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Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
A
Supplemental Figure 1
170
N
O
+ HO
H+
N
+ H
O
N
Pr
O
O
O
Pr
M+
B
O
Pr
N
H
O
O
HN
Pr
O
O
O
Lys
Pr
M+
C
HN
156
+
O
H+ N
Pro
Pr
O
HN
+O
O
O
O
Pr
M+
D
Pr
H
N
O
O
O
HN
O
O
O
Pr
Orn
Pr
M+
E
authentic Pip
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 1. Mass spectral identification of the initially unknown substance
detected in extracts of P. syringae-inoculated plants as pipecolic acid.
(A) Mass spectrum of the unknown substance after derivatization with propyl chloroformate
converting amino groups into propyl carbamate and carboxyl groups into propyl ester
derivatives. The mass spectrum was not present in our mass spectral library containing 45
standard amino acids and amines. Containing the fragment series m/z 198, 170, 128, and
84, the spectrum showed similarities to the spectrum of the lysine derivative (B). Moreover,
the m/z 257 ion appeared to be the molecular ion (M+) of the unknown substance. We
recognized that a homologous relationship existed to the spectrum of the proline derivative
(C) in a way that each of the fragments m/z 257 (M+), 198, 170, 128, and 84 of the unknown
substance spectrum (A) was replaced by a fragment reduced by 14 mass units: m/z 243
(M+), 184, 156, 114, and 72, respectively (C). The spectra of the derivatives of lysine
(“homoornithine”) (B) and ornithine (D) exhibited a similar 14 mass unit fragment shift, which
is consistent with the presence of an additional methylene group in lysine compared to
ornithine. These observations and deduced structures of fragment ions (A, C) were
consistent with the assumption that the unknown compound is pipecolic acid (homoproline),
the methylene homologue of proline. (E) Authentic pipecolic acid yielded an identical mass
spectrum than the extracted substance (A) after propyl chloroformate derivatization.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 2
Supplemental Figure 2. The plant-derived substance identified as Pip and authentic Pip
have identical GC retention times.
Overlay of GC ion chromatograms (m/z 158, before t = 10.6 min; m/z 170 after t = 10.6 min)
derived from a pure plant extract sample (blue) and the same sample supplemented with 5
ng of authentic Pip demonstrate co-elution of extract peak and authentic Pip standard
(retention time = 11.5 min). Note that NorVal (m/z 158, retention time = 10.4 min), which is
used as an internal standard and was added to the plant extract before the work-up
procedure, has similar abundance in the supplemented and original sample.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 3
H 2N
CO2H
Lys
NH2
aminotransferase
H2N
CO2H
lysine ketoglutarate
reductase
LKR/SDH
-ketoglutarate
CO2H
HO2C
HN
CO2H
NH2
saccharopine
dehydrogenase
Glu
LKR/SDH
-amino--keto
caproic acid
O
CO2H
-amino adipic
semialdehyde
NH
O
2
dehydrogenation
sarcosine
oxidase
reduction
HO2C
N
saccharopine
CO2H
1-piperideine-
2-carboxylic acid
N
H
CO2H
pipecolic acid
(Pip)
N
CO2H
1-piperideine-
6-carboxylic acid
CO2H
NH2
-aminoadipic acid
(Aad)
Supplemental Figure 3. Proposed scheme for pathogen-inducible Pip and Aad
biosynthesis in plants via lysine catabolism.
The scheme is based on earlier (e.g. Galili et al., 2001; Gupta and Spenser, 1969; Song et
al., 2004b; Goyer et al., 2004) and present findings (e.g. Fig. 3). Our presented findings
demonstrate that the lysine aminotransferase ALD1 mediates pathogen-induced pipecolic
acid biosynthesis. Whether -amino--ketocaproic acid and/or 1-piperideine-2-carboxylic
acid are direct reaction products of an ALD1-catalysed transamination reaction still needs
experimental verification.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 4
A
**
40
1° leaves, 1 dpi
30
Pip (µg g-1 FW)
MgCl2
**
35
***
25
Psm avrRpm1
***
20
15
*
10
•••
5
0
°° •••
Col-0
B
ald1
6,0
fmo1
ics1
pad4
**
***
5,0
Pip (µg g-1 FW)
2° leaves, 2 dpi
npr1
4,0
3,0
2,0
1,0
0,0
treatment of 1° leaves
MgCl2
Psm
Psm
avrRpm1
Supplemental Figure 4. Psm avrRpm1-induced Pip accumulation in inoculated and distal
leaves.
(A) Accumulation of Pip in Psm avrRpm1-inoculated leaves of wild-type Col-0 and different
defense mutant plants at 1 dpi. Details as described in the legend to Fig. 2.
(B) Pip accumulation in upper (2°) leaves following inoculation of lower (1°) leaves with Psm
or Psm avrRpm1 at 2 dpi. Details as described in the legend to Fig. 3.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 5
*
MgCl2 Psm
E
1,2
0,4
0,2
25
20
15
10
5
0
MgCl2 Psm
*
3,0
2,5
2,0
1,5
1,0
0,0
H
G 0,6
F
JA (ng ml -1 leaf-1)
0,4
0,3
0,2
0,1
1,4
1,2
1,0
0,8
0,6
0,4
0,0
MgCl2 Psm
I
1,6
MgCl2
Psm
3,5
3,0
1,2
1,0
0,8
0,6
0,4
0,0
*
1,8
0,2
2,5
2,0
1,5
1,0
0,5
0,2
MgCl2 Psm
MgCl2 Psm
1,6
1,4
0,5
AZA (ng ml -1 leaf-1)
***
30
0,5
MgCl2 Psm
**
35
MeSA (ng ml -1 leaf-1)
SAG (ng ml -1 leaf-1)
SA (ng ml -1 leaf-1)
0,6
45
40
3,5
0,8
0,0
C
4,0
1,0
0,0
50
45
40
35
30
25
20
15
10
5
0
Phe (ng ml -1 leaf-1)
B
Camalexin
(ng ml -1 leaf-1)
D
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
Lys (ng ml -1 leaf-1)
Aad (ng ml -1 leaf-1)
A
MgCl2
Psm
0,0
MgCl2
Psm
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 5. Metabolite levels in petiole exudates of leaves collected between
6 to 48 h post Psm- or MgCl2-treatment.
Mean values are given in ng ml-1 exudate leaf-1 ± SD from at least three replicate samples.
Asterisks denote statistically significant differences between P. syringae- and MgCl2samples (***: P < 0.001; **: P < 0.01; *: P < 0.05; two-tailed t test). The corresponding
values for Pip are depicted in Fig. 3D.
(A) -Amino adipic acid (Aad). (B) Lysine. (C) Phenylalanine. (D) Free salicylic acid (SA).
(E) Conjugated salicylic acid (SAG). (F) Methyl salicylate (MeSA). (G) Azelaic acid (AZA).
(H) Jasmonic acid (JA). (I) Camalexin.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 6
B
A
200
MgCl2
Psm
***
25
Relative LKR expression
Relative ALD1 expression
250
***
150
***
100
50
0
C
MgCl2
Psm
***
20
15
***
10
***
5
0
Col-0
ald1
lkr-1
Col-0
lkr-2
ald1
lkr-1
lkr-2
6
Relative ALD1 expression
in 2° leaves
*
5
1° leaves: MgCl2
1° leaves: Psm
4
3
2
1
0
Col-0
fmo1
ald1
Supplemental Figure 6. Pathogen-induced ALD1 and LKR expression in wild-type Col-0
and different mutant plants.
(A) and (B) Psm-induced ALD1 (A) and LKR (B) expression in inoculated leaves of Col-0,
ald1, lkr-1 and lkr-2 plants at 24 hpi.
(C) Relative expression of ALD1 in upper (2°) leaves upon Psm-inoculation of lower (1°)
leaves (2 dpi).
Transcript levels were assessed and data analyses were performed as described in the
legend to Fig. 4A.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 7
A
B
Soil application
H 2O
**
12
Soil application
**
10 µmol Pip
2,5
***
6
4
2
Col-0
1,8
2,0
•
1,5
***
1,0
0,5
°°
0,0
ald1
***
Col-0
ald1
Soil application
1,6
Leaf content
(µg g-1 FW)
10 µmol Pip
°
0
C
Aad leaf content
(µg g-1 FW)
Pip leaf content
(µg g-1 FW)
10
8
H 2O
1,4
H 2O
1,2
10 µmol Aad
1,0
0,8
0,6
0,4
0,2
0,0
Aad
Pip
D
Leaf content
(µg g-1 FW)
250
***
8
7
*
***
16
H 2O
6
200
150
4
8
3
100
2
50
*
1
0
4
0
0
BABA
10 µmol BABA
12
5
Pip
Aad
Soil application
Lys
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 7. Metabolite levels in leaves following Pip, Aad, and -amino
butyric acid (BABA) application via the root in Col-0 and ald1 plants.
(A) and (B) Exogenous pipecolic acid supplied via the root is transported to the shoot and
leads to an enhancement of Aad levels. Leaf contents of Pip (A) and Aad (B) in Col-0 and
ald1 plants one day after supplying 10 µmol Pip via the roots (as outlined in Fig. 5).
(C) Leaf contents of Aad and Pip in Col-0 plants one day after supplying 10 µmol Aad via
the roots.
(D) BABA treatment induces Pip accumulation in Col-0 plants.
Leaf contents of BABA, Pip, Aad, and Lys one day after supplying plant pots with 10 µmol
BABA. Mean values are given in µg g-1 fresh weight (FW) ± SD from at least three replicate
samples. Asterisks denote statistically significant differences between samples from Pip-,
Aad-, or BABA-fed and non-fed control plants (***: P < 0.001; **: P < 0.01; *: P < 0.05, twotailed t test). In (A) and (B), open (closed) circles indicate statistically significant differences
of a H2O- (Pip-) supplied ald1 sample to the H2O- (Pip-) supplied wild-type sample (twotailed t test).
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
A
***
1000
Supplemental Figure 8
B
H 2O
Pip
100
10
1
root
application
Psm (cfu cm-2 * 10-4)
Bacterial numbers 3 dpi
Psm (cfu cm-2 * 10-4)
Bacterial numbers 3 dpi
5000
***
1000
***
500
***
100
10
µmol Pip
leaf
infiltration
0
0.1
***
100
10
1000
5 µmol
a
Psm (cfu cm-2 * 10-4)
Bacterial numbers 3 dpi
b
c
100
10
1
H 2O
Pip
Aad
1
10
**
**
s: H2O; 1°: MgCl2
s: Pip; 1°: MgCl2
s: H2O; 1°: Psm avrRpm1
s: Pip; 1°: Psm avrRpm1
100
10
1
H2O D,L-Pip D-Pip L-Pip
0.1
ald1
*
Psm (cfu cm-2 * 10-4)
1000
Bacterial numbers 3 dpi
Psm (cfu cm-2 * 10-4)
Bacterial numbers 3 dpi
1000
10 µmol
0
**
***
5000
E
10
ns
D
***
1
Col-0
C
500
***
Col-0
ald1
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 8. Concentration dependency of resistance induction by exogenous
pipecolic acid, resistance-enhancing activity of L- but not D-Pip, and chemical
complementation of ald1 defects in Psm avrRpm1-induced SAR by exogenous Pip.
(A) Leaf resistance to Psm after application of 1 mM Pip via the root (as outlined in Fig. 5)
or after infiltration into leaves. Bacterial inoculation was performed one day after Pip
treatment.
(B) Concentration dependency of resistance induction by exogenous pipecolic acid in Col-0
and ald1 plants.
Plant pots were supplied with different amounts of D,L-Pip one day prior to inoculation.
Experimental details were as outlined in the legend to Fig. 5. Asterisks denote statistically
significant differences relative to the water control within each genotype (***: P < 0.001;
two-tailed t test).
(C) Resistance-enhancing activity of L- but not D-Pip.
Indicated amounts of racemic D,L-Pip, D-Pip, or L-Pip were fed to plants one day prior to
Psm inoculation, and bacterial numbers were scored at 3 dpi. Details as described in the
legend to Fig. 5. Asterisks denote statistically significant differences relative to the water
control (***: P < 0.001; two-tailed t test).
(D) Psm growth in upper leaves following SAR induction by Psm avrRpm1 in lower leaves
one day after exogenous Pip or water treatments via the root. Experimental details were as
described in Fig. 6A, except that incompatible Psm avrRpm1 instead of compatible Psm
was used as the SAR-inducing pathogen.
(E) Col-0 resistance to Psm upon root application of water, 5 µmol L-Pip or 5 µmol L-Aad.
Details as described in the legend to Fig. 5. Different letters above the bars denote
statistically significant differences between pairwise compared samples (P < 0.05, twotailed t test).
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Figure 9
A
***
Pip (µg g-1 FW)
20
18
H 2O
16
SA
14
12
10
8
6
4
*
2
0
8 hpi
24 hpi
Psm
24 hpi
B
4,0
H 2O
4500
SA
Relative PR-1 expression
Relative ALD1 expression
4,5
3,5
3,0
2,5
**
2,0
1,5
1,0
0,5
**
***
H 2O
SA
4000
3500
3000
2500
2000
1500
1000
500
0,0
0
4 hpi
24 hpi
4 hpi
24 hpi
Supplemental Figure 9. Effect of exogenous SA on leaf Pip levels, ALD1-transcript
levels, and PR-1-transcript levels.
(A) Pip levels in leaves at indicated times after infiltration with water, 0.5 mM SA, or a
suspension of Psm (OD 0.005). Details as indicated in the legend to Fig. 2.
(B) Relative expression of ALD1 and PR-1 in Col-0 leaves at indicated times after
infiltration with water or 0.5 mM SA. Transcript levels were assessed and data analyses
were performed as described in the legend to Fig. 4A.
Supplemental Data. Návarová et al. (2012). Plant Cell 10.1105/tpc.111.103564
Supplemental Table 1
Primer name
ald1‐fw
ald1‐rv
lkr‐1‐fw
lkr‐1‐rv
lkr‐2‐fw
lkr‐2‐rv
ALD1‐FW
ALD1‐RV
LKR‐FW
LKR‐RV
FMO1‐FW
FMO1‐RV
PR‐1‐FW
PR‐1‐RV
PTB‐FW
PTB‐RV
Primer sequence (5’ to 3’)
TTACGATGCATTTGCTATGACC
TTTTAAATGGAACGCAAGGAG
TCATTCTGCCTTCTCCATCAG
AGCAACAACGATATTTCGTGG
CGCTTCGATCATATCAAGAGC
CCCCTATGACTTTCTGTGCAG
GTGCAAGATCCTACCTTCCCGGC
CGGTCCTTGGGGTCATAGCCAGA
CATGTTGATGGGAAGAATCTC
AATATCGTTGTTGCTTCGCT
TCTTCTGCGTGCCGTAGTTTC
CGCCATTTGACAAGAAGCATAG
GTGCTCTTGTTCTTCCCTCG
GCCTGGTTGTGAACCCTTAG
GATCTGAATGTTAAGGCTTTTAGCG
GGCTTAGATCAGGAAGTGTATAGTCTCTG
Supplemental Table 1. Primers used in this study.
Usage
Genotyping of T‐DNA lines
Genotyping of T‐DNA lines
Genotyping of T‐DNA lines
Genotyping of T‐DNA lines
Genotyping of T‐DNA lines
Genotyping of T‐DNA lines
qRT‐PCR
qRT‐PCR
qRT‐PCR
qRT‐PCR
qRT‐PCR
qRT‐PCR
qRT‐PCR
qRT‐PCR
qRT‐PCR; reference gene
qRT‐PCR; reference gene