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Anatomical and FTIR analyses of phloem and xylem of Tetracentron sinense

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DOI: 10.1007/s11676-017-0425-4

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J. For. Res.
DOI 10.1007/s11676-017-0425-4
ORIGINAL PAPER
Anatomical and FTIR analyses of phloem and xylem
of Tetracentron sinense
Jiangtao Shi1 • Feng Wang1 • Yaoli Zhang1
Received: 4 November 2015 / Accepted: 5 November 2016
Ó Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2017
Abstract The fast growth of Tetracentron sinense is a
potential valuable timber resource, but whether its anatomy
and chemical components are suitable for timber is
unknown. We used light microscopy and SEM to examine
the anatomical structure and FITR to measure the chemical
components of the phloem and xylem of this tree. Radial
variations in growth ring width and tracheid dimensions
were also evaluated. The sieve tube, phloem parenchyma
cell and sclereids clusters were the main cells in phloem,
and the tracheid was the fundamental cell in xylem. An
unusual tracheid type, fiber-tracheids or vessel-liked ele-
ments was visible. Wood rays nonstoried, uniseriate and
multiseriate, including heterogeneous II, occasionally I,
and usually 3–6 cells wide. The mean growth-ring width
was 2.53 ± 0.46 mm, and the percentage of late wood was
over 60%. For radial variation, growth-ring width increased
at an early growth stage, and reached the largest increment
during years 11–15, then decreased. The maximum growth-
ring width was 5.313 mm. During late growth
(60–85 years), trees also maintained a high radial growth
increment. Radial variation in the percentage of late wood
Project funding: This work was financially supported by the Youth
Science and Technology Innovation Fund of NJFU (CXL2015018)
and the Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
The online version is available at http://www.springerlink.com
Corresponding editor: Yu Lei.
& Jiangtao Shi
shijt@njfu.edu.cn
1 suggested that Tetracentron is a vessel tree species. In
Collage of Material Science and Engineering, Nanjing
Forestry University, Nanjing 210037, People’s Republic of
China
was uniform, about 50–70%, throughout the growth years.
Growth patterns in the length and width of early and late
wood were similar as the trees aged. From the FTIR results,
the chemical components differed significantly between
xylem and phloem, hemicellulose in particular was higher
in the xylem than in the phloem, where it was apparently
absent. All of these suggest that the composition of phloem
in T. sinense is very similar to that of hardwood, and it has
higher growth ratio and uniform wood properties.
Keywords Phloem  Radial variation  Tracheid 
Tetracentron sinense  Xylem
Introduction
Originated during the Cretaceous or early Triassic and
distributed in East Asia countries such as China, Nepal and
India, Tetracentron sinense Oliv. is a broad-leaf tree spe-
cies in terms of its leaf and tree morphology. Over
100 years ago, however, the genus Tetracentron was
described as lacking vessel elements in the secondary
xylem (Harms 1897; Thompson and Bailey 1916; Li and
Jin 1989), which resulted in the classification of its wood as
a softwood. However, other characteristics of T. sinense
wood, such as its late wood having more ray cells and
bordered pits on the tracheid cell walls, are just like general
hardwood. In contrast, Suzuki et al. (1991) reported the
presence of ‘‘unusual tracheids’’ in mature wood and
branch wood of Tetracentron, similar to vessel elements in
hardwood. Recently, Ren et al. (2007) and Li et al. (2011)
defined these particular cells as vessel-like elements and
addition, its chemical composition has elements shared by
softwoods and hardwoods. For example, the content of
123

polymannose and polyxylose is similar to that in hardwood,
but polygalactose and polyarabinose are similar to that in
softwood (Fang et al. 1992). Furthermore, it also contains
tiny amounts of polyrhamnose as found in birch and beech
wood, whereas it has more guaiacyl monomers, which is
common in softwood. Thus, the categorization of T.
sinense wood is controversial.
Although the structure and chemistry of secondary
xylem has been the primary focus for such categorizations,
phloem also offers vital clues. Phloem, particularly func-
tional secondary phloem, plays a key role in tree physiol-
ogy. The appearance of the bark of T. sinense is also
similar to hardwood bark. As far as we know, however,
details on its anatomical structure and chemical compo-
nents have never been reported. Herein, we hypothesized
that (1) the cell structure and chemical group in T. sinense
phloem will show a marked hardwood tendency; (2) the
annual growth in T. sinense wood will be greater than for
coniferous wood in general and its wood characteristics
more homogeneous than other deciduous tree wood.
Materials and methods
Plant materials and location
Three wild-growing, apparently healthy T. sinense were
collected from Qinling Mountains (N33°490 22.0000 ,
E107°360 47.2900 ) Huangbaiyuan, Taibai Town, Shaanxi
Province, in northwestern China. All samples contained
xylem, cambium and secondary phloem and were har-
vested from stems at the breast height and immersed
immediately into formalin (5%)–acetic acid (5%)–alcohol
(90%) (FAA). Then, they were taken back to the lab and
stored at -20 °C.
Light microscopy
Samples (approximately size: 20 mm 9 20 mm 9 8 mm)
were fixed in 3–4% formalin (35% formaldehyde in water)
at least 24 h, then they were cut into small blocks (ap-
proximately size: 4 mm 9 5 mm 9 5 mm) and subjected
to a vacuum until they sank. After an ethanol dehydration
series (50, 70, 85, 95 and 100%, each for 24 h), the small
blocks were infiltrated with a series of collodion (Wako
Pure Chemical Industries, Ltd.) (2, 4, 6, 8 and 10%, each
for 48 h) at 50 °C. Finally, sections (15–20 lm thickness)
were cut with semi-auto microtome, placed on slides and
double-stained with 1% w/v safranin in water (30 min) and
0.5% (w/v) fast green in absolute ethyl alcohol (for 2 min).
After the slides had air-dried, the sections were observed
by 10 9 and 40 9 with an Olympus BX51 microscope.
123
J. Shi et al.
Scanning electron microscopy
Small blocks of primary sample were selected for SEM.
Their surface was smoothed before vacuum freezing drying
at 65 °C for 24 h. Then the samples were Gold sprayed
immediately and observed with a scanning electron
microscope (Hitachi TM 1000).
Cell maceration
Xylem and phloem were separated and cut into small
pieces (3 mm 9 3 mm 9 10 mm). All samples were
placed in glass tube with water and water bathed in 90 °C
for 2 h. Next, the original solution replaced with 1:1 (v/v)
acetic acid: hydrogen peroxide and water bathed in 75 °C
until wood tuned white. Lastly, the tissues were washed
with distilled water until pH 7.0, then dipped briefly into
the 1% safranin solution. A slide was then dipped into the
mixture, and the samples were observed by 40 9 and
100 9 with an Olympus BX51.
Image analysis and data processing
The size of macerated cells and sclereid clusters was
measured using Image Pro Plus 6.0 (Olympus, Japan). Fifty
cells were randomly chosen from each tree sample and
averaged. Sclereid cell mass size and tissue proportion
were averaged from five sections. All data were analyzed
by Excel 2007 (Microsoft, Redmond, WA, USA) and
plotted by Origin 8.0 (Originlab, USA).
Results
Secondary structure and cell morphology
Secondary phloem of T. sinense consists of sieve tube,
phloem parenchyma cell, and sclereid clusters alternatively
in the axial direction and phloem rays in the radial direc-
tion. Sieve tube and phloem parenchyma cells were diffi-
cult to distinguish in cross sections with the light
microscope (LM), but distinguishable with SEM. Sieve
plates were found in cross sections and tangential sections,
which proved that these cells were sieve tubes rather than
not sieve cells (Fig. 1). Their length and width was
554.36 ± 44.54 lm (n = 50) and 43.51 ± 2.58 lm
(n = 50), respectively. Phloem parenchyma cells were in
strands (4–5 cells) in longitudinal directions and round in
cross section. Average length and width of parenchyma
cells was 631.76 ± 116.35 lm and 37.18 ± 1.11 lm,
respectively. Sclereids were easily distinguished with the
LM after double-staining with safranin and fast green
(Fig. 1) with the proportion of 32.9 ± 2.6% in secondary
Anatomical and FTIR analyses of phloem and xylem of Tetracentron sinense
Fig. 1 Cells in phloem. LM of cross sections (a); SEM (b–d);
macerated cell (e). Ca: cambium (red line with two arrows in a), P:
parenchymal cell (longer red line in a, red line in b and shorter red
phloem. They were much more lignified compared with
sieve tubes and phloem parenchyma cells; thus, they were
stained red. With LM, some sclereids showed in purple
(Fig. 1a), which appeared ‘‘white aggregate’’ with SEM
(Fig. 1b, c). Width of sclereid clusters was
0.66 ± 0.09 mm in tangential section.
The xylem of T. sinense consists of tracheids, unusual
tracheids in the axial and rays in the radial direction. In
cross section, secondary xylem was obviously distin-
guishable in early wood and late wood. Elongated bordered
pits in scalariform arrangement were present in the cell
wall of tracheids in early wood while circular bordered pits
occurred in cell wall of trachieds in late wood (Fig. 2).
Tracheid size varied between early wood and late wood.
Tracheid mean length was 2852.57 ± 121.97 lm, mean
width was 44.69 ± 2.65 lm and length to width ratio (L/
W) in early wood was 67.34 ± 4.1, while tracheids in late
wood had the mean length of 3063.66 ± 127.09 lm, the
mean width of 39.14 ± 1.85 lm and mean L/W of
81.21 ± 2.14. Unusual trachieds, vessel-liked elements or
fiber-trachieds, were reported for T. sinense (Suzuki et al.
1991) and are similar to the tracheids in some hardwoods
such as Celtis occidentalis, Phellodendron amurense, and
Sophora arizonica (Wheeler et al. 1989). Their average
length was 260.32 ± 39.13 lm (much shorter than normal
tracheids), and average width was 51.87 ± 11.17 lm.
Rays were nonstoried, uniseriate and multiseriate,
line in c), Ph: phloem, PR: phloem ray, S: sieve (shorter red line in a,
red line in b and longer red line in c), Scl: sclereid, Xy: xylem
including heterogeneous type II, occasionally type I, and
width was usually 3–6 cells.
Radial variation in wood properties
Growth-ring width and percentage of late wood
The mean growth-ring width of T. sinense was
2.53 ± 0.46 mm, of which 60.1 ± 1.6% was late wood
(Table 1). The growth-ring width radial variable is shown in
Fig. 3a. For the first 20 growth years, tree-ring width
increased with increasing age and reached the maximum
increment during years 11–15 (Tree 2 reached 3.002 mm in
14th year), then decreased. Growth-ring width then increased
during the next growth 20–50 years; maximum increment
was 5.313 mm. For growth during the 60th–85th years, trees
also maintained a high radial growth increment. After
90 years, the increment decreased but was consistent with the
first growth years, which may be the result of high-efficiency
photosynthesis in the deciduous tree crown and suggests that
T. sinense is a potential fast-growth timber resource.
Generally, a higher percentage of late wood is regarded
as increments from more cell walls and conferring better
strength properties to the wood (Fig. 3b). The radial vari-
ation in the late wood percentage in T. sinense was rela-
tively stable, about 50–70%, over all growth years. The
radial pattern is similar to that of growth ring width. Late
123
 J. Shi et al.

Fig. 2 Cells in xylem. LM of cross sections (a, b); radial section (d); tangential section (e). SEM of radial (c) and tangential (g) section;
macerated cell (f). In a, c E: early wood, L: later wood; U: unusual tracheid

Table 1 Means ± SD of variables for growth rings and tracheids

Tree Growth-ring Later wood Early wood Later wood
width (mm) percent (%)
Length (lm) Width (lm) Length to Length (lm) Width (lm) Length to
width ratio width ratio

Tree 1 2.64 ± 1.15 58.3 ± 8.6 2968.76 ± 486 47.05 ± 8.34 65.24 ± 9.92 3206.55 ± 364 41.10 ± 5.05 80.07 ± 5.64
Tree 2 2.10 ± 1.26 61.3 ± 10.1 2863.41 ± 412 41.81 ± 9.93 72.1 5 ± 12.41 3021.19 ± 409 37.44 ± 7.35 83.68 ± 10.51
Tree 3 3.02 ± 1.34 60.7 ± 5.6 2725.54 ± 436 45.22 ± 7.96 64.61 ± 14.30 2963.23 ± 464 38.87 ± 3.35 79.86 ± 9.71
Mean 2.53 ± 0.46 60.1 ± 1.6 2852.56 ± 121 44.69 ± 2.65 67.34 ± 4.19 3063.66 ± 127 39.14 ± 1.85 81.20 ± 2.12

Fig. 3 Radial variation in

a growth ring width and
b latewood percentage

wood percentage depends on the number of cells and the
tracheid width in late wood. Growth of many tree species
can differ dramatically among years, a phenomenon that is
strongly linked to temperature, rainfall and other environ-
mental factors. Because the late wood percent was over
50%, T. sinense wood is likely harder and has greater
bending strength than most softwood.
Tracheid length and width
Because of the importance of tracheid size in wood quality
and wood products characteristics, macerations were
accurately measured. Figure 4 shows the relation of radial
tracheid length and width to growth year. The length and
width in both early wood and late wood followed similar

123
Anatomical and FTIR analyses of phloem and xylem of Tetracentron sinense
patterns and increased with growth years. Early in growth,
tracheid length and width increased rapidly in early wood,
mean length went from 1102.78 to 2902.07 lm and mean
width from 20.71 to 59.65 lm. Then they slowed slightly,
then continued to increase with growth year, remaining
similar after growth year 45. Mean length increased from
2211.04 to 3786.71 lm, mean width from 30.46 to
52.86 lm.
The mean ratio of length and width was closely related
to wood physical properties. The combined capacity of
tracheids was better while a larger ratio would reflect
greater cell wall strength and tree stiffness, which would
also significantly improve the quality of paper and wood-
based panels. The ratio of length and width was
67.34 ± 4.19 in early wood and 81.20 ± 2.14 in late wood
(Table 1). The radial length and width of tracheids
increased during the first growth years and was similar to
the changes in tracheid length and width (Fig. 4).
FTIR
FTIR is an effective, widely used method for chemical
analysis of wood, which consists of cellulose, hemicellu-
lose, lignin and other multitudinous extractives, such as
polyphenols, carboxylic acid and lipids and so forth. All of
these biomolecules have various functional groups, which
present conspicuous infrared spectrum characteristics.
Sometimes the intensity of the band can be used for semi-
quantification of the wood chemicals. The FTIR spectrum
of T. sinense xylem differed significantly from that of the
secondary phloem (Fig. 5). Xylem and secondary phloem
both had a strong band at 3423 cm-1 and a weak band at
Fig. 4 Radial measures of
tracheid length and width over
time
2930 cm-1, attributed, respectively, to O–H stretching
vibration and C–H stretching vibration, although we cannot
attribute the source definitively because the O–H and C–H
stretching vibration rarely exist in the entire molecule. The
band at 1738 cm-1 for the xylem was assigned to uncon-
jugated C=O in xylans (hemicellulose) (Pandey 1999).
However, this band doesn’t appear in the secondary
phloem spectrum and indicates that hemicellulose content
may be rare in phloem. A well-defined narrow band at
1635 cm-1 was attributed to acid amide, which might be in
some nitrogen natural polymers such as proteins, alkaloids
and so on. In the spectra, the relative abundance of the
nitrogen polymer in secondary phloem was higher than that
in xylem. The band at 1510 cm-1 was assigned to the
benzene skeletal bending vibration in lignin and is obvi-
ously higher in the secondary phloem than in the xylem.
Another lignin-related band at 1227 cm-1, assigned as a
C-O stretching vibration in phenolic hydroxyl, is also dis-
tinct to the xylem and absent in the phloem. These suggest
that the secondary phloem has less lignin than in the xylem.
Discussion and conclusion
In this study, characteristics of secondary phloem and
xylem of T. sinense wood were observed and measured and
their chemical nature analyzed. Radial variation in annual
growth and percentage of late wood over time was inves-
tigated. The tracheid shape on cross section is rectangle or
irregular in T. sinense and hexagonal or polygonal in other
softwood. Richter et al. (2004) defined tracheid length as
shorter (\3000 lm, Taxus baccata), medium
123

Fig. 5 FTIR of secondary xylem and phloem
(3000–5000 lm, Larix decidua) and longer ([5000 lm,
Araucaria angustifolia). Our results showed that in T.
sinense tracheid length in early wood and in late wood is
shorter than in normal softwood, but it has longer fiber
length than in hardwood, which was less B900 lm, med-
ium 900–1600 lm, longer C1600 lm (Wheeler et al.
1989). Moreover, there are special cells, reported by
Suzuki et al. (1991) as ‘‘unusual tracheids’’ that are like
vascular tracheids in hardwood. Recently, Sperry et al.
(2007) and Hudson et al. (2010) called them ‘‘primitive
vessels’’, and they may be related to the rates of photo-
synthesis and growth. This result is in accordance with the
result for Araceae, which are entirely vesselless (Carlquist
and Schneider 2014). These results indicate that T. sinense
is more advanced evolutionarily and annual growth amount
is greater than many other conifer tree species. It might
have potential as good resource for a fast-growth
plantation.
Growth ring width not only shows radial increments in
tree growth but indirectly shows changes in the environ-
ment. Also, it is positively correlated with wood density.
According to the above results, the radial annual incre-
ments and late wood percentage in T. sinense was more
larger than in many conifer trees, although it was relatively
smaller than in some plantation tree species, e.g., Metase-
quoia (Qi et al. 2014), Populus (Wang et al. 2006a), Eu-
calyptus (Luo et al. 2015), suggesting that T. sinense tree
has higher annual growth and wood with uniform proper-
ties. Tracheid shape is affected by cells in the cambium,
tree-age, and growth conditions and so on and varies at
different growth stages. The radial variation in tracheid
length of T. sinense can be considered as type of Panshin I,
which shows horizontal line after it changed from pith to
bark. It is similar to that in Calocedrus macrolepis wood
(Cui et al. 2012). On the other side, the radial variation in
fiber length and width in some broad-leaf trees, such as
Liriodendron chinense 9 L. tulipifera (Pan et al. 2005),
123
J. Shi et al.
Populus tremula (Yanchuk 1984), increased with growth
years and did not increase more at particular years.
Cell types and arrangement in phloem differ signifi-
cantly between softwood and hardwood. In softwood, sieve
cells, phloem parenchyma cells and phloem fibers are the
main cells types, with sclereids occasionally found in
specific species. The phloem in the hardwood consists of
sieve tubes, companion cells, parenchyma cells and phloem
fibers. Ryberg et al. (2007) found that sieve and fibers
tangentially arranged and alternating in Cycas revoluta. A
similar cell distribution was also reported for Sabina chi-
nensis (He et al. 1999). Our study showed sieve tubes,
phloem parenchyma cells, and sclereids in the phloem of T.
sinense. Sieve plates including the sieve area was clearly
shown in SEM (Fig. 1). Sclereids were distinct after
staining in the phloem. Wang et al. (2006b) extracted
compounds from T. sinense bark that had cytotoxic activity
against human leukaemia cells. All of these results indicate
that the phloem of T. sinense is very similar to the phloem
in hardwood and has some components that might be used
as medicine. Thus, T. sinense wood can be classified as
hardwood and should be an excellent raw commercial
material.
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