Download Figure legends Figure 1. Biosynthesis and catabolism of NAD+ in

Figure legends
Figure 1. Biosynthesis and catabolism of NAD+ in higher plants. De novo NAD+ synthesis starts
in the chloroplast with quinolinate synthesis from aspartate by aspartate oxidase (AO) and
quinolinate synthase (QS). Quinolinate is metabolised by QPT (EC 2.4.2.19), which catalyses the
transfer of the phosphoribosyl moiety from phosphoribosyl pyrophosphate to quinolinate
yielding nicotinic acid mononucleotide (NaMN), pyrophosphate and CO2, to maintain the de
novo source of NaMN for NAD+ biosynthesis. Further steps are carried out in the cytosol and are
shared between the biosynthesis and recycling of molecules derived from NAD +. NaMN is
subsequently adenylylated to nicotinic acid adenine dinucleotide (NaAD) by nicotinate
mononucleotide adenyl transferase (NaMNAT). The final amidation of NaAD to NAD+ is achieved
by NAD synthetase (NADS) (Ashihara et al., 2005). nadC gene encodes QPT from E.coli. NaPT:
Nicotinic acid Phosphoribosyl Transferase, NAM: nicotinamide, PARP: Poly-ADP-Ribosylation
Proteins. The reversibility of the NaPT reaction remains unclear in plants (Noctor et al., 2006).
Figure 2. Overexpression of Escherichia coli nadC gene in Arabidopsis transgenics. A,
Construction used for generating Arabidopsis nadC-overexpressing transgenic plants. The
coding sequence of E. coli nadC gene was cloned between the CaMV 35S promoter and
terminator using the PCW162 vector, which provided kanamycin resistance to transformed
plants. B, PCR gel blot analysis of the expression of the nadC transgene. Genomic DNA was
prepared from the vector control 162.7.17 (Ctrl) or the nadC lines (4.11, 15.3). PCR primers
specific to the nadC transgene were used. A PCR fragment of 935 pb is observed in nadC lines
4.11 and 15.3 indicating overexpression of nadC gene from E. coli in Arabidopsis. C, QPT specific
activity. Both the 4.11 and 15.3 lines exhibit increased QPT activity compared to Ctrl. Samples
were taken from 3-week-old plants.
Figure 3. Metabolic responses of increased NAD+ contents in Arabidopsis. Foliar discs of
Arabidopsis Ctrl and nadC overexpression lines were incubated 48 h in the dark, in a MOPS
buffer with or without 100 µM quinolinate (Q). After incubation, total cellular contents of NAD +
(A), NADP+ (B), GSH (C) (closed bars) and NADH, NADPH and GSSG (open bars) were quantified
using a modified colorimetric assay. Non-targeted (GCMS) and targeted (HPLC) metabolite
profiling was also carried out (D to I). Amounts of NAD + (A) as well as the total pool of NADP(H)
(B) in the presence of quinolinate were drastically increased in both nadC lines compared to
Ctrl. A slight increase of NAD/P(H) was also observed in Ctrl upon quinolinate treatment. The
reduction state of NAD and NADP was not changed. Total cellular contents of glutathione (GSH
and GSSG) were not modified whereas GSSG levels were higher in quinolinate-treated Ctrl (C).
Asparagine, aspartate and methionine levels substantially increased in the nadC lines in the
presence of quinolinic acid (E, F and G). Treatment with quinolinate caused the accumulation of
nicotinic acid in all lines (H) and enrichment of xylose in nadC discs (I). Results are the means of
three to six biological replicates with standard deviation. * P<0.05, ** P<0.01, *** P<0.001
compared with the quinolinate-treated Ctrl (by Student’s t test).
Figure 4. Transcript profiling of vector control and nadC 15.3 lines. A, Representation of the
hybridisation scheme to depict experimental designing. Each pool of probes derived from
mRNAs was hybridised as described by the black arrows. B, Hierarchical clustering of statistically
different fragments identified by CATMA analysis of A (nadC + Q vs nadC), B (nadC + Q vs Ctrl +
Q), C (Ctrl + Q vs Ctrl) and calculated condition D (nadC vs Ctrl) using MultiExperimentViewer
(MeV) software. C, Verification of selected differentially expressed sequenced fragments.
Histograms show qPCR confirmation of relative expression to ACT2 responses. For qPCR
analysis, data are means of three independent extracts with standard deviation. * P < 0.05.
Figure 5. Increased NAD levels induce resistance to the bacterial pathogen Pseudomonas
syringae pv. tomato Pst-AvrRpm1. A, leaves of Ctrl (closed bars) and nadC 15.3 line (open bars)
were infiltrated with 5 mM quinolinate (Q), pH 6. After 48 h incubation, the same leaves were
infected with 105 cfu/mL of bacterial strain Pst-AvrRpm1 (A) or MgCl2 as a control (data not
shown). Bacterial growth kinetic was carried out every 24 h until 72 hpi. Growth of the avirulent
strain Pst-AvrRpm1 fell down 48 hpi and remained lower 72 hpi in quinolinate-treated nadC
leaves compared to non-treated leaves. B, quinolinate toxicity on bacterial growth in leaves of
Ctrl and nadC lines. After 48 h of incubation, quinolinate-infiltrated (closed bars) or uninfiltrated
leaves (open bars) were inoculated with 105 cfu/mL of Pst-AvrRpm1 strain or control MgCl2 (data
not shown). Growth of Pst was measured 48 hpi. In Ctrl, quinolinate treatment did not modify
bacterial growth but led to a lower number of bacteria in both the nadC 4.11 and 15.3 lines.
Data, expressed in log10, are the means of three to six samples with standard deviation. The
experiment was repeated three times with similar results.
Figure 6. In folia contents of pyridine nucleotides upon quinolinate infiltration and PstAvrRpm1 infection. A, B, C and D, measurements of NAD/P(H) levels using a plate-reader assay
on untreated and quinolinate-treated leaves of Ctrl and nadC lines from - 48 hpi to 48 hpi (PstAvrRpm1). A similar pattern of NAD(P) enrichment reported by the foliar disc system was
observed in planta when nadC leaves were infiltrated with quinolinate. Upon quinolinate
infiltration, NAD(P) levels increased slightly in Ctrl, raised in the nadC 15.3 line at - 24 hpi and
reached a maximum at 0 hpi, corresponding to 48 hours of quinolinate treatment (A and C).
After treatment with mock or Pst-AvrRpm1 (24 and 48 hpi), the NAD+ and NADP+ contents
decreased in the time course but remained higher in the quinolinate-treated nadC line
compared to Ctrl in the presence of quinolinate. Reduced forms NADH (B) and NADPH (D) also
increased after quinolinate application in the Ctrl and nadC lines. Upon bacterial infection, the
NADH content remained stable in nadC line in the presence of quinolinate but it rose under
other conditions (B). Infection with Pst-AvrRpm1 lead to increased NADPH levels, which were
higher in quinolinate-infiltrated nadC line (D). Results are the means of three biological
replicates with standard deviation. Asterisks indicate statistical significance versus the
appropriate control in each case (* P < 0.05, Student’s t test).
Figure 7. NAD derivatives and relative gene expression 48 hpi. Alternative sampling of bacterial
inoculation was performed 48 hpi with Pst-AvrRpm1 on untreated (closed bars) and quinolinatetreated leaves (open bars) of the Ctrl and the nadC 15.3 line to analyse NAD derivatives by a
targeted LCMS profiling approach (A and B). Nicotinamide and nicotinate accumulate in
response to quinolinate treatment. Additionally, the relative levels of pathogen related and NAD
metabolism transcripts were also examined by RT-qPCR, using ACT2 as an internal control. AO
was induced during bacterial infection (C). For PR1, the relative transcript levels detected in the
different lines rose with the infection, but the highest induction was observed in nadC leaves
treated with quinolinate (D). The expression profile of ICS1 correlated with that of PR1 (E). The
values shown are means of three repeats with standard deviation (indicated by error bars).
Experiments were repeated and showed comparable results. Asterisks indicate statistical
significance versus the appropriate control in each case (* P < 0.05, Student’s t test).
Figure 8. Total and free SA accumulate in one nadC line overproducing NAD. Quantification of
salicylic acid (SA) in mock-infiltrated and bacteria-inoculated leaves of the Ctrl and nadC 15.3
lines, with (closed bars) or without (open bars) quinolinate, 48 hpi. SA was extracted as
described previously (Simon et al., 2010). A, total pool of SA and B, free SA. Infection with PstAvrRpm1 leads to increased total and free SA levels. In nadC leaves pre-infiltrated with
quinolinate, total and free forms of SA significantly rose compared to Ctrl in the presence of
quinolinic acid. Data shown are means of three replicates with standard deviation (indicated by
error bars). Experiments were repeated and showed comparable results. C, 3D plot of NAD
content, Pst-AvrRpm1 bacterial growth and free SA pool 48 hpi. Results were summarised for
the Ctrl and nadC 15.3 lines, with or without quinolinate. Remarkably, quinolinate-treated nadC
samples (circled plots) stood out from other plots, indicating a strong correlation between the
increase in NAD and resistance to avirulent pathogen Pst-AvrRpm1, associated with increased
levels of free SA. Data correspond to three biological replicates.
Supplemental Table T1. Various metabolomic responses with the nadC inducible foliar system.
Amino acids were quantified as described in Bathellier et al. (2009). ''Total'' stands for adding all
detected amino acids except proline, which was not detected with such a method. Results are
the means of three biological replicates with standard deviation.
Supplemental Table T2. 333 significantly expressed genes in CATMA microarray analysis. A
stands for nadC+Q vs nadC, B for nadC+Q vs Ctrl + Q, C for Ctrl+Q vs Ctrl and D for calculated
condition nadC vs Ctrl from the log2 ratio.
Supplemental Table T3. Leaf ATP contents after quinolinate infiltration and bacterial infection.
ATP levels were quantified as described in Djebbar et al., (2011) in the Ctrl and nadC lines. No
substantial changes were observed for the different tested conditions. Results are the means of
three biological replicates with standard deviation.
Supplemental Data S1. Graphical representations of the microarray data for the inducible
foliar system. A, Venn diagram showing overlap between gene lists that show significant
changes in transcript abundance. The threshold is set to 0.5. A stands for nadC+Q vs nadC, B for
nadC+Q vs Ctrl+Q and C for Ctrl+Q vs Ctrl. B, Functional categories of microarray results.
MapMan® software was used to subdivide significantly expressed genes into functional
categories, according to the software nomenclature, and using average data points.
Supplemental Data S2. Hierarchical clustering between the NAD transcriptome response and
CATMA database. HCL was performed using MeV software with Pearson correlation between
our conditions A (nadC+Q vs nadC), B (nadC+Q vs Ctrl + Q), and C (Ctrl+Q vs Ctrl) and the
available CATMA database (see CATdb).
Supplemental Data S3. Closer experiments with similar global expression pattern to
conditions A and B. A, focus on S3 HCL. B, table of relative close clusters showing similar global
expression patterns with conditions A and B. Experiments names refer to CATdb nomenclature.
Distance was calculated using Pearson correlation. Biotic stress-related experiments are shown
in red.
Supplemental Data S4. nadC line 15.3 challenged with virulent Pst DC3000. A, bacterial growth
of Pst DC3000. Leaves of Ctrl (closed bars) and nadC lines (open bars) were infiltrated with
5 mM quinolinate (Q) pH 6. After 48 h of incubation, the same leaves were infected with
105 cfu/mL of bacterial strain Pst-DC3000 (A) or MgCl2 as a control (data not shown). Bacterial
growth kinetic was carried out every 24 h until 72 hpi. Growth of the virulent strain Pst-DC3000
increased from 24 hpi to 72 hpi in all lines treated or not with quinolinic acid.
Supplemental Data S5. NAD+/NADH and NADP+/NADPH ratios after quinolinate infiltration
and bacterial infection. A and B, redox ratios of NAD/P(H) were calculated from data presented
in Figure 6. Both for mock and bacterial infiltration, redox ratios of NAD/P(H) increased in the
nadC line in the presence of quinolinic acid. Experiments were repeated and showed
comparable results. Asterisks indicate statistical significance versus the appropriate control in
each case (* P < 0.05, Student’s t test).
Supplemental Data S6. nadC line 15.3 accumulates glycosylated salicylic acid upon quinolinate
treatment. Quantification of glycosylated salicylic acid (G-SA) in mock-infiltrated and bacteriainoculated leaves of Ctrl and nadC 15.3 lines, with (closed bars) or without (open bars)
quinolinate, 48 hpi. Free and total SA were extracted as described in Simon et al. (2010). G-SA
corresponds to total SA minus free SA. Infection with Pst-AvrRpm1 leads to increased G-SA
levels. In nadC leaves pre-infiltrated with quinolinate, G-SA significantly increase compared to
Ctrl treated with quinolinic acid. Data shown, expressed in log10, are means of three replicates
with SD (indicated by error bars). Experiments were repeated and showed comparable results.
Asterisks indicate statistical significance versus the appropriate control in each case (* P < 0.05,
Student’s t test).