ARTICLE IN PRESS
Flora 201 (2006) 555–569
www.elsevier.de/flora
Anatomical and chemical characteristics of foliar vascular bundles in four
reed ecotypes adapted to different habitats
Kun-Ming Chena,b, Feng Wanga, Yu-Hua Wanga, Tong Chena,
Yu-Xi Hua, Jin-Xing Lina,
a
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China
b
Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China
Received 21 July 2005; accepted 14 December 2005
Abstract
We investigated the anatomical and chemical characteristics of the foliar vascular bundles in four ecotypes of
common reed (Phragmites communis Trin.) inhabiting the desert region of northwest China: swamp reed (SR), low-salt
meadow reed (LSMR), high-salt meadow reed (HSMR), and dune reed (DR). The cell walls of the vascular systems of
all four ecotypes exhibited bright autofluorescence. Compared to SR, the three terrestrial ecotypes, LSMR, HSMR
and DR, had higher percentages of bundle sheath cell areas, lower percentages of xylem and phloem areas, lower
xylem/phloem ratios, and higher frequencies of leaf veins. In addition to differences in the autofluorescence intensity
and the morphology of the detached cell walls of the vascular bundle sheath, the three terrestrial ecotypes also
exhibited anatomical differences in the outerface tangential walls of the bundle sheath and higher frequencies of pit
fields in the walls in comparison to SR. The Fourier transform infrared (FTIR) microspectroscopy spectra of the
vascular bundle cell walls differed greatly among the tissues of the different ecotypes as well as within different tissues
within each ecotype. Histochemical methods revealed that although pectins were present in all bundle tissue cell walls,
large amounts of unesterified pectin were present in the phloem cell walls, especially in the salt reed ecotypes LSMR
and HSMR, and large quantities of highly methyl-esterified pectin were present in the xylem and sclerenchyma cell
walls of the SR and DR ecotypes. Differences were observed in the lignification and suberization of the xylem and
sclerenchyma cell walls of the four ecotypes, but the phloem and bundle sheath cell walls were generally similar. These
results suggest that the adaptation of common reed, a hydrophytic species, to saline or drought-prone dunes triggers
changes in the anatomical and chemical characteristics of the foliar vascular bundle tissues. These alterations,
including higher percentages of bundle sheath areas and lower percentages of xylem and phloem areas and their ratios,
changes in the chemical compositions and modifications of the cell walls of different vascular bundle tissues, and
differences in the deposition of major cell wall components in the walls of different vascular bundle tissues, could
contribute to the high resistance of reeds to extreme habitats such as saline and drought-prone dunes.
r 2006 Elsevier GmbH. All rights reserved.
Keywords: Anatomical and chemical plasticity; Cell wall; Different habitats; Foliar vascular bundle; Histochemistry; Reed
(Phragmites communis Trin.) ecotypes
Corresponding author. Fax: +86 10 62590833.
E-mail address: [email protected] (J.-X. Lin).
0367-2530/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.flora.2005.12.003
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Introduction
The plant vascular system is responsible for the
transport of water, ions, carbohydrates, and other
nutrients. It has been the subject of numerous studies,
because it may also constrain the distribution of
resources within a plant (Orians and Jones, 2001; Orians
et al., 2002; Zwieniecki et al., 2003). Multiple characteristics of vascular structures have been investigated, such
as modifications to the wall architecture, ion composition, protein expression, and alteration of the xylem/
phloem ratio, all of which are thought to be involved in
the resistance of the plant to environmental stresses
(Child et al., 2003; Equiza and Tognetti, 2002; Saijo et
al., 2001; Zwieniecki et al., 2003). Wang et al. (1997)
found that salinity influenced the patterns of lignification, peroxidase activity, and extension deposition in the
cortex and vascular tissues in Atriplex prostrata Boucher
and the changes in cell wall composition occurred at
different salinities. Kawashima et al. (2005) reported
that all genes of a serine acetyltransferase gene family
were predominantly localized in the vascular system of
Arabidopsis and a complex array of the compartmentspecific isoforms with distinct enzymatic properties and
expression patterns was developed to respond to
developmental and environmental changes. However,
most investigations have focused on the structure of the
root cylinder (Colmer, 2003; Hose et al., 2001; Steudle,
2000), and few have targeted the leaf vascular bundles,
especially during environmental stresses. Therefore, the
anatomical and chemical characteristics of foliar vascular bundles of plants growing under different environmental conditions require more investigation.
Plant cell walls play essential roles in growth,
development, response to environmental factors, and
interactions with pathogens and symbionts (Carpita
and Gibeaut, 1993; Emma and Herman, 2003; Kaczkowski, 2003). Cell walls are also a source of signal
molecules in self- and non-self-recognition (Brownlee,
2002; Esquerré-Tugayé et al., 2000; Kaczkowski, 2003).
These diverse functions require that cell walls have
complex and variable structures and composition
(Emma and Herman, 2003; Enstone et al., 2003;
Kaczkowski, 2003). Variations in the cell wall during
the development of the plant provide an excellent model
system for studies of the mechanisms that determine
growth regulation and adaptation to different environmental conditions (Carpita and Gibeaut, 1993; Enstone
et al., 2003; Moore et al., 2002; Sabba and Lulai, 2002;
Tan et al., 1991).
The cell wall consists of a complex matrix of
carbohydrate polymers, proteins, phenolic compounds,
and other components, which determine the plasticity of
the walls through cross-links and/or interactions
(Brownlee, 2002; Enstone et al., 2003; Equiza and
Tognetti, 2002; Kaczkowski, 2003; Marga et al., 2003).
Several recent studies have shown that modifications of
cell wall polymers such as polyphenolics (lignin), longchain aliphatic polymers (suberin), pectins, and proteins
help to create barriers to water, solutes, gases, and
pathogens in plants exposed to unfavorable biotic and
abiotic stress conditions (Enstone et al., 2003; Hartmann
et al., 2002; Hose et al., 2001; Moore et al., 2002; Sabba
and Lulai, 2002). For example, Casparian bands and
suberin lamellae, the major components of apoplastic
barriers in plant roots, are laid down in radial transverse
and tangential walls in response to different habitat
conditions such as drought, anoxia, salinity, and heavy
metal and nutrient stresses. These barriers including
aliphatic and aromatic suberin and lignin in different
amounts and proportions are established based on the
growth regime (Hose et al., 2001). Shannon et al. (1994)
found that salinity promotes the suberization of the
hypodermis and endodermis paralleled by a development of the Casparian strip closer to the root tip than in
roots grown under normal conditions. Reinhardt and
Rost (1995) showed that the absolute amounts of
suberin and lignin in the exodermal cell walls of cotton
seedling roots are markedly increased by external
stresses such as salt stress, osmotic stress, and heavy
metal stress, even though the qualitative compositions
are not altered. Increased rates of suberization and
lignification in the exodermis may lead to greater
resistance to the entry of materials into the root apoplast
(Hose et al., 2001). In addition, Sabba and Lulai (2002)
used histochemical analysis to show that in potato
tubers, the significantly different strengths of phellogen
cell walls in native and wounded periderm are related to
the degree of pectin de-esterification. Based on the
results from droughted roots of Eucalyptus camaldulensis, Lemcoff et al. (2002) suggested that an increase in
cell-wall elasticity, which are largely established by thick
and stiff cell walls, is involved in plant drought
resistance by maintaining water uptake. However, these
results were almost all obtained under artificial stress
conditions or in plant roots; less is known about the
roles of the chemical composition and modifications to
the cell wall under natural environmental stress conditions and in leaf vascular systems.
The common reed (Phragmites communis Trinius) is a
hydrophytic species with typical habitats of fresh and
brackish swamps, riverbanks, and lakesides. However,
reeds can also adapt to adverse terrestrial habitats:
various ecotypes with exhibiting genetic differences have
evolved resistance to drought, salinity, and low temperatures (Haslam, 1970, 1975; Matoh et al., 1988;
Zheng et al., 2000). In general, genetic variation within a
species is brought about by ecological adaptation to
various habitats. Thus, genetic variation strongly
depends on the habitats. In addition to swamp reed
(SR), the desert regions of northwest China are home to
three terrestrial reed ecotypes: low-salt meadow reed
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(LSMR), high-salt meadow reed (HSMR), and dune
reed (DR). In the course of our long-term studies, these
four reed ecotypes have shown stable variations in
morphological, physiological, and genetic characteristics
in response to drought and salinity (Chen et al., 2003;
Cheng et al., 2001; Wang et al., 1998; Zheng et al., 2000;
Zhu et al., 2001). The physiological mechanisms of the
adaptation of these plants to their respective habitats
have been well documented. In studying the leaf
anatomy and ultrastructure of the four reed ecotypes, Zheng et al. (2000) found that the ecotypes
show distinct variations in the shapes of the bundle
sheath chloroplasts and the abundance of the bundle
sheath organelles. However, to date, the anatomical
characteristics of the foliar vascular bundles and the
plasticity of the cell walls of the four reed ecotypes,
which likely hold important information on the adaptation of plants to habitats such as saline and droughtprone dunes, have not been investigated. In this study,
we examined the anatomical characteristics of the foliar
vascular bundles, the chemical composition and histochemistry of the cell walls of different bundle tissues,
and the characteristics of the bundle sheath cell walls of
the four reed ecotypes. Hence, the areas of the different
bundle tissues and their percentages, the morphology
and distribution of plasmodesmata and the alignment of
microfibrils in the cell walls of the bundle sheath, and
the chemical composition and modifications in the cell
walls of different vascular bundle tissues, were investigated. The ecophysiological significance of the alterations in the anatomical and chemical characteristics of
the foliar vascular bundles of the different ecotypes was
discussed.
557
Materials and methods
Plant material and sampling sites
Four ecotypes of the reed P. communis Trin., referred
to as SR, LSMR, HSMR, and DR based on their
respective habitats (Chen et al., 2003; Wang et al., 1998;
Zheng et al., 2000), grow in Pingchuan, Linze County,
Gansu Province, China. This region, which has a typical
desert landscape, is part of the Linze Research Area of
the Cold and Arid Regions Environmental and Engineering Research Institute of the Chinese Academy of
Sciences (39140 –240 N, 991250 –350 E; elevation 1300 m).
The mean annual precipitation is 118 mm, and the
annual potential evaporation is 2392 mm. The relative
humidity usually does not exceed 50%. The air
temperature is characterized by large daily fluctuations
with annual maximum and minimum temperatures of 39
and 27 1C, respectively. The four reed ecotypes were
sampled from different habitats in the same region. SR
grows in pools with depths greater than 2 m, with a
0.35% salt content in the root zone. In contrast, the
terrestrial reed ecotypes, LSMR, HSMR, and DR, grow
in low-lying edges of corn fields (with 35.4% water
content and 0.49% salt content in the root zone), lowlying salt flats (with 48.8% water content and 0.71% salt
content in the root zone), and sand dunes (with 13.7%
water content and 0.09% salt content in the root zone),
respectively (Table 1). Although found in areas with
varying soil water and salt states, these four reed
ecotypes share similar meteorological conditions since
all of the sampling sites are located within a narrow
region of about 6.5 km2.
Table 1. Water and salt conditions of different habitats, leaf water status, and soluble protein and three organic solutes of four reed
ecotypes
Basic parameters
SR
LSMR
HSMR
DR
Soil water content (%)
Soil salt content (%)
Leaf water content (%)
Leaf water potential (MPa)
Ratio of dry weight to fresh weight
Biomass (g DW m2)a
Soluble protein content (mg g1 FW)b
Soluble sugar (mg g1 DW)b
Proline (mg g1 DW)b
Betaine (mg g1 DW)b
Saturated soil
0.15a
90.9a
0.45a
0.379a
2530a
25.0670.52a
80.5574.68a
3.6770.21a
1.0270.24a
35.4a
0.49b
88.4b
0.50b
0.352b
790b
ND
ND
ND
ND
48.5b
1.31c
82.8c
1.32c
0.402c
1175c
19.6570.85b
83.3177.21a
12.2370.92b
6.1270.36b
13.7c
0.09d
71.9d
2.17d
0.455d
224d
17.8170.21c
151.9379.88b
9.0670.45c
5.2370.17b
Soil water content and soil salt content were measured around noon, on June 20, 2003, and the values (7SD, Pp0.05) are the means of three
measurements. Leaf water content and water potential were determined at 15:00 on June 18, 2003. Each value is the mean (7SD, Pp0.10) of 10–13
leaves. One-way analysis of variance was used for comparisons between the means. Values with different letters are significantly different at Pp0.05.
SR, swamp reed; LSMR, light salt meadow reed; HSMR, heavy salt meadow reed; DR, dune reed.
a
Data come from Ren et al. (1994).
b
Data come from Zhu et al. (2003); ND ¼ no data.
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From 15 to 20 June 2003, the second leaves from the
top of the four reed ecotypes were simultaneously
collected at midday and frozen in liquid N2 until
analysis. Soil water content and salt content in the root
zone were determined as previously described by Zheng
et al. (2000). Leaf water potential was measured on
leaves with a pressure chamber. The dry matter of leaves
was determined after drying for 72 h at 80 1C, and the
difference between fresh and dry weight was used to
evaluate the water content of the leaves (Chen et al.,
2003).
Isolation of foliar vascular bundles and cell walls of
bundle sheath cells
The foliar vascular bundles and walls of the bundle
sheath cells were isolated enzymatically as described by
Hartmann et al. (2002). Samples of about 0.5 cm were
incubated separately in an enzymatic buffer solution
containing 0.25% (w/v) cellulase (Onozuka R-10; Serva)
and 0.25% (w/v) pectinase (Macerozyme R-10; Serva) in
10 mM sodium acetate at pH 4.5 and 25 1C. After
approximately 2 weeks of maceration, the leaf vascular
bundles were mechanically separated under a binocular
microscope. Integrated vascular bundle sections with
bundle sheath cells were subjected to scanning electron
microscopy (SEM). Bundle sheath cell walls were
separated from the vascular bundle sections for FTIR
and histological analysis.
Field emission scanning electron microscopy
(FE-SEM)
The isolated vascular bundles were fixed in FAA
solution (50% ethanol, 5% acetic acid, 10% formalin).
The samples were dehydrated in an ascending ethanol
series followed by critical-point drying with carbon
dioxide. The dried samples were then fixed on aluminum
slides using double-sided adhesive tape. Finally, the
samples were coated with gold and examined by FESEM (XL30 S-FEG; FEI, The Netherlands).
Light microscopy and histology
Leaf blocks of about 0.5 0.3 cm from the four reed
ecotypes were fixed for at least 2 h in FAA solution and
then hand-sectioned with a razor blade. Sections of
about 20–40 mm were either directly examined or
subjected to staining with 0.02% ruthenium red to
detect unesterified (acidic) pectin, with hydroxylamineferric chloride to detect methyl-esterified pectin (Sabba
and Lulai, 2002), or were de-esterified in 0.1 M Na2CO3
overnight at 4 1C before staining with ruthenium red to
detect total pectin (acidic pectin plus methyl-esterified
pectin). To detect suberin and lignin, sections were
stained with Sudan red III or phloroglucin, respectively
(Wang et al., 1997; Zeier et al., 1999), followed by
bright-field viewing. Autofluorescence was examined
using UV epifluorescent illumination with an exciter
filter (365 nm). Bright-field and fluorescence microscopy
were carried out on an Axioplan microscope (Zeiss).
Photographs were taken with a Zeiss Q500 IW light
microscope, and digital images were captured using an
AxioCam MRc camera (Carl Zeiss). Three independent
experiments were performed for each staining analysis.
To determine the cross-sectional area of different
tissues, at least 50 vascular bundles of 4–6 different leaf
slices were analyzed with an image analyzer (Axio
Vision 4.1; Carl Zeiss).
FTIR microspectroscopy analysis
To analyze the xylem and phloem sectors using FTIR
microspectroscopy analysis, transverse sections of approximately 20 mm were washed six times with deionized
water and then dehydrated in an ascending ethanol
series. For the analysis of bundle sheath cell walls,
isolated cell walls were washed with deionized water and
then dried in a layer on a barium fluoride window
(13 mm diameter 2 mm) at 40 1C. Analysis was performed using a MAGNA 750 FTIR spectrometer
(Nicolet Corporation, Tokyo, Japan) equipped with a
mercury–cadmium–telluride (MCT) detector. Spectra
were recorded between 2000 and 800 cm1 with a
resolution of 8 cm1. Each spectrum was automatically
normalized to obtain the relative absorbance. Three
replicate measurements were carried out for each buddle
section of the four reed ecotypes.
Results
Habitat conditions, leaf water status and stress
responses
As shown in Table 1, the largest differences between
the habitats of the four reed ecotypes were in the
moisture and salt contents of their root zones. The DR
possessed the lowest water and salt contents in root
zones as well as the lowest water content and water
potential in the leaves among the four reed ecotypes,
indicating that a high water deficit stress existed in this
reed ecotype. The HSMR had the highest salt content in
root zones and, although it had a much higher moisture
in root zones, it also exhibited water deficit in the leaves
because of low leaf water content and water potential. A
slight water deficit status also existed in the leaves of
LSMR as compared to the SR. In addition, the ratio of
dry weight to fresh weight was obviously higher in the
three terrestrial reed ecotypes than that in SR while
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biomass was much higher in the SR than in the
terrestrial ecotypes. Although the soluble protein content of leaves was markedly higher in SR than in HSMR
and DR, the contents of three organic solutes, soluble
sugar, proline and betaine, which are tightly correlated
with the responses of plants to environmental stresses,
were significantly higher in the two terrestrial reed
ecotypes than those in SR.
559
exhibited higher percentages of bundle sheath cell areas
than did SR, as well as lower percentages of xylem and
phloem areas. The xylem/phloem ratios were highest in
SR, followed by DR, LSMR, and HSMR. The
foliar vein frequencies in the four ecotypes also differed
(Table 3). Of the four ecotypes, SR possessed the
thickest layers of mesophyll cells between the veins and
the largest interveinal distances, DR exhibited the
thinnest layers of mesophyll cells between the veins,
and HSMR exhibited the shortest interveinal distances.
Anatomical characteristics of foliar vascular bundles
Two types of foliar vascular bundles, small and large,
were observed in the four reed ecotypes (Fig. 1). Both
types consisted of about four sectors, the xylem, phloem,
sclerenchyma, and bundle sheath, and no obvious
differences in morphologies were observed among the
ecotypes. The cell walls of the foliar vascular bundle
tissues of all of the ecotypes exhibited bright autofluorescence, and phloem cell walls showed even more intense
autofluorescence. The foliar vascular bundles of the four
ecotypes exhibited significant anatomical differences in
the dimensions of the bundles and their percentages in
different sectors (Table 2). Since the percentages of the
small vascular bundle tissues in each ecotype were
similar (Table 2), only the characteristics of the large
vascular bundles were selected for comparison. As
shown in Table 2, the whole vascular bundles of the
DR ecotype had the largest areas, followed by those of
SR, LSMR, and HSMR. The three terrestrial ecotypes
Anatomical characteristics of the cell walls of foliar
bundle sheath
Detached cell walls of foliar bundle sheath cells from
the four reed ecotypes treated with cellulose and
pectinase were observed with UV epifluorescent illumination (Fig. 2). The autofluorescence of the cell walls
appeared more intense in the three terrestrial ecotypes
than in SR, with the most intense autofluorescence in
DR. Furthermore, the morphologies of the detached cell
walls differed among the four ecotypes, with relatively
long radial walls observed in SR and LSMR (Fig.
2A, B).
The outerface characteristics of the bundle sheath cell
walls between the bundle tissues and mesophyll cells
in the four ecotypes were investigated using FE-SEM
(Fig. 3). In addition to differences in the frequencies of
pit fields in the outerface tangential walls of the bundle
sheath cells, which were highest in DR, intermediate in
the two salt reed ecotypes, and lowest in SR (Table 3),
the distributions of plasmodesmata in the pit fields
differed among the four ecotypes (Fig. 3B, E, H, K). In
LSMR and HSMR, the plasmodesmata were distributed
in a more concentrated manner (Fig. 3E, H), whereas
in the drought-prone DR, they were more dispersed
(Fig. 3(K). In contrast, in SR, no plasmodesmata were
observed in the pit fields under these experimental
conditions (Fig. 3B). Lastly, the alignment of microfibrils in the cell walls also differed among the four
ecotypes (Fig. 3C, F, I, L).
FTIR analysis of the cell walls of bundle tissues
Fig. 1. The morphology of leaf vascular bundles in four reed
ecotypes and the autofluoresence of different bundle tissues
under UV epifluorescent illumination, demonstrating the
different bright autofluoresence of cell walls of bundle tissues
between the ecotypes. Bars ¼ 100 mm. Ph, phloem; Xy, xylem;
BSC, bundle sheath cell; Sc, sclerenchyma. (A) Swamp reed
(SR). (B) Light salt meadow reed (LSMR). (C) Heavy salt
meadow reed (HSMR). (D) Dune reed (DR).
We observed significant differences in the FTIR
spectra of the cell walls of the different vascular bundle
tissues of the four ecotypes (Table 4, Fig. 4). The spectra
of bundle sheath cell walls showed several broad bands
at around 1720, 1635, 1512, 1427, 1373, 1319, 1242,
1157, and 1034 cm1 (Fig. 4A). The peak at 1635 cm1,
which was assigned to the amide-stretching bands of
proteins (Ivanova and Singh, 2003), was present in all
four ecotypes, but the absorbance was highest in LSMR,
intermediate in HSMR and DR, and lowest in SR. The
three other peaks at 1720, 1512, and 1242 cm1, which
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Table 2. The area of different tissues and their percentage within a single vascular bundle in the leaves of four reed ecotypes
growing in the different habitats
Ecotypes
Whole vascular bundle
Area (mm2)
The big vascular bundle
SR
0.45370.040a
Bundle sheath cells
Area (mm2) (%)
Xylem
Area (mm2) (%)
Phloem
Area (mm2) (%)
Ratio of xylem/phloem
0.10070.013a
(22.372.7)a
0.15670.019a
(34.572.9)a
0.08970.010a
(19.372.0)a
1.870.25a
LSMR
0.24070.013b
0.08570.009b
(35.573.5)b
0.05870.008b
(24.373.1)b
0.03970.007b
(16.472.8)b
1.5170.25b
HSMR
0.21570.022c
0.08470.010b
(39.273.3)c
0.04670.006c
(21.572.4)c
0.03670.009c
(16.573.4)b
1.3770.39c
DR
0.65870.047d
0.19170.018c
(29.071.8)d
0.17870.016d
(27.171.9)b
0.11470.013d
(15.271.5)c
1.6570.26d
0.09870.0171
(57.975.1)e
0.01770.005e
(9.772.3)e
0.01270.004e
(7.072.0)e
1.5270.62bd
The small vascular bundle
SR
0.17070.026e
LSMR
0.14970.013f
0.07770.010d
(52.276.6)e
0.01570.003f
(10.172.0)e
0.01170.003e
(7.771.8)e
1.3870.46bc
HSMR
0.13870.017
0.07870.008d
(56.575.3)e
0.01470.003f
(10.171.6)e
0.01170.004e
(7.672.3)e
1.4570.50bc
DR
0.38370.073
0.20770.037c
(54.373.3)e
0.03870.012
(9.872.0)e
0.02470.008f
(6.272.4)e
1.6770.47d
The values are means7SD. For cross section area determination of different tissues, at least 50 vascular bundles of six different leaf slices from three
independent experiments were calculated. One-way analysis of variance was used for comparisons between the means. Values with different letters
are significantly different at Pp0.05. SR, swamp reed; LSMR, light salt meadow reed; HSMR, heavy salt meadow reed; DR, dune reed.
Table 3. Leaf vein frequency and pit field frequency at interface between mesophyll cells and bundle sheath cells of four reed
ecotypes growing in the different habitats
Ecotypes
SR
LSMR
HSMR
DR
Pit field frequency (mm2)
Leaf vein frequency
Mesophyll cells between veins (layer)
Interveinal distance (mm)
3–6
2–3
2–3
2
322.1721.7a
247717.0b
229722.5c
294716.4d
0.02070.002a
0.03670.005b
0.03670.006b
0.04570.004c
The values are means7SD. For leaf vein frequency, at least 50 interveinal distances were determined from six different leaves of three independent
experiments. For pit field frequency, 10 isolated vascular bundle sections from five different leaves of two independent experiments were used for
calculation. One-way analysis of variance was used for comparisons between the means. Values with different letters are significantly different at
Pp0.05. SR, swamp reed; LSMR, light salt meadow reed; HSMR, heavy salt meadow reed; DR, dune reed.
were assigned to the C–O-stretching vibration v(C ¼ O)
in carboxylic ester or acid groups (Zeier and Schreiber,
1999), the aromatic skeleton of lignin (Pandey and
Pitman, 2003), and the syringyl ring and C–O-stretch in
lignin and xylan (Pandey and Pitman, 2003), respectively, behaved similarly to the 1635 cm1 peak in the
four ecotypes. However, the peaks at 1427, 1373, and
1319 cm1, which were assigned to the C–H deformation
in lignin and carbohydrates, the C–H deformation in
cellulose and hemicellulose, and the C–H vibration in
cellulose and the C1–O vibration in syringyl derivatives,
respectively (Pandey and Pitman, 2003), were highest in
DR, intermediate in HSMR and LSMR, and lowest in
SR. In contrast, the peaks at 1157 and 1034 cm1,
representing the C–O–C-stretching and C–O-stretching
vibrations in carbohydrates (Galichet et al., 2001;
Pandey and Pitman, 2003), respectively, were highest
in HSMR, followed by LSMR, DR, and then SR.
The FTIR spectra of xylem cell walls showed more
differences among the ecotypes (Table 4, Fig. 4B).
Except for the peaks at 1034 cm1, all the peaks in the
bundle sheath cell wall FTIR spectra were present in the
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561
As a whole, the FTIR microspectroscopy spectra of
the vascular bundle cell walls differed greatly in both
peak pattern and absorbent intensity among the tissues
of the different ecotypes as well as within different
tissues within each ecotype.
Histochemical analysis of the cell walls of bundle
tissues
Fig. 2. The autofluoresence of detached cell walls of the
vascular bundle sheath cells in four reed ecotypes growing in
the different habitats, showing the deposition of suberin in the
walls of vascular bundle sheath cells and their morphology
between the ecotypes. Bars ¼ 50 mm. Tw, transverse wall; Rw,
radial wall; (A) Swamp reed (SR). (B) Light salt meadow reed
(LSMR). (C) Heavy salt meadow reed (HSMR). (D) Dune
reed (DR).
spectra from xylem cell walls. The xylem wall spectra
also contained other peaks, e.g., at 1458 and 1250 cm1,
which, respectively, correspond to the C–H deformation
in lignin and carbohydrates and the syringyl ring and
C–O-stretch in lignin and xylan (Pandey and Pitman,
2003). The frequencies at around 1200–800 cm1, which
are dominated by the stretching vibrations of C–O,
C–C, ring structures, and the deformation vibrations of
CH2 groups, were found to be useful for the identification of polysaccharides (Hori and Sugiyama, 2003;
Pereira et al., 2003). In this region, except for the peak at
1157 cm1, which was present in all four ecotypes, peaks
at 1065, 1034, and 995 cm1 were found in SR; peaks at
1041, 995, and 903 cm1 appeared in LSMR; peaks at
1088, 1049, 1018, and 895 cm1 were found in HSMR;
and peaks at 1095, 1026, 1003, and 895 cm1 were
present in DR. In addition, the absorbances of the FTIR
spectra of cell walls in the xylem sectors were higher in
the two salt reed ecotypes than in SR and DR.
The walls of phloem cells also exhibited more complex
spectral patterns in the 1200–800 cm1 region (Table 4,
Fig. 4C). Peaks at 1157, 1065, 1034, 995, 895, and
850 cm1 were present in SR; peaks at 1164, 1111, 1088,
1049, 995, 903, and 850 cm1 appeared in LSMR; peaks
at 1157, 1126, 1095, 1065, 1034, 903, and 841 cm1 were
found in HSMR; and peaks at 1157, 1103, 1034, 903,
and 841 cm1 were observed in DR. Moreover, the
absorbances of almost all of the peaks were significantly
higher in LSMR and HSMR than in SR and DR, except
for the 1034 cm1 peak, assigned to b(1-4)-glucans
(Galichet et al., 2001), which was significantly higher
in DR.
The pectin deposition in walls of the bundle tissues of
the four reed ecotypes was visualized by staining with
ruthenium red and hydroxylamine-FeCl2 (Fig. 5).
Hydroxylamine-FeCl2 weakly stained the walls of
phloem and bundle sheath cells, but intensely stained
the walls of xylem and sclerenchyma cells, especially in
SR and DR (Fig. 5A, D, G, J). Ruthenium red stained
the walls of phloem tissues, with more intense staining
(deeper red) appearing in the two salt reed ecotypes than
in SR and DR, and the dye weakly stained the walls of
other tissues in the vascular bundles (Fig. 5B, E, H, K).
Chemical de-esterification with 0.1 M Na2CO3 resulted
in an increase in the overall staining with ruthenium red;
all the bundle cell walls were stained with ruthenium red
following sodium carbonate treatment (Fig. 5C, F, I, L).
Histochemical analysis was also conducted using
phloroglucin and Sudan red III to detect lignin and
suberin, respectively (Fig. 6). Phloroglucin stained the
walls of xylem and sclerenchyma cells in all of the reed
ecotypes, but only weakly stained the walls of bundle
sheath cells and phloem (Fig. 6A–D). The staining was
more intense in SR and DR than in the salt reed
ecotypes. However, Sudan red III stained the xylem and
sclerenchyma cell walls nacarat in SR, saffron in the two
salt reed ecotypes (LSMR and HSMR), and yellow in
DR (Fig. 6E–H).
Discussion
As described previously, SR grows in brackish areas
of swamps, which are considered typical habitats for
reeds. However, due to the moisture and salt contents of
the root zone soils in the habitats of the three terrestrial
reed ecotypes, LSMR and HSMR were subjected to
long-term saline stress and DR was subjected to longterm drought stress (Table 1). Considering the accumulations of osmoregulatory solutes (Table 1) and the
higher activities of leaf and chloroplast-localized antioxidant enzymes (our unpublished data) in the terrestrial reed ecotypes, the relatively low water content and
water potential of the leaves indicates that the three
terrestrial reed ecotypes have been subjected to different
degrees of water deficit stress. This point was also
demonstrated in some previous studies (Chen et al.,
2003; Wang et al., 1998; Zheng et al., 2000; Zhu et al.,
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Fig. 3. FE-SEM (field emission scanning electron microscopy) of the detached vascular bundles of four reed ecotypes growing in the
different habitats, showing the outerface differences of the tangential walls of bundle sheath cells in four reed ecotypes. Pt, pit field;
Pl, plasmodesma. (A)–(C) Swamp reed (SR). (D)–(F) Light salt meadow reed (LSMR). (G)–(I) Heavy salt meadow reed (HSMR).
(J)–(L) Dune reed (DR). (A), (D), (G), and (J) show different frequencies of the pit field on the outerface tangential walls of four
reed ecotypes. (B), (E), (H), and (K) show the diverse distribution of plasmodesma in the pit fields between the ecotypes. (C), (F), (I),
and (L) show the alterable architecture of the bundle sheath cell walls in which the alignment of microfibrils in bundle sheath cell
walls is looser in the three terrestrial reed ecotypes than in SR.
2001, 2003). Consequently, these four ecotypes are a
suitable system for studies of plant mechanisms of
adaptation to natural long-term environmental stresses
such as drought and salinity. However, the anatomical
and chemical characteristics of foliar vascular bundles in
these reed ecotypes had not been investigated. This work
is the first combining structural and chemical information to provide a clearer picture of the role of the foliar
vascular bundle system in the adaptation of reed
ecotypes to their long-term adverse habitats.
Anatomical characteristics of foliar vascular bundles
Vascular systems are responsible for the transport of
water and solutes in plants. However, because of its
special anatomical structure, the vascular system also
functions as an apoplastic barrier for plants in the
acquisition of water and solutes (Hose et al., 2001;
Steudle, 2000). Although some studies have suggested
that changes in the vascular architecture, such as
modifications to the wall architecture, ion composition,
and protein expression, are involved in the resistance of
plants to environmental stresses (Child et al., 2003;
Cholewa and Griffith, 2004; Engloner et al., 2003;
Equiza and Tognetti, 2002; Orians and Jones, 2001;
Saijo et al., 2001; Zwieniecki et al., 2003), information is
lacking on the functions of anatomical and chemical
modifications of foliar vascular bundles in plant
resistance to these stresses.
He and Zhang (2003) reported that in the shrub Sabina
vulgaris, which grows in the semi-arid Mu Us Sandland
of China, the size of the vascular bundle is strongly
negatively correlated with the soil water content,
whereas net photosynthesis, night respiration, and
stomatal conductance are highly positively correlated
with soil water content. Ogle (2003) found that photosynthetic carbon reduction is primarily restricted to
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Table 4.
Wave
numbers
(cm1)
563
FTIR spectra and their assignment of cell walls of the different vascular bundle tissues in four reed ecotypes
Bundle sheath cells
SR
LR
HR
Xylem
DR
1728
Pholem
Assignment
SR
LR
HR
DR
SR
LR
HR
DR
+
+
+
+
+
+
+
+
1720
+
+
+
+
1635
1512
1458
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1450
+
+
1427
+
+
+
+
+
+
+
+
+
+
+
+
1373
+
+
+
+
+
+
+
+
+
+
+
+
1319
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1250
+
1242
+
+
+
+
+
1157
+
+
+
+
+
+
1126
1111
1103
1095
1088
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1065
+
+
+
1049
1041
1034
1026
1018
1003
995
903
895
850
841
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Carboxylic ester or acid groups (esterlinked compounds)
Carboxylic ester or acid groups (esterlinked compounds)
Amide-stretching bands of proteins
Aromatic skeletal in lignin
C-H deformation in lignin and
carbohydrates (suberin)
C-H deformation in lignin and
carbohydrates (suberin)
C-H deformation in lignin and
carbohydrates (suberin)
C-H deformation in cellulose and
hemicellulose
C-H vibration in cellulose and C1-O
vibration in syringyl derivatives
Syringyl ring and C-O-stretch in
lignin and xylan
Syringyl ring and C–O-stretch in
lignin and xylan
C–O–C vibration in cellulose and
hemicellulose
Aromatic skeletal and C–O stretching
b(1–3)-glucans
b(1–3)-glucans
Acetylglucomannan
C–O and C–C stretching and C–O–H
deformations
C–O and C–C stretching and C–O–H
deformations
C–O stretch in cellulose and
hemicellulose
b(1–3)-glucans
b(1–4)-glucans
b(1–4)-glucans
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
b(1-6)-glucans
Mannans
C–H deformation in b-glucans
D-galactose-4-sulfate
D-galactose-4-sulfate
SR, swamp reed; LR, light salt meadow reed; HR, heavy salt meadow reed; DR, dune reed.
specialized bundle sheath cells, and suggested that a
smaller interveinal distance would increase the number of
veins and thus increase the density of bundle sheath cells,
thereby potentially enhancing photon capture. In our
study, the sizes and percentages of the different sectors
within the bundles differed significantly, although the
morphologies of the leaf vascular bundles of the four reed
ecotypes were quite similar (Fig. 1, Table 2). The
terrestrial reed ecotypes LSMR, HSMR, and DR
exhibited higher percentages of bundle sheath cell areas
and higher frequencies of leaf veins than SR (Tables 2
and 3), implying that more water and solutes are
exchanged and photosynthesis is more efficient in reeds
that are normally under saline and drought stresses.
The xylem and phloem are responsible for the uptake
of ions and water and the transport of assimilates,
respectively, and the vascular bundle functions as an
apoplastic barrier in the distribution of water and
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0.25
LSMR
SR
HSMR
0.20
Absorbance
Anatomical properties of the cell walls of vascular
bundle sheath
1034
A
0.15
1635
0.10
1720
1512
0.05
1157
1427
1373 1242
1319
903
0.00
1.40
1018
1049
1242 1088
B
Absorbance
1.20
1.00
1728
1427 1250 1157
1373
1635
1041
1319
0.80
1512
0.60
903
0.40
0.20
0.00
1.80
1.60
Absorbance
1.40
1.20
C
1111
1157
1250
1373
1319
1427
1728 1635
1034
995
1.00
1512
0.80
0.60
903
850
0.40
0.20
0.00
–0.20
1800
1600
1400
1200
1000
800
Wavenumbers (cm–1)
Fig. 4. FTIR spectra of cell walls of the different vascular
bundle tissues in four reed ecotypes growing in the different
habitats. Values are the means of three replicate measurements
for each ecotypes. Standard errors were less than 5%. (A)
Vascular bundle sheath cells. (B) Xylem. (C) Phloem. Swamp
reed (SR); light salt meadow reed (LSMR); heavy salt meadow
reed (HSMR); dune reed (DR).
solutes (Cholewa and Griffith, 2004; Hose et al., 2001;
Steudle and Frensch, 1996; Steudle, 2000). In this study,
the three terrestrial reed ecotypes exhibited a lower
percentage of xylem and phloem and a lower xylem/
phloem ratio than SR did (Table 2), indicating that
different mechanisms for water and solute distribution
exist in the terrestrial ecotypes, possibly leading to
higher stress tolerance in these ecotypes. Changes in the
areas of xylem and phloem and their ratios have also
been reported in wheat leaves at different temperatures
(Equiza and Tognetti, 2002).
The foliar vascular bundle sheath has been subjected
to much study because of its essential roles in photosynthesis and the exchange of substances between
mesophyll tissues and the vascular system. Based on
the anatomy, ultrastructure, and physiology of the
bundle sheath cells (Zheng et al., 1999, 2000, 2002),
reeds inhabiting swamp environments have been classified as C3-like ecotypes, those found in saline environments as C3-like C3–C4 intermediates, and those in
dune-desert environments as C4-like C3–C4 intermediates. Modifications to the wall architecture are involved
in the responses of plants to environmental stresses
(Engloner et al., 2003; Orians and Jones, 2001; Wang et
al., 1997). Bernards and Lewis (1998) suggested that the
autofluorescence of cell walls is due to aromatic suberin
polymers, which function as a barrier to water and
solutes (Hartmann et al., 2002). Using FE-SEM,
Sugimoto et al. (2000) showed that the orientation and
alignment of cortical microtubules and cellulose microfibrils in the cell walls of Arabidopsis roots differ at
different developmental stages. Comparable differences
were also observed between an Arabidopsis cellulosedeficient mutant and wild-type plants grown at high
temperatures (Sugimoto et al., 2001). The bright
autofluorescence of bundle sheath cell walls in the four
reed ecotypes (Fig. 2) implies that aromatic suberin is
also present in this type of cell wall, especially in the DR
ecotype. The demonstration by FE-SEM that the
alignment of microfibrils in bundle sheath cell walls is
looser in the three terrestrial reed ecotypes than in SR
(Fig. 3) suggests that the alterable architecture of the
bundle sheath cell walls might be involved in the
adaptation of reed plants to different habitats, such as
drought and salinity. This hypothesis should be tested in
the further experimentation.
We observed a higher frequency of pit fields in
the outerface tangential walls of vascular bundle
sheath cells in the three terrestrial reed ecotypes
(Table 3, Fig. 3), suggesting that in these plants, the
exchange of photosynthetic metabolites and assimilates
between the mesophyll cells and bundle sheath cells is
more efficient. Differences in the distribution of
plasmodesmata in the pit fields of the outerface
tangential walls of the vascular bundle sheath cells were
also observed in the four reed ecotypes (Fig. 3). The
frequency of plasmodesmata in maize foliar vascular
bundles has been reported to change under chilling
stress (Sowinski et al., 2003); therefore, the different
distributions of plasmodesmata in the pit fields may be
another adaptive response of the reed ecotypes to their
different habitats, although the ecophysiological significance of this phenomenon has not yet been clarified
in detail.
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565
Fig. 5. Cross sections of vascular bundle tissues in four reed ecotypes stained with ruthenium red (indicating unesterified pectins)
and hydroxylamine-FeCl2 (indicating highly methyl-esterified pectins). Notice the differing deposition of pectins in different
vascular bundle tissues between the four reed ecotypes. Bars ¼ 20 mm. Ph, phloem; Xy, xylem; BSC, bundle sheath cell; Sc,
sclerenchyma. (A)–(C) Swamp reed (SR). (D)–(F) Light salt meadow reed (LSMR). (G)–(I) Heavy salt meadow reed (HSMR).
(J)–(L) une reed (DR). (A), (D), (G), (J) Hydroxylamine-FeCl2 staining. (B), (E) (H), (K) Ruthenium red staining. (C), (F), (I), (L)
De-esterified with 0.1 M Na2CO3 before ruthenium red staining. In the printed black-and-white version of the figure, the red stained
part can be identified as the distinctly darker part of the sclerenchyma or phloem sector.
Chemical characteristics of the cell walls of foliar
vascular bundles
The chemical nature of cell walls has a great influence
on their mechanical function (Carpita and Gibeaut, 1993;
Chen et al., 1998; Hori and Sugiyama, 2003; Marga et al.,
2003). FTIR microspectroscopy is an extremely rapid,
non-invasive vibrational spectroscopic method that can
quantitatively detect a range of functional groups,
including carboxylic esters, phenolic esters, protein
amides, and carboxylic acids, providing a complex
fingerprint of carbohydrate constituents and their orga-
nization. Therefore, the analysis of FTIR spectra is a
robust method to identify a broad range of structural and
architectural alterations in cell walls during developmental regulation and environmental response or upon
genetic modification (Chen et al., 1998). In this study,
the FTIR spectra of the cell walls obviously differed not
only between the same tissues in the different ecotypes,
but also between different tissues within individual
ecotypes (Table 4, Fig. 4). This suggests that the
characteristics of the cell walls of these tissues developed
when the reed ecotypes adapted to their long-term
habitats of saline and drought-prone dunes.
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In bundle sheath cell walls, the relatively high
absorbance of almost all of the peaks in the FTIR
spectra of the three terrestrial reed ecotypes LSMR,
HSMR, and DR, especially the peaks at 1635, 1242, and
1034 cm1, showed that many types of wall components,
including wall-bound proteins, lignin and suberin, and
cellulose, might be upregulated when the habitats of
reed plants change from water to drought-prone dunes
or salinity. The FTIR spectral patterns of xylem cell
walls, in which the absorbance of most peaks was higher
in the salt reed ecotypes LSMR and HSMR and lower in
DR than in SR, indicated that different alterations in
the cell wall structure and architecture are involved in
the responses of the xylem to salinity and drought.
Differences in pectins (the peak at 1728 cm1), proteins
(the peak at 1635 cm1), lignin and suberin (the peaks at
1512, 1427, and 1250 cm1), and polysaccharides (the
peaks at 1200–800 cm1) were observed. Since the xylem
is responsible for water transport (Steudle, 2000), the
differences could result in different capabilities or
mechanisms of water transport in the xylem tissues of
the four reed ecotypes. In contrast, in phloem, which is
responsible for the transport of assimilates (Cholewa
and Griffith, 2004), markedly higher absorbance of the
FTIR peaks and more complex spectral patterns were
observed in the two salt reed ecotypes than in SR and
DR, suggesting that modifications in the wall plasticity
of the reeds related to salt response or tolerance rather
than drought response. It is evident that the chemical
compositions and modifications of the cell walls in the
different sectors within a vascular bundle and the
differences in the different ecotypes might be essential
for the salt and drought tolerance of these reeds. The
changed deposition of proteins and other key components in the vascular bundle cell walls may be related to
the adaptation of these ecotypes to long-term extreme
habitats that feature drought or salinity. Greater
amounts of wall-bound proteins were also observed in
HSMR and DR using another approach in which the
proteins were extracted from isolated wall materials
using salt (data not shown).
Fig. 6. Cross sections of vascular bundle tissues in four reed
ecotypes stained with phloroglucin and Sudan red III for
detecting the lignin and suberin, respectively. Notice the
different compositions of lignin and suberin between the
different vascular bundle tissues of four reed ecotypes growing
in the different habitats. Bars ¼ 50 mm. Ph, phloem; Xy, xylem;
BSC, bundle sheath cell; Sc, sclerenchyma. (A), (E) Swamp
reed (SR). (B), (F) Light salt meadow reed (LSMR). (C), (G)
Heavy salt meadow reed (HSMR). (D), (H) Dune reed (DR).
(A)–(D) Phloroglucin staining. (E)–(H) Sudan red III staining.
In the printed black-and-white version of the figure, the red
stained part can be identified as the distinctly darker part of
the xylem near the adaxial surface of the leaf.
Histochemical characteristics of the cell walls of
foliar vascular bundles
Pectin is an important cell wall component and plays
a major role in the elastic behaviors of plant cell walls
(Jarvis, 1992; Marga et al., 2003). A more elastic pectin
network permits more rapid adaptation to mechanical
stress and therefore alleviates stress gradients across the
wall (Jarvis, 1992; Marga et al., 2003; Wilson et al.,
2000). As described by Sabba and Lulai (2002), pectins
in plant cell walls can be stained with ruthenium red and
hydroxylamine-FeCl2, which stain unesterified (acid)
pectin and highly methyl-esterified pectins, respectively.
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K.-M. Chen et al. / Flora 201 (2006) 555–569
Unesterified pectin imparts rigidity to the cell wall by
cross-linking via calcium bridges to form calcium
pectate, whereas esterified pectin is responsible for the
elastic behavior of the cell walls (Jarvis, 1984; Thakur et
al., 1997). In this study, we found that the levels of
unesterified pectin were highest in the phloem cell walls,
especially in the salt reed ecotypes, whereas the greatest
amounts of highly methyl-esterified pectin were present
in the xylem and sclerenchyma cell walls, especially in
SR and DR. However, chemical de-esterification with
sodium carbonate resulted in an increase in the overall
staining with ruthenium red, but almost no differences
in staining between the different bundle tissues (Fig. 5).
This indicated that pectins were present in all the bundle
tissue cell walls, such as the bundle sheath cell walls,
although the walls stained with neither ruthenium red
nor hydroxylamine-FeCl2. These results suggest that the
differing deposition of pectins has an ecophysiological
significance for reed plants in different habitats,
although the detailed mechanisms are unknown.
Further studies will be necessary to understand the
predominance of unesterified pectins in phloem cell
walls and methyl-esterified pectins in xylem cell walls.
Lignin and suberin are two other important components of plant cell walls that have been intensely
investigated in root cells because of their function as
apoplastic barriers (Bernards, 2002; Hose et al., 2001;
Steudle, 2000; Zeier et al., 1999). Wang et al. (1997) used
phloroglucinol staining to show that the lignification of
cell walls of Atriplex prostrata is reduced under saline
growth conditions. Zeier et al. (1999) observed the
distributions of suberin in the cell walls in Pisum sativum
L., Cicer arietinum L., and Ricinus communis L. with
Sudan red III staining, and the results corresponded
strongly with the results of chemical degradation
analyzed by chromatography. In our study, we used
phloroglucin and Sudan red III staining to demonstrate
greater lignification and suberization of the walls of
xylem and sclerenchyma cells in reeds, with the
exception that less lignification occurred in xylem and
sclerenchyma cell walls of salt-habitated ecotypes than
in SR or DR. Staining with Sudan red III also revealed
differences in the suberization of the cell walls of
different vascular bundle tissues of each of the reed
ecotypes (Fig. 6). Considering the importance of lignin
and suberin in establishing an effective apoplastic
transport barrier, the high lignification and suberization
of the vascular cell walls, especially those of xylem and
sclerenchyma, may be an important finding. These
results suggest that, by influencing the apoplastic
transport of water and assimilates, variations in the
deposition of components such as lignin and suberin in
different vascular bundle cell walls may be involved in
the adaptation of plants to various long-term habitats.
This conclusion is also supported by the results of the
FTIR analysis. An essential role of chemical deposition
567
in plant resistance responses has also been reported in
root cell walls (Hartmann et al., 2002; Hose et al., 2001;
Soukup et al., 2002; Zeier et al., 1999; Steudle, 2000).
Conclusions
The findings described in this paper suggest that the
anatomical and chemical characteristics of the foliar
vascular bundles are different when the common reed, a
hydrophytic species, adapted in the long term to
different habitats with severe stress factors such as
salinity or drought. These differences, including the area
of the xylem and phloem and their ratios, the chemical
composition and modifications in the cell walls of
different vascular bundle tissues, and the deposition of
major components of cell walls such as pectins, lignin,
and suberin, between the different vascular bundle
tissues, could contribute to the high resistance of reeds
to salinity and drought by influencing the apoplastic
transport of water and assimilates.
Acknowledgements
This research was supported by the Nature Science
Foundation of China (Nos. 30470274 and 30270238)
and by the National Science Fund of China for
Distinguished Young Scholars (No. 30225005). We
thank Dr. Arthur Benson and other anonymous
botanists for their critical comments on the first draft
of this manuscript. The authors thank Dr. Shi-fu Wen
for his technical assistance with the FTIR microspectroscopy at the Department of Chemistry, Peking University, China.
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