PA treatment induces genome-wide transcriptional changes
consistent with IR
To gain insights into the molecular mechanisms behind PA-IR, mRNA from
roots and shoots of PA-treated tomato plants was sequenced. By
sequencing root and shoot samples, both local responses (shoots) and
systemic (roots) transcriptional responses were analyzed. Principal
component analysis and hierarchical clustering showed that PA treatment
led to substantial transcriptome shifts in both tissues (Figure
3 ). When comparing PA-treated and mock-treated plants, 164 genes were
differentially expressed (DE) in shoots (133 up, 31 down) and 309 in
roots (86 up, 223 down). A full list of transcripts and their expression
changes can be found in Supplementary Table S2.
Local transcriptional response to PA treatment
Gene ontology (GO) analysis identified 69 enriched GO terms in
PA-treated shoots compared to mock-treated shoots, of which 23 were
related to immunity (e.g., systemic acquired resistance andregulation of immune response; Supplementary Table S3 ),
11 to abiotic stress and four to photosynthesis. InterPro domain
analysis also identified enrichment of immunity- and
photosynthesis-related domains (Supplementary Table S3 ).
Among immunity-related genes, 15 genes annotated as encoding
pathogenesis-related (PR) proteins were DE in PA-treated shoots, all of
which were upregulated. Some of these have been used as IR markers in
tomato, e.g. PR1a (Schuhegger et al. 2006; Martínez-Medinaet al. 2017). Other genes involved in IR are also upregulated in
shoots after PA treatment, including four NIM1-INTERACTING2-like
(NIMIN2-like) genes and the tomato ortholog of Arabidopsis
thaliana SAD4 (SAR-DEFICIENT 4). NIMIN2 is an interactor of NPR1
(Nonexpressor of PR genes) and has a role in IR establishment (Zwicker,
Mast, Stos, Pfitzner & Pfitzner 2007), and SAD4 is involved in the
biosynthesis of the immunity regulator L-pipecolic acid (Shan & He
2018). The effect of PA on shoot immunity is further reflected by the
induction of six genes encoding WRKY transcription factors.
Both NIMIN2-like and PR1a are SA-inducible, but no genes
known to be involved in SA biosynthesis were DE. There were no clear
expression changes in genes involved in abscisic acid, auxin or jasmonic
acid biosynthesis and signaling, but two1-aminocyclopropane-1-carboxylate oxidase-like genes possibly
involved in ethylene biosynthesis were upregulated in shoots.
PA also appears to affect shoot reactive oxygen species (ROS)
metabolism, as four genes encoding PEROXIDASES and six encoding
GLUTHATIONE-S-TRANSFERASES were upregulated.
Although PA is a well-characterized C4H inhibitor (Schalk et al.1998; Schoch et al. 2002), our data show no change in shoot
expression of C4H , nor in that of PAL or 4CL, two other
early phenylpropanoid pathway (PPP) gene families. However, several
downstream PPP-related genes were upregulated: seven genes involved in
flavonoid biosynthesis and glycosylation (two putativeFLAVANONE-3-HYDROXYLASES, one putativeISOFLAVONE-2’-HYDROXLYASE and fourUDP-GLYCOSYLTRANSFERASES acting on flavonoids, includingTOMATO WOUND-INDUCED 1 ) and three genes involved in lignification
and cell wall reinforcement (Caffeoyl-CoA-O-methyltransferase 1 ,
a cinnamyl alcohol dehydrogenase and THT1-3, an
N-hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase) .
Several other genes involved in cell wall development were also
upregulated, including a CELLULOSE SYNTHASE and twoGLYCINE-RICH CELL WALL PROTEINS. Together, these results suggest
active cell wall remodeling upon PA treatment.
Systemic transcriptional response to PA treatment -
To investigate the effect of foliar PA application on gene expression in
systemic (untreated) tissues, mRNA-sequencing was performed on roots of
PA-treated tomato plants. GO analysis of DE genes in roots is consistent
with an IR phenotype, 20 out of 45 enriched GO terms were related to
plant immunity and five to abiotic stress response
(Supplementary Table S3 ).
As in shoots, foliar PA treatment strongly induced PR gene expression in
roots: 16 genes annotated as possible PR genes were DE, of which 14 were
upregulated. Again as in shoots, these PR genes include IR markers such
as PR1a . ROS metabolism might also be altered in roots upon PA
treatment: four PEROXIDASES , one LACCASE and oneCATALASE (but no GLUTHATIONE-S-TRANSFERASES) were
upregulated.
In contrast to PR- and ROS-related genes, WRKY and ERF transcriptions
factors show a divergent expression pattern between root and shoot after
PA treatment. While five WRKY genes were upregulated in shoots
after PA treatment, seven were downregulated in roots and none
upregulated. Genes encoding ETHYLENE RESPONSE FACTORS (ERFs), another
major class of transcription factors involved in biotic stress response,
also show a contrasting expression pattern between root and shoot: in
shoots one such gene is upregulated, whereas in roots nine ERFgenes were downregulated and three upregulated.
The expression of PPP and cell wall remodeling genes upon PA treatment
also differs between shoot and root. One PAL gene
(Solyc09g007900.5) is downregulated in roots after PA treatment, but no
other early PPP genes are DE. One Hydroxycinnamoyl-CoA-quinate
transferase involved in chlorogenic acid biosynthesis and oneSINAPATE GLUCOSYLTRANSFERASE were upregulated, but – in marked
contrast to shoots – no genes related to flavonoid biosynthesis were
DE. Several cell wall-related genes were DE in roots of PA-treated
plants: two cellulose synthases and THT1-3 were
downregulated, whereas Xyloglucan endotransglucosylase/hydrolaseand two pectinesterases were upregulated.