全 文 ：Received 19 Jul. 2003 Accepted 20 Oct. 2003
Supported by the Ministry of Science and Technology of China (2001BA506b04), the State Forestry Administration of China (98-04-08) and
the Guangdong Provincial Natural Science Foundation, China (021581).
* Author for correspondence. Tel: +86 (0)20 87032612; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 560-564
Photosynthetic Pathway of Three Rattan Species in South China
LI Rong-Sheng1*, YIN Guang-Tian1, XU Huang-Can1, YANG Jin-Chang1, ZHAO Xia1,
CHEN He-Ming2, YANG Hua1
(1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou 510520, China;
2. Forestry School, Fujian Agriculture and Forestry University, Nanping 353001, China)
Abstract: Incorporating C3 plants with C4 photosynthesis traits by genetic engineering are available for
improving plant productivity. Although rattans are a special and big group of plants including 13 genera and
ca. 600 species in the world and have been studied since the mid-nineteenth century, the knowledge of its
photosynthetic pathway is still lacking. Therefore , it is meaningful to determine the photosynthetic
pathway of rattans. It was reported that chlorophyll a/b value, ratio of stomata occurrence on upper to
lower epidermis, activities of phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase
(PPDK), and stable carbon isotope ratio in leaves of C3 plants were lower than those of C4 plants, thus this
study tested them with seedlings and adult plants of the following three rattan species, Daemonorops
margaritae (Hance) Becc., Calamus simplicifolius C. F. Wei and C. tetradactylus Hance occurring in South
China. The results showed that values of these indices were rather low, falling into the ranges of other C3
plants, so it was concluded that the three rattan species were C3 plants.
Key words: chlorophyll; stomata; phosphoenolpyruvate carboxylase (PEPC); pyruvate phosphate dikinase
(PPDK); stable carbon isotope ratio
Plants are mainly grouped into C3 and C4 plants by the
primary product of photosynthesis. Generally speaking, the
photosynthesis of C4 plants is higher than that of C3 plants
for differences in physiological, anatomical, and biochemi-
cal characteristics (Noggle and Fritz, 1976; Kramer and
Kozlowski, 1979). As the photosynthesis proces s is the
base for the formation and growth of all plant organs, there
is an alternative way to increase the production by improv-
ing photosynthesis in the fields of agriculture and forestry.
For C3 plants, attempts to enhance their photosynthetic
efficiency by incorporating C4 photosynthetic traits into
them were started since the 1960s, up to now, there have
been some success examples of such attempts on rice by
genetic engineering (Ku et al., 1999; Huang et al., 2002).
The ach ievements by Ku et al. (1999) and Huang et a l.
(2002) implied that it is possible to improve the production
of cultivated C3 plants other than rice by genetic engineer-
ing technique.
Rattans are climbing palms in tropical area of the Old
World, numbering 13 genera with 600 known species (Uhl
and Dransfield, 1987). They are well known as the source of
cane for the well-developed rat tan industry of curren tly
worth some 6.5 billion US dollars annually (Prebble, 1997;
Sunderland and Dransfield , 2002). As rattan resource in
wild has been decreasing rapidly during last decades for its
over-harvest and mass des truction of its habitat— tropi-
cal forests, rattan plantations are being paid more and more
attention and their areas are expanding consequently. Up
to date, cultivated technologies for improving the produc-
tion of rattan plantations have been well developed (Mohd
et al., 1992) and it is the right time to apply other ways such
as genetic improvement to promote the production of rat-
tan plantation (Rao and Rao, 1995). As stated above, incor-
porating cultivated rattan species with C4 photosynthetic
traits are the alterative way to improve the product ion of
rattan plantations if rattans are C3 plants. Although photo-
synthesis pathway of many plants is well determined, there
is a dearth of knowledge on the photosynthetic pathway of
rat tans throughout the world, not mention to rat tans in
China. That is to say, the photosynthetic pathway of rat-
tans should be iden tified prior to adopt ing any genetic
modification.
In a dd it io n to t he d if ferenc e in t he p rimary
photos ynthesis , there are differences in phys iological,
anatomical, and biochemical characteristics such as CO2
compensation point, net photosynthetic rate in full sunlight,
stomata density, ratio of chlorophyll a/b, activities of phos-
phoenolpyruvate carboxylase (PEPC), pyruvate phosphate
LI Rong-Sheng et al.: Photosynthetic Pathway of Three Rattan Species in South China 561
dikinase (PPDK) and stable carbon isotope ratio (Holden,
1973; Noggle and Fritz, 1976; Kramer and Kozlowski, 1979;
Hocking and Anderson, 1986; Lin et al., 1986; Ye et al.,
1993; 1998). These differences not only exp lain why C4
plants have a higher photosynthetic capacity than C3 plants,
but also can serve as indices for identifying the photosyn-
thesis pathway. Some of them are employed in this paper to
recognize the photosynthesis pathway of three rattan spe-
cies in South China.
1 Materials and Methods
1.1 Plant materials and site descriptions
One-year-old seedlings and adult plants of three rattan
species in South China, Calamus simplicifolius C. F. Wei,
Calamus tetradactylus Hance and Daemonorops
margaritae (Hance) Becc. were grown at Research Insti-
tute of Tropical Forestry, Chinese Academy of Forestry, an
inst itution located in northeas tern Guangzhou City. The
seedlings were sheltered with a 50% trans paren t plas tic
shelter. The adult plants were grown with indigenous broad-
trees being supporter in mid-1980s and have bloss omed
out s everal times. Guangzhou where tested rattan p lants
growing belongs to southern sub-tropical areas, the wet
season runs from April to September, the dry season from
October to next March. The mean temperature in a year is
21.8 ℃ and 28.4 ℃ in the hottest month (July), 13.3 ℃
in the coldest month (January). The mean precipitation in a
year is 1700 mm (Huang et al., 1994).
1.2 Sampling procedure and measurements
Measurements were carried out from 2002 to 2003. Two
to four leaflets at the middle of the second to fourth leaf on
the top of three rattan species plants were collected, then
mixed and selected randomly to be used. Three replications
were tested for all indices.
1.2.1 Chlorophyll measurements Sections detached
from the middle of the leaflets were cut into slips of about 1
cm in leng th and 0.1 cm in width. Naught point one g of
slips was put into a 25 mL vo lume-fixed bottle with the
extracting solution mixed by ethanol: acetone: distilled wa-
ter (4.5:4.5:1 by volume). The bottle was kept in the dark
until the leaf tissue became colorless fully, then the solu-
tion was filtered and set volume to 25 mL again. Chloro-
phyll absorption was measured by a spectrophotometer at
663 nm and 645 nm (PE UV/ VIS spectrophotometer, Lambda
25), and the concentration was calculated following Chen
and Chen (1984).
1.2.2 Measurements of stomata density Stomata occur-
rence on upper epidermis and beneath epidermis was mea-
sured in a way modified from the method described by Lin
et al. (1986). Two sections of leaf about 0.5 cm×0.5 cm in
area were excised from the middle of the same leaflet beside
the central vein. They were pasted on copper platform, one
with upper epidermis up, the other one with beneath epi-
dermis up. After open-air drying for 10 d, they were plated
by gold-silver alloy , then the occurrences o f stomata on
each epidermis were examined on a JSM-6330F field-emis-
sion scanning election microscope at the analyst center of
Zhongshan University. For each section, ten fields of vi-
sion were examined randomly and the mean of stomata oc-
currence of them was calculated.
1.2.3 Assay the activities of PEPC and PPDK Activities
of PEPC and PPDK were measured according to Sayer and
Kennedy (1979), and the method described by the Society
of Plant Physiology of Shanghai, China (1985), respectively.
Naught point five g of leaves material was excised from
leaflets and quickly ground in 5 mL extracting buffer with
liquid nitrogen . The buffer for extracting PEPC was 100
mmol/L Tris-HCl (pH 7.0) containing 1 mmol/L EDTA, 20
mmol/L d ith io threito l (DTT), 2% (W /V) Po lyv iny l
pyrrolidone-10 and 10% (W/V) glycerol. The one for ex-
tracting PPDK was 50 mmol/L Tris-HCl (pH 8.3) containing
5 mmol/L DTT and 2 mmol/L Mg2SO4. After complete
homogenization, the crude extract was centrifuged at 4 500g,
4 ℃ for 20 min, and the supernatant was used immediately
for assay of enzyme activities. Determination of all enzyme
activ ity was conducted by a PE UV/VIS s pectrophotom-
eter Lambda 25 at 340 nm.
The total volume of reaction mixture for PEPC measure-
ment was 3.0 mL, which contained 0.1 mL crude extract, 0.1
mol/L Tris-HCl (pH 8.0), 10 mmol/L NaHCO3, 10 mmol/L
MgCl2, 0.1 mg NADH, excessive malate dehydrogenase,
and overdose of phosphoenolypyrute (PEP). The change
of O.D. (DA) every 30 s at the wavelength of 340 nm was
recorded and the change of O.D. per minute was calculated.
For PPDK measurement the volume of the mixed solu-
tion for measurement was 3 mL, containing 0.1 mL crude
extract, 0.83 mmol/L NADH, 0.31 mL distilled water, 6 unit
lactate dehydrogenase (LDH), 0.06 mol/L Tris-HCl (pH 8.3),
6 mmol/L Mg2SO4, 6 mmol/L DTT, 6 mmol/L NH4Cl, 1.2
mmol/L PEP, and 0.5 mmol/L Na4P2O4·10H2O. Measure-
ment spect ropho tometrically was started by add ing
Na4P2O4·10H2 O s olu tion . The same mixtu re withou t
Na4P2O4·10H2O solution was used as blank and the room
temperature was set at 22 ℃. The change of O.D. (DA)
every 30 s at the wavelength of 340 nm was recorded and
the change of O.D. per minute was calculated.
Concentration of protein in the extract was determined
at 595 nm by the method described by Li and Jiao (1980).
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004562
BSA was used as protein standard.
1.2.4 Measurement of carbon isotope ratio Sampled
leaflets were oven-dried at 80 ℃ for 48 h and ground to fine
powder which passed through a 60-mesh screen per inch in
the laboratory . Six mg of the powder was sampled to be
au tomatic ass ayed by the Element Analyses cum the
DELTAPL USXL Isotop ic Mass-spect rometer (Finn igan
Mass -spect rometric Company) at the State Key Labora-
tory of Organic Chemistry in Guangzhou Global Chemistry
Institute. The mass-spectrometer was checked by the na-
t io na l fi r s t -c la s s s t an da r d ma te ria l mo no co l
(STDCHARCOAL), the gas calibrated by the international
material of NBS-22 was determined as reference. Values are
reported as d13C (‰ ) relat ive to Pee Dee Belemnite (PDB)
standard.
2 Results
2.1 Chlorophyll content
As shown in Tab le 1, although s eed lings and adu lt
plants o f the three rat tan s pecies had a high content of
chlorophyll a, b, and a+b, their ratios of chlorophyll a to b
were less than that of C4 plant like sugar cane, while similar
to that of such C3 plants as Cymbidium plants (Ye et al.,
1993; Ye et al., 1998). According to Holden (1973), the ratio
of chlorophyll a to b in C4 plants was higher than that of C3
plant, it could be known that the three rattan s pecies had
C3 plants trait regarding chlorophyll.
2.2 Stomata occurrence
As seen in Table 2, regardless of s eedlings or adu lt
plants, stomata were rarely found on the upper epidermis
of the three rattan species, occurrence of stomata was not
found on the upper epidermis of D. margaritae leaves. The
density of stomata on the beneath epidermis of rattan ma-
terials fallen in the range of those C3 and C4 plants reported
by Lin et al. (1986), however, the ratio of stomata density
on the top epidermis to the beneath epidermis was small,
falling in the range of those C3 plants but less than that of
those C4 plants reported by Lin et al. (1986), which was an
characterist ic of C3 p lants (Das and Santakumari, 1977;
Pathan and Nimbalkar, 1982; Lin et al., 1986).
2.3 Activities of PEPC and PPDK
The enzymes of PEPC and PPDK were the key enzymes
of C4 plants, activities of two enzymes were much higher in
C4 plants than those in C3 plants (Edwards et al., 1985;
Hocking and Anderson, 1986). As shown in Table 3, re-
gardless of seedlings or adult plant, activities of PEPC of
the three rattan species are similar to that of C3 plants Cym-
bidium (Ye et al., 1993; Ye et al., 1998) and soybean (Li et
al., 2001) , but much less than that of the C4 plant like sugar
cane (Ye et al., 1993). So did activities of PPDK of the three
rattan species, they were similar to the act ivities of PPDK
of Cymbidium, but less than that of sugar cane.
2.4 Carbon isotope ratio
Data on the s table carbon iso tope ratio (d13C) of the
Table 1 Chlorophyll contents in the leaves of three rattan species

Species Material
Chl a Chl b Chl a+b
Chl a/b
(mg/g FW) (mg/g FW) (mg/g FW)
Daemonorops margaritae Seedlings 3.06±0.20 0.96±0.34 4.03±0.15 2.85±0.22
Adult plants 3.18±0.38 1.04±0.12 4.23±0.23 3.06±0.12
Calamus simplicifolius Seedlings 3.19±0.23 1.16±0.13 4.35±0.35 2.75±0.13
Adult plants 2.70±0.17 0.99±0.07 3.69±0.24 2.73±0.02
C. tetradactylus Seedlings 2.57±0.10 0.86±0.04 3.44±0.14 2.97±0.01
Adult plants 2.62±0.93 0.94±0.45 3.55±0.89 2.84±0.19
Cymbidium* 0.96-2.22 0.36-1.34 1.31-4.00 1.24-2.73
Sugar cane* 1.42±0.02 0.34±0.01 1.74±0.04 4.22±0.02
*, data were means ± SE, which were cited from Ye et al. (1993; 1998).
Table 2 Stomata density on the upper and beneath epidermis of the three rattan species
Species Material
Upper epidermis Beneath epidermis
Upper/beneath
(Stomata/mm2) (Stomata/mm2)
Daemonorops margaritae Seedlings 0.0±0.0 541.5±35.2 0.0±0.0
Adult plants 0.0±0.0 536.8±86.4 0.0±0.0
Calamus simplicifolius Seedlings 17.0±4.6 357.8±48.5 0.05±0.02
Adult plants 9.5±5.2 655.1±87.2 0.01±0.01
C. tetradactylus Seedlings 15.1±6.3 729.9±82.5 0.02±0.02
Adult plants 24.6±9.7 869.0±8.1 0.03±0.01
C3 plants* 0-358 33-646 0-0.91
C4 plants* 0-269 20-435 0(rare)-3.34
*, data were cited from Lin et al. (1986).
LI Rong-Sheng et al.: Photosynthetic Pathway of Three Rattan Species in South China 563
three rattan species were shown in Table 3. Carbon Isotope
ratio was a well-known key way to recognize the photosyn-
thet ic pathway of plan ts, because its value in C4 plan ts
varied from -9.2‰ to -19.3‰ , and from -21.1‰ to
-35.0‰ in C3 plants (Lin et al., 1988). As shown in Table 4,
the values of carbon isotope ratio of the th ree rattan spe-
cies were around -28‰ , which fell within the range of car-
bon isotope ratio for C3 plants.
3 Conclusion
As stated above, the ratio of chlo rophyll a/b, stomata
density on top epidermis/beneath epidermis, the activities
of PEPC and PPDK, and carbon isotope ratio of the three
rattan species were low and fallen in the ranges of C3 plants
while less than those o f C4 plants reported before. So it
could be concluded that three rattan species tested were
C3 plant and it is expected to incorporate rattan plants with
C4 photosynthetic trait by genetic engineering to improve
rattan production in near future.
The conclusion can be checked indirect ly by data on
photos ynthetic rate by Mohamad (1992). As reported by
Mohamad, the photosynthesis of two rattan species of C.
manan Miq and C. tumidus Furtado in Malaysia was 1 mg
CO2·m-2·s-1, the value was less than those C3 plants like
tobacco, egg plant and hors ebean (Lin et al., 1986). Re-
garding stomata occurrence, Mohamad concluded that there
was no s tomata on the upper ep idermis of rattan leaves
after observing stomata occurrence on leaf epidermis of
two rattan species in Malaysia and, it was not consistent
with the pres ent s tudy on C. simplici fol ius and C.
tetradactylus and the paper by Li et al. (2002).
Acknowledgements: We are grateful for Mrs LI Xue-Mei
and WANG Xi-Mei from Zhongshan University; Dr. LI Chao
from Guangzhou Institu te of Geochemistry , The Chinese
Academy of Sciences; Prof. LIN Zhi-Fang , Mr. OU Zhi-
Ying and Mrs. CHEN Shao-Wei from South China Institute
of Botany, The Chinese Academy of Sciences for their as-
sistance in measurements.
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