Supplemental Data. Návarová et al. (2012). Plant Cell …€¦ · authentic Pip M+ M+ Supplemental Figure 1 ... The plant-derived substance identified as Pip and authentic Pip

N

OO

O

OPr

Pr

M+

170

H+

NH+

+N

OOH

NH

OO

O

OPr

Pr

NH

O

OPrLys

OO

O

OPr

Pr

N

156

H+

NH+

N

OO

H+

Pro

NH

OO

O

OPr

Pr

NH

O

OPr

Orn

B

A

C

D

M+

E

authentic Pip

M+

M+

Supplemental Figure 1

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 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.



NH2

NH2 CO2H

Lys

-ketoglutarate NH2

NH CO2H

HO2C CO2H

Glu

LKR/SDHsaccharopine

LKR/SDH

lysine ketoglutaratereductase

saccharopinedehydrogenase

NH2

O CO2H-amino adipicsemialdehyde

-aminoadipic acid(Aad)

NH2 CO2H

O

-amino--ketocaproic acid

N CO2H

pipecolic acid(Pip)

N CO2H NH2

CO2HHO2C

1-piperideine-2-carboxylic acid

1-piperideine-6-carboxylic acid

reduction

dehydrogenation

NH

CO2H

amino-transferase

sarcosineoxidase


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.


0,0

1,0

2,0

3,0

4,0

5,0

6,0

A

Pip

(µg

g-1

FW

) 2°

leav

es, 2

dpi

B

MgCl2 Psm PsmavrRpm1

*****

treatment of 1° leaves

MgCl2Psm avrRpm1

Pip

(µg

g-1

FW

) 1°

leav

es, 1

dpi

Col-0 ics1fmo1 pad4npr1ald10

5

10

15

20

25

30

35

40

*

°° •••

•••

*** ***

**

**


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.


0

5

10

15

20

25

30

35

40

45

05

101520253035404550

0,00,10,20,30,40,50,60,70,80,91,0A

MgCl2 Psm MgCl2 Psm MgCl2 Psm

SA

(ng

ml -

1 le

af-1

)

MgCl2 Psm

B C

D

MgCl2 Psm MgCl2 Psm

MgCl2 Psm MgCl2 Psm

E F

G H

*

***

**

**

I

MgCl2 Psm

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

SA

G (

ngm

l -1

leaf

-1)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

MeS

A(n

gm

l -1

leaf

-1)

0,0

0,1

0,2

0,3

0,4

0,5

0,6

AZ

A (

ngm

l -1

leaf

-1)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

JA (

ngm

l -1

leaf

-1)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

Cam

alex

in(n

gm

l -1

leaf

-1)

Aad

(ng

ml -

1 le

af-1

)

Lys

(ng

ml -

1 le

af-1

)

Phe

(ng

ml -

1 le

af-1

)



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 MgCl2-

samples (***: 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.


0

1

2

3

4

5

6

0

50

100

150

200

250A B

Col-0 lkr-1ald1 lkr-2

Rel

ativ

e A

LD1

expr

essi

on

Rel

ativ

e LK

R e

xpre

ssio

n

MgCl2Psm

MgCl2Psm

C

Rel

ativ

e A

LD1

expr

essi

onin

2°

leav

es

Col-0 fmo1 ald1

1° leaves: MgCl2

1° leaves: Psm

*

0

5

10

15

20

25

Col-0 lkr-1ald1 lkr-2

***

***

***

***

***

***


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.


0

2

4

6

8

10

12

0

1

2

3

4

5

6

7

8

Leaf

con

tent

(µ

g g-

1 F

W)

Pip Aad Lys

***

*

*

0

50

100

150

200

250

BABA0

4

8

12

16

***

H2O

10 µmol BABA

Soil application

Pip

leaf

con

tent

(µ

g g-

1 F

W)

H2O

10 µmol Pip

Soil applicationA

D

Col-0 ald1

**

***

°

0,0

0,5

1,0

1,5

2,0

2,5

B

H2O

10 µmol Pip

Soil application

Aad

leaf

con

tent

(µ

g g-

1 F

W)

Col-0 ald1

**

***

°°

•

C

H2O

10 µmol Aad

Soil application

Leaf

con

tent

(µ

g g-

1 F

W)

PipAad

***

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8



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, two-

tailed 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 (two-

tailed t test).


1

10

100

1000

10

100

1000

10

100

1000

1

10

100

1000

H2O D,L-Pip

Bac

teria

lnum

bers

3 dp

i

Psm

(cfu

cm-2

*

10-4

)

L-PipD-Pip

B

C

500

5000

5 µmol10 µmol

Bac

teria

lnum

bers

3 dp

i

Psm

(cfu

cm-2

*

10-4

)

0 0.1 1 10 0 0.1 1 10µmol Pip

Col-0 ald1

5000

***

***

***

***500

******

D

Bac

teria

lnum

bers

3 dp

i

Psm

(cfu

cm-2

*

10-4

)

H2O Pip Aad

c

A

Bac

teria

lnum

bers

3 dp

i

Psm

(cfu

cm-2

*

10-4

)

***H2OPip

leafinfiltration

rootapplication

Bac

teria

lnum

bers

3 dp

i

Psm

(cfu

cm-2

*

10-4

)

s: H2O; 1°: MgCl2

s: Pip; 1°: MgCl2

s: H2O; 1°: Psm avrRpm1

s: Pip; 1°: Psm avrRpm1

Col-0 ald1

E

*****

**

*

**

ns

ba

1

10

100

1000



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, two-

tailed t test).


0

500

1000

1500

2000

2500

3000

3500

4000

4500

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

0

2

4

6

8

10

12

14

16

18

20

AP

ip(µ

g g-

1 F

W)

H2O

SA

8 hpi 24 hpi

***

Psm24 hpi

*

BH2O

SA

**

** H2O

SA

***

4 hpi 24 hpi 4 hpi 24 hpi

Rel

ativ

e A

LD1

expr

essi

on

Rel

ativ

e P

R-1

expr

essi

on

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 Table 1

Supplemental Table 1. Primers used in this study.

Primer name Primer sequence (5’ to 3’) Usage

ald1‐fw TTACGATGCATTTGCTATGACC Genotyping of T‐DNA lines

ald1‐rv TTTTAAATGGAACGCAAGGAG Genotyping of T‐DNA lines

lkr‐1‐fw TCATTCTGCCTTCTCCATCAG Genotyping of T‐DNA lines

lkr‐1‐rv AGCAACAACGATATTTCGTGG Genotyping of T‐DNA lines

lkr‐2‐fw CGCTTCGATCATATCAAGAGC Genotyping of T‐DNA lines

lkr‐2‐rv CCCCTATGACTTTCTGTGCAG Genotyping of T‐DNA lines

ALD1‐FW GTGCAAGATCCTACCTTCCCGGC qRT‐PCR

ALD1‐RV CGGTCCTTGGGGTCATAGCCAGA qRT‐PCR

LKR‐FW CATGTTGATGGGAAGAATCTC qRT‐PCR

LKR‐RV AATATCGTTGTTGCTTCGCT qRT‐PCR

FMO1‐FW TCTTCTGCGTGCCGTAGTTTC qRT‐PCR

FMO1‐RV CGCCATTTGACAAGAAGCATAG qRT‐PCR

PR‐1‐FW GTGCTCTTGTTCTTCCCTCG qRT‐PCR

PR‐1‐RV GCCTGGTTGTGAACCCTTAG qRT‐PCR

PTB‐FW GATCTGAATGTTAAGGCTTTTAGCG qRT‐PCR; reference gene

PTB‐RV GGCTTAGATCAGGAAGTGTATAGTCTCTG qRT‐PCR; reference gene


Documents

Supplemental Data. Návarová et al. (2012). Plant Cell …€¦ · authentic Pip M+ M+ Supplemental Figure 1 ... The plant-derived substance identified as Pip and authentic Pip