson 2018). Further evidence
for altered ROS metabolism after PA treatment is provided by induction
of peroxidase-encoding genes and higher shoot guaiacol peroxidase
activity, the latter of which has also been reported after exposure to
the IR inducer COS-OGA (van Aubel et al. 2016). In addition, PA
treatment also induces the expression of several IR-associated genes,
including PR1a (Schuhegger et al. 2006; Martínez-Medinaet al. 2017), NIMIN2-like (Zwicker et al. 2007) andSAD4 (Shan & He 2018).
The accumulation of (often glycosylated) PPP derivatives is a hallmark
of IR, which has been observed with diverse IR stimuli in several plant
species (Mhlongo, Steenkamp, Piater, Madala & Dubery 2016; Gamiret al. 2020; Ameye et al. 2020; Huang et al. 2021).
While it is known that PPP perturbation is a consequence of IR
induction, our research suggests that PPP perturbation, in this case by
transient inhibition of C4H, can also be a cause of IR. Based on
our results, we propose a model of PA-IR outlined in which transient C4H
inhibition by PA leads to an initial PPP perturbation, which results in
the accumulation of (unidentified) PPP-derived immune-signaling
metabolites and, eventually, IR establishment. The PA-IR state includes
primed lignification and phenolic compound accumulation, induction of PR
genes and accumulation of (likely PPP-derived) nematistatic metabolites.
In this model, PPP-derived metabolites contribute to PA-IR in multiple
ways. First, PPP derivatives are involved in PA-IR signaling, a role
consistent with the known role of flavonoids and other PPP-derivatives
in plant immune regulation (Mandal, Chakraborty & Dey 2010; Saijo &
Loo 2020). Second, PPP derivatives contribute to the PA-IR phenotype
through their roles as phytoanticipins/phytoalexins (Yadav et al.2020; Desmedt et al. 2020) and building blocks for cell wall
reinforcement (Vogt 2010). Finally, their potent antioxidant activity
(Rice-Evans, Miller & Paganga 1997; Agati, Azzarello, Pollastri &
Tattini 2012) might help the plant cope with the oxidative stress
involved in pathogen attack (Barna et al. 2012; Lehmann, Serrano,
L’Haridon, Tjamos & Metraux 2015; Segal & Wilson 2018). With regards
to the role of PPP derivates as phytoalexins and phytoanticipins, it is
noteworthy that we found that PA-IR induces accumulation of metabolites
with a nematistatic effect on M. incognita . A similar
accumulation of nematistatic compounds upon IR induction has been
reported in oat (Avena sativa) (Soriano, Asenstorfer, Schmidt &
Riley 2004). In addition to direct induction of defense metabolites upon
PA treatment, we also found evidence that PA primes local lignification
and phenolic compound accumulation in roots infected by M.
incognita . Local lignification upon pathogen exposure has previously
been implicated in both induced and genetic resistance against various
pathogens (Yadav et al. 2020), including P. syringae (Leeet al. 2019) and M. incognita (Veronico et al.2018).
When considering potential metabolites involved in initial IR
establishment upon PPP perturbation, it is tempting to look at the
defense hormone SA. SA accumulates in PA-treated shoots, and SA
accumulation is a known IR marker in dicots (Pieterse et al.2014). However, we found that PA-IR was largely preserved in tomato
plants impaired in SA accumulation. SA-independence has also been shown
for IR stimuli including COS-OGA and beta-aminobutyric acid in various
pathosystems (Cohen, Vaknin & Mauch-Mani 2016; Singh et al.2019).
Metabolites involved in PA-IR signaling might instead be found amongst
(glycosylated) flavonoids and phenylpropanoids, as several such
metabolites show a very large increase in abundance after PA treatment.
Several genes encoding glycosyltransferases acting on flavonoids and
phenylpropanoids were induced by PA treatment, including TOMATO
WOUND-INDUCED 1 (Twi1). Twi1 encodes a promiscuous glycosyltransferase
acting on various benzoic acids, flavonoids and coumarins (Camposet al. 2019). Twi1 silencing perturbs flavonoid metabolism
and increases susceptibility to tomato spotted wilt virus (Camposet al. 2019). Although Twi1 is SA-inducible, the continued
ability of elicitors to induce Twi1 in tomato plants expressing
the NahG construct suggests the existence of SA-independentTwi1 regulation (O’Donnell et al. 1998). Interestingly,Twi1 is already upregulated 6 hours after PA treatment in shoots
and roots and remains upregulated by 72 hours in both tissues,
indicating that Twi1 induction is an early, systemic and
relatively long-lived response to PA treatment. In support of a
regulatory role for phenylpropanoid and flavonoid glycosylation in plant
immunity, it was recently shown that the A. thalianaglycosyltransferaseUGT73C7 , which glycosylates
phenylpropanoids including para- coumaric acid and ferulic acid,
plays a major role in A. thaliana immunity against P.
syringae and that its overexpression leads to constitutive PR gene
induction and SA accumulation (Huang et al. 2021).
A key feature of IR is that it is a systemic as well as a local
phenomenon (Vlot et al. 2020). A systems biology view of PA-IR
reveals considerable differences between (systemic) roots and (directly
treated) shoots. Whereas the root metabolome is much less affected by PA
treatment than the shoot metabolome, the transcriptional response (as
measured by the number of differentially expressed genes) to PA
treatment is comparable in magnitude. This suggests that PA-IR involves
systemic transcriptional changes, while direct accumulation of
(defense-related) metabolites is mostly confined to local tissues. By
contrast, metabolome analysis of tomato plants foliarly treated with
oligogalacturonides found greater metabolic changes in distal root
tissues than in directly treated leaves (Gamir et al. 2020).
Our data are compatible with at least two - non-exclusive - explanations
for the systemic spread of PA-IR. The first is that metabolites with IR
signaling functions, most likely PPP-derived, are transported from
treated to distal tissues. In tentative support of this hypothesis, it
can be noted that the metabolites which accumulate in systemic root
tissues during PA-IR form a small subset of those accumulating in
directly treated shoot tissues. A second possibility is that the small
quantities of PA found in roots after foliar PA treatment are sufficient
for establishing PA-IR in roots. Grafting experiments combined with
further metabolomic analysis might elucidate the role of metabolite
transport in PA-IR.
Although the precise mechanisms behind PA-IR induction and spread remain
to be fully elucidated, our research shows that PA-IR is broad-spectrum
and that the PA-IR phenotype appears to be conserved amongst flowering
plants. While further multi-season and multi-location field trials as
well as ecotoxicological assessments will be required before PA can be
used in agriculture, our results – which include data from a field
trial in naturally nematode-infested soil - indicate the potential of PA
as a sustainable crop protection product.