PA treatment induces extensive metabolic reprogramming
Untargeted UPLC-MS/MS profiling of shoots and roots of tomato seedlings
24h after the second PA treatment was used to further investigate the
molecular basis of PA-IR.
After pre-processing, 4692 features were retained in shoots, of which
1672 were differentially abundant (DA). 985 features were more abundant
in PA-treated than in mock-treated shoots, whereas 687 were less
abundant. A substantial number of DA features were unique to either mock
(17)- or PA-treated (396) samples. In roots, 2549 features were
retained, of which 186 were significantly DA between PA- and
mock-treated samples - a much smaller proportion than in shoots. There
was a clear bias towards overaccumulation after PA treatment in roots:
177 features were significantly more abundant, while 9 were less
abundant. The smaller effect of PA on the root metabolome is reflected
by principal component analysis and hierarchical clustering, both of
which show complete separation between PA- and mock-treated shoot
samples, but only partial separation for root samples (Figure
6 ).
Pathway analysis identified five metabolic pathways enriched upon PA
treatment in tomato shoots: phenylpropanoid biosynthesis (P
< 0.001), stilbenoid and diarylheptanoid biosynthesis(P < 0.001), flavonoid biosynthesis (P = 0.020),riboflavin metabolism (P < 0.001) and thiamine
metabolism (P = 0.030). In roots, four pathways were enriched:phenylpropanoid biosynthesis (P < 0.001),stilbenoid and diarylheptanoid biosynthesis (P <
0.001), flavonoid biosynthesis (P = 0.040) and galactose
metabolism (P = 0.030). A full list of pathways is shown inSupplementary Table S4 .
PA itself was found in both shoot and root samples from PA-treated
plants, both as free PA and as a hexose conjugate. This indicates that
PA is taken up, conjugated and possibly systemically transportedin planta . The hexose conjugate was eleven times more abundant
than free PA in shoots, and thirteen times more abundant in roots. Both
free and conjugated PA were approximately ten times more abundant in
shoots than in roots.
A second feature of interest is the defense hormone SA, which was 6.2
times more abundant in PA-treated than in mock-treated shoots (P
< 0.001). By contrast, SA abundance in roots was unaffected by
foliar PA treatment. To confirm the effect of PA treatment on SA seen
after untargeted metabolome profiling, and to verify the absence of
responses to other major plant hormones suggested by our transcriptome
data, we also performed targeted UPLC-MS/MS measurement of SA, JA,
abscisic acid and indole-3-acetic acid. We observed a similar SA
accumulation in tomato shoots after PA treatment (4.8-fold increase) in
this experiment, and found no major changes in the level of the other
measured phytohormones (Supplementary Table S6 ).
Since foliar PA treatment induces SA accumulation in shoots, we
investigated whether SA accumulation is necessary for PA-IR by testing
the efficacy of PA in tomato plants expressing the NahGtransgenic construct, which converts SA to catechol (Brading,
Hammond-Kosack, Parr & Jones 2000). Plants expressing NahG were
more susceptible to M. incognita (+14%, P = 0.037),confirming the role of SA in defense against M. incognita(MartÃnez-Medina et al. 2017). However, PA was almost as
effective in NahG plants (-36%, P < 0.001) as in wild
type plants (-43%, P < 0.001; Figure 6 ), which
suggests that PA-IR against M. incognita is at least partially
SA-independent.
Besides PA and SA, 59 other DA features were annotated with a high
degree of confidence by matching against an in-house database using mass
and retention time. A further 226 DA features were putatively annotated
to a metabolite class by matching their m/z against the METLIN database
(Guijas et al. 2018). Both features identified against the
in-house database and annotated using METLIN were notably enriched in
PPP derivatives and flavonoids. PPP derivates identified via the
in-house database include para- coumaroyl quinic acid (4-fold
increase after PA treatment, P < 0.001), syringin (12-fold
increase, P < 0.001), feruloyl hexose (3-fold reduction, P
< 0.001), benzoic acid (66-fold increase, P < 0.001)
and benzoyl hexose (16-fold increase, P < 0.001). The PPP
precursor phenylalanine was two times more abundant in PA-treated than
in mock-treated shoots (P < 0.001). Several PPP derivatives
were also DA in roots, but generally with smaller fold changes. Examples
include para- coumaroyl quinic acid (2.3-fold increase, P
< 0.001), syringin (2.2-fold increase, P = 0.031) and benzoyl
hexose (12.6-fold increase, P = 0.002).
Among DA features, 153 had m/z values whose only METLIN matches were
flavonoids or flavonoid glycosides and whose retention times were
compatible with these metabolite classes. Of these 153 features, 67 were
less and 86 were more abundant in PA-treated than in mock-treated
shoots. Remarkably, 50 of the 86 upregulated flavonoids were unique to
PA-treated samples; these flavonoids were either absent in mock-treated
plants, or present below the limit of detection. No putative flavonoids
were unique to mock-treated samples. In root samples, twelve putative
flavonoids were DA, all of which were upregulated and six of which were
unique to PA-treated samples.
There is a strong overlap between DA features in root and shoot: 156 out
of 186 DA features (84%) in roots were also DA in shoots. Putatively
annotated features that were more abundant in both PA-treated roots and
shoots include two benzoic acid hexosides, a cinnamoyl hexoside,para- coumaroyl quinic acid, a hexose conjugate of
5-hydroxyferulic acid, syringin, seven unidentified flavonoid glycosides
and three unidentified flavonoids. A full list of DA features, alongside
their empirical formulas and putative annotations, is provided inSupplementary Table S5 .