[Link].

com Plant Signaling & Behavior 1

Plant Signaling & Behavior 7:6, 1-4; June 2012; 2012 Landes Bioscience
SHORT COMMUNICATION
*Correspondence to: Vctor Flors Herrero; Email: fors@[Link]
Submitted: 01/26/12; Accepted: 03/05/12
[Link]
Introduction
Increasing evidences are shown suggesting additional effects of
nitrate transporters. We recently demonstrated that NRT2.1
may act as a transceptor. This term comprises a dual role as a
high-afnity nitrate transporter and also as a signal deliverer in
order to coordinate other metabolic plant features. NRT1.1 and
NRT2.1 can both perceive changes in the external amounts of
nitrate and transmit signals throughout the plant to coordinate
growth and nutrition.
1
Additionally, NRT2.1 represses responses
to biotrophyc pathogens probably favoring abiotic stresses
resistance, probably as an attempt to save energy for the plant.
Consequently, nrt2 mutant displays reduced sensitivity to the
bacterial pathogen P. syringae, and this phenotype is attributed
to faster responses of the SA-dependent signaling and a reduced
sensitivity to the bacterial effector coronatine.
2
The implication of hormonal regulated pathways in the basal
and primed defense of Arabidopsis against P. syringae have been
widely studied.
3,4
Despite these advances, recent metabolomic
approaches have revealed an implication of indolic and phenolic
compounds in the basal defense of Arabidopsis against the bacte-
rium.
5
In addition to this metabolite families, rapid alterations in
the abundance of amino acids and other nitrogen-containing com-
pounds, as well as glucosinolates, disaccharides and molecules that
inuence the prevalence of reactive oxygen species have been related
to plant responses against P. syringae. However, among these last
set of compounds, these research also comprises the superimposed
effect of pathogens effectors on defense suppression. Pathogens
have been shown to recongure host metabolism through Effector
A deletion in the high afnity nitrate trasporter NRT2.1 in Arabidopsis results in a reduced susceptibility to Pseudomonas
syringae by two diferent mechanisms, the SA priming and an interference in the efector triggered susceptibility. In the
present research we further characterized the metabolic and genetic profles of the mutant nrt2 in the interaction with
P. syringae. Despite the priming found in the SA-dependent pathway, the metabolic changes in nrt2 compared with
wild-type plants are more remarkable prior infection. This is associated mainly to a pre-existing over representation of
signals attributed to aromatic amino acids and phenylpropanoids in the nrt2. Genomic analysis confrms the implication
of aromatic aminoacids and phenylpropanoids, but additionally, suggests a new role in ribosomal proteins as the major
changes observed in nrt2 upon infection by the bacterium.
A deletion in the nitrate high afnity transporter
NRT2.1 alters metabolomic and trasncriptomic
responses to Pseudomonas syringae
Gemma Camaes, Victoria Pastor, Miguel Cerezo, Pilar Garca-Agustn and Victor Flors Herrero*
rea de Fisiologa Vegetal; Departamento de Ciencias Agrarias y del Medio Natural; ESTCE; Universitat Jaume I; Castelln, Spain
Keywords: NRT2, Pseudomonas syrnigae, metabolomics, disease resistance
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Triggered Susceptibility to provide the adequate environment in
the plant apoplast to sustain growth of bacterial populations.
6

Therefore, it must be considered that most metabolomic analysis
may have a contribution of the response of the plant and the host
manipulation by the pathogen. In this regard, a complementary
transcriptomic study in resistant genotypes is very helpful to dis-
criminate responses of the plant contributing to resistance.
Pre-existing Metabolic Changes in nrt2 Mutant May
Contribute of its Reduced Sensitivity
We showed previously that SA-dependent responses are primed in
nrt2 upon P. syringae infection, this response is critical to hold a less
susceptible phenotype since the mutant nrt2-sid2 losses the resis-
tance showing wild-type levels again.
2
Some previous reports by Fan
et al. showed that pre-existing enhanced ABA levels in cds1 compro-
mise its resistance against biotrophs while contributes positively to
resist A. brassicicola. Interestingly, in a LC-Q-TOF analysis of the
metabolome of nrt2 vs. Ws, major differences in nrt2 compared with
Ws are found in the absence of infection. Therefore some responses
in the mutant can be attributed to pre-existing changes. This is
observed clearly in the heatmap and also in pc-1 and pc-2 compo-
nents of the PCA analysis in both positive and negative Electrospray
ionization (ESI) analysis (Fig. 1A and B). Additionally, 48 h after
infection, the metabolic changes in Ws and nrt2 merge to a similar
prole although still cluster differently in both positive and negative
as it is shown in the PCA analysis (Fig. 1B).
In a tentative approach to identify the main metabolic changes
we used the exact mass data for a search in the metabolic database
2 Plant Signaling & Behavior Volume 7 Issue 6
treated or infected with P. syringae were
obtained using Arabidopsis ATH1 array
of Afmetrix. Bioinformatic analy-
sis was performed by Progenika Co.,
using the PartekGenomics Suite, dChip
([Link]) and the software Affy
and affyPLM form the consortium
BioConductor ([Link].
org). As we showed previously there
are sets of genes differentially expressed
in the mutant that were not changed
in Ws, while many genes were up or
downregulated in Ws and remained
unaltered in nrt2 as a consequence of
infection. A gene ontology (GO func-
tion analysis) by categories of differ-
entially expressed genes of Ws vs. nrt2
plants upon infection showed that at
the Biological Process level, most genes
belong to response to biotic or abiotic
stress or stimulus and defense (Fig. 2A).
At the molecular function level, main
changes have been observed for oxido-
reductase enzymes, ribosomal proteins
and logically in membrane transporters
(Fig. 2B).
Kegg pathway analysis has shown
that infection with P. syringae causes
alterations of two major pathways in nrt2
compared with Ws (Fig. 2C). These are
the synthesis of phenolic amino acids
and modication in the gene expression
codifying for 15 ribosomal proteins.
The biosynthetic pathway of Phe and
Tyr and the indolic amino acid Trp have
a clear link to defense signaling since the
synthesis of SA has a second branch in
which phenylalanine ammonia lyase has
been proposed as a key enzyme. In addi-
tion, some Trp derivatives such as glu-
cosinolates are also relevant mediators
of PAMP-triggered callose accumula-
tion
8
and defense against bacterial pathogens.
9
This is in agree-
ment with the results observed in the metabolome since aromatic
aminoacids are also overrepresented in the mutant compared
with the wild type. Contrastingly, much less is known about the
implication of ribosomal proteins in plant disease resistance. The
silencing of two ribosomal proteins (L12 and L19) in Nicotiana
benthamiana suppresses the non host resistance to bacterial
pathogens but does not affect the host resistance.
10
Recent nd-
ings in, proteomic analysis of rice have demonstrated that sev-
eral ribosomal proteins are upregulated after a Nilaparvata lugens
infection.
11
Interestingly, the L10 ribosomal protein is upregu-
lated in nrt2 after P. syringae infection compared with Ws. This
protein has been shown to be a component in the nuclear shuttle
protein-interacting kinase of Arabidopsis, that participates in
METLIN ([Link]/). Four main metabolite families
are overrepresented in nrt2 vs. Ws upon infection; they belong to
the aromatic aminoacids, nucleotides and phenylpropanoids. In
the other hand, a metoxiavone, a dinucleotide and glucosilated
avonoids are less represented in the mutant upon infection. This
suggests that both priming of SA and aromatic aminoacids and
phenylpropanoids may prepare the plant to perform better once
the pathogen is present.
Trasncriptomic Profling of nrt2 Infected with
Pseudomonas syringae
To further characterize the molecular response of nrt2 to P. syrin-
gae, whole-genome transcriptional proles of Ws and nrt2 mock
Figure 1. The multivariate analyses applied include hierarchical cluster analysis (HCA) and principal
component analysis (PCA). (A) Heatmaps of nrt2 vs. Ws either in the presence or in the absence of the
infection. Heatmaps were generated by using Multi-experiment Viewer
14
after a t-test by applying
a standard Bonferoni correction and a hyerarchical clustering. Data are represented in a log
2
scale.
Detection of peaks was performed in positive or negative electrospray (ESI) ionization mode and
subsequent separation of the ions in a Quadrupole-Time-of-Flight (model Premier of Waters). (B)
PCA plot were generated to specifcally identify m/z changes with infection or plant genotype. PCA
plots were constructed for P. syringae infected material for each genotype (WS and nrt2). Ginkgo
Analysis System (1.7) ([Link] was used to obtain PCA score
plots of the LC-MS in negative and positive mode. PCA plot obtained showing two major sources of
variability among Ws and nrt2 after LC-MS. Percentage of variability is given on each axis.
[Link] Plant Signaling & Behavior 3
explanation to its lower sensitivity to the bacterial effector coro-
natine, which is also contributing to the nrt2 reduced susceptibil-
ity to the pathogen.
2
Concluding, it is clear that the alteration in the NRT2.1 gene
produces global changes, affecting other plant responses that are
not solely linked to nutrient transport rather than to responses to
environmental changes, indeed this include responses to patho-
gens. Therefore these observations reinforces the hypothesis that
the NRT2.1 may be acting also as a transceptor, coordinating
perception of external signals with plant responses to abiotic and
biotic stresses.
Disclosure of Potential Conicts of Interest
No potential conicts of interest were disclosed.
Acknowledgements
We thank the Serveis Centrals de Instrumentaci Cientca
of the Universitat Jaume I for its technical support. We
also thank the funding provided by Generalitat Valenciana
GV/2007/099, Plan Promocin Bancaja-UJI P1.1A2007-07
and P1.1B2007-42.
antiviral signaling.
12
Although there are many other factors that
could contribute to the enhanced resistance of the mutant, our
results suggest a putative implication of ribosomal proteins in
resistance against bacterial diseases in Arabidopsis.
Interestingly, a study of gene vs. stimulus performed with
the Genevestigator application ([Link]),
13
has
shown a reasonably good correlation in the response to stimulus
between the NRT2.1 (At1g08090) and NRT2.2 (At1g08100).
Stimuli such as iron deciency, OPDA, SA and MeJA downregu-
late all these genes but on the contrary cold, nematodes, the elici-
tors Hrpz, LPS and FLG22 strongly upregulate their expression
in a coordinated manner. This suggests that they act coordinately
not only under nitrogen deprivation, but also upon different
abiotic and biotic stimuli. Finally, another study of response to
stimuli performed on the Genevestigator database of the gene
NRT2.1 compared with some marker genes of SA, JA and ABA
signaling pathways, has shown a low correlation in the induc-
tion or repression. This may indicate that perception of stimulus
activates differentially NRT2.1 and hormonal controlled defense
pathways.
Unfortunately, the metabolic and genomic analysis of
nrt2 infected by P. syringae, still has not revealed a denitive
Figure 2. Microarray analysis and Kegg pathway and Go categorization of wild-type and nrt2 genes diferentially expressed after inoculation with
P. syringae using PartekGenomics Suite. (A) Number of genes diferentially expressed in nrt2 vs. wild-type upon infection categorized by metabolic
pathways. (BD) Number of genes diferentially expressed in nrt2 vs. wild-type upon infection categorized by biological process, cellular component
and molecular function. Percentages relate to total number of genes with an ontology at each analysis. Only categories with higher percentages of
total number of genes are shown.
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