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