3800 3600 3400 3200 3000 2800 26001.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

PM

-IR

RA

S s

ignals

, arb

. units

Wavenumber (cm-1)

1900 1800 1700 1600 1500 1400 1300 1200 1100 10002.5

3.0

3.5

4.0

4.5

5.0

PM

-IR

RA

S s

ignals

, arb

. units

Wavenumber (cm-1)

3050 3000 2950 2900 2850 2800 27503.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

PM

-IR

RA

S s

ignals

, arb

. units

Wavenumber (cm-1)

1500 1490 1480 1470 1460 1450 1440 14304.6

4.7

4.8

4.9

5.0

PM

-IR

RA

S s

ignals

, arb

. units

Wavenumber (cm-1)

A B

1734

ν(C=O)

ν(CO)

1171

1124

1080

(a)

(b)

(a) Before immersion

(b) After immersion

(a)

(b)

1039

(a)

(b)

δ(CH2)

1472

1462

3410

ν(OH)

3450

3003

ν(CH)

2916

2848νa(CH2) νs(CH2)

1514

ν(C=C)

(a) Before immersion

(b) After immersion

(a)

(b)

(a) Before immersion

(b) After immersion

(a) Before immersion

(b) After immersion

1464

1472

1462

C D

MCT

PEM +

polarizer

Lens

(BaF2)

Parabolic

mirror

Mirror

IR light from FT-IRMirror

Leaf

T. Hama,1,2 K. Seki,3 A. Ishibashi,2 A. Miyazaki,2 A. Kouchi,2 N. Watanabe,2 T. Shimoaka,4 T. Hasegawa4

1 Komaba Institute for Science and Department of Basic Science, UTokyo 2 Institute of Low Temperature Science, Hokkaido Univ.

3 Nagano Vegetable and Ornamental Crops Experiment Station 4 Institute for Chemical Research, Kyoto Univ.

Probing the molecular structure of the intact leaf cuticle

by polarization modulation-infrared reflection-absorption spectroscopy

Experimental Methods

Introduction

Conclusions

Results and Discussion

The multifunctional interface between the plant and the environment.

Critical for the development and survival of plants.

Barrier to protect plants against (1) Dehydration, (2) UV radiation,

(3) Atmospheric oxidants (OH, O3) (4) Pathogen and insect attacks

Cuticle: lipid membrane on the plant surface

Very little is known about the molecular arrangement

(conformation, crystallinity, and orientation).

The key for the physicochemical properties.

(1)Wax: Organic solvent soluble.

Aliphatic hydrocarbons (e.g., alkanes, alcohols)

Carbon chain lengths of C20–C40

(2)Cutin: Organic solvent insoluble polymer (polyester).

Chain lengths of C16 and C18 cross-linked by ester bonds.

(3)Polysaccharides: Pectins, and hemicelluloses.

Yeats and Rose, (2013) Plant Physiol. 163: 5–20.TEM of Arabidopsis stem. SEM of Arabidopsis leaf.

5 m

PEM：Photo-elastic modulator

PM-IRRAS of a wild cabbage (Brassica oleracea L.)

PM-IRRAS: Polarization-modulation infrared reflection absorption spectroscopyDouble modulation FT-IR spectroscopy based on the difference in IR reflectance between the p- and s-polarizations

IAC

IDC

(1) Rapidly switched measurements of

Rp and Rs (e.g., 50 kHz) using a PEM

↓

(2) The electric signal is separated into

the direct current form (IDC),

and the alternate current form (IAC).

↓

(3) The ratio spectrum (S) is obtained.

[The ratio of IAC to IDC (IAC/IDC)]

↓

IR spectra of thin sample films can be obtained on a metallic substrate

and even on a dielectric substrate (e.g., silicon, liquid water, and a plant leaf).

p

s

Surface normal

θ Air

Dielectric substrate

Sample

FT-IR

Parabolic mirror

PolarizerPEM Lens

(BaF2)

Mirror

MCT

Single modulation

Double

modulation

Dielectric substrate

Positive

Negative

Positive

Negative

Wavenumber

S

In situ analysis of a living specimen is possible

without sample pretreatment

1. Background-free measurement↔ Conventional IR measurements need background and sample measurements.

Impossible to measure a leaf surface (substrate) without the cuticle (sample).

2. Nondestructive analytical technique↔ Structural analysis methods using electrons, ions, X-rays. or lasers.

3. Average molecular orientation can be determinedSimilar surface selection rule to that of external reflection spectroscopy

using a dielectric substrate (p-polarization).

Surface-parallel

vibration

→ Positive peak*

Surface-normal

vibration

→ Negative peak*

Blaudez et al., (1996) Faraday Trans. 92: 525. Itoh et al., (2010) Appl. Spectrosc. 64: 1374.

νa(CH2)

2916 cm-1

positive

ν(OH)

3410 cm-1

negative

νs(CH2)

2848 cm-1

positive

n

O

H

H2C

CH3

CH2

H2C

CH2

H2C

CH2

H2C

Leaf

Doublet by

the factor group splitting

↓

Crystalline having

orthorhombic subcell

(CH2)

1472 cm-1

positive

(CH2) 1462 cm-1

positive

H

H

H

H

C

C

a

bc

Orthorhombic or Monoclinic: 1472, 1462 cm-1

Triclinic: 1473 cm-1

Hexagonal: 1468 cm-1

Amorphous: a broad peak at 1467 cm-1

The thickness of the cuticular wax (50-375 nm),

The cuticle thickness of wild cabbage: 3-6 m.

↓

PM-IRRAS probes the outer cuticle region less than

about 100-500 nm surface region.

Leaf

O

O

R’

ν(C=O)1734 cm-1

Positive

ν(C-O)

Negative

R

C

Leaf

ν(C-O)

Negative

Probing an inner region by ATR-IR spectroscopy

Pectin peaks at 1241, 1147, 1102, 1067, 1050, 1016 cm-1

No hemicellulose peaks (dashed gray lines)

12

41

11

47

11

60

11

02 10

16

17

37

14

72

14

631

62

0

H2O

15

18

10

50

10

67

1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900

0.00

0.05

0.10

0.15

Wavenumber (cm-1)

AT

R a

bsorb

ance u

nits

1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900

0.0

0.5

1.0

1.5

2.0

2.5

Pe

ne

tra

tio

n d

ep

th (m

)

Wavenumber (cm-1)

Cutin Wax

The thickness of cuticle: 0.1 to 10 m or more

(depending on the species)

500 nm

Epidermal cell

Cuticle layer:

(polysaccharide-rich inner region)

Epicuticular wax:

Coating the outermost cuticle surface

Cuticle proper:

Polysaccharide-free outer region

Hama et al., JPC B, 121, 11124 (2017) Hama et al., Plant Cell Physiol., in press.

(1) PM-IRRAS is an easy-to-use approach for studying the plant cuticle

No need for sample pretreatment or background measurements

(2) The positive a(CH2), s(CH2) and (CH2) bands:

The all-trans zigzag alkyl chains of the epicuticular wax.

Packed in the orthorhombic subcell.

Oriented perpendicular to the leaf surface.

(3) Polysaccharides are widely distributed across the leaf cuticle.

Hemicelluloses in the outer cuticle region (less than about 100-500 nm).

Pectins in the inner 2 m region and are more abundant than hemicelluloses.

500 nm

Current cuticle model: A surface sensitive (nm-scale) spectroscopy is necessary.

A

B

Figure: Ratio spectra of a wild cabbage leaf at (A) 3800–2600 cm-1 and (B) 1900–1000 cm-1.

(a) before solvent treatment and (b) after immersion in chloroform. (C) and (D) Magnification of spectra in (A) and (B).

SEM images of leaf surfaces.

(A) Untreated control.

(B) After immersion in chloroform.

10 m

10 m

PM-IRRAS

(≤ 0.1-0.5 m)

ATR-IR

(≤ 2 m)Pectins，wax，cutin, phenolics

Hemicelluloses(Xylan, xyloglucan),

wax，cutin, phenolics

Epidermal cell

Epicuticular wax:

“In situ observation” was technically difficult.

(i) Sample pretreatment, and (ii) Sample damage

Epicuticular wax was removed

after immersion in chloroform.

Dashed gray guidelines

are at 2924, 2916,

2855 and 2848 cm-1.

*When the incidence angle (76) is larger than Brewster angle of the air/leaf interface (55 )

The positive ν(C=O) band at 1734 cm-1:

the non hydrogen-bonding character of cutin.

1713 cm-1 or lower when hydrogen bonded.

The intense negative ν(CO) bands:

1171, 1124 cm-1: Glycosidic linkage of xylan

1080, 1039 cm-1: Ring vibration of xyloglucan2916, 2848 cm-1

the ordered

all-trans zigzag

2924, 2855 cm-1

Disordered

The presence of waxes, cutin, and hemicelluloses in the outer cuticle region.

↔ The cuticle model (the cuticle proper as the outer region free from polysaccharides)?

Phenolics

Phenolics