全 文 ：Received 24 Feb. 2004 Accepted 13 Jul. 2004
Supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars ((2003)406), State Education Ministry and
the Science Foundation of Education Department of Shaanxi Province (03JC39).
* E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1242-1248
Secretory Structures and Their Relationship to Accumulation of
Camptothecin in Camptotheca acuminata (Nyssaceae)
LIU Wen-Zhe*
(College of Life Sciences, Northwest University, Xi’an 710069, China)
Abstract: Camptotheca acuminata Decne. (Nyssaceae) is a major source of the anticancer camptothecin
(CPT). It is important to understand how CPT accumulates in C. acuminata in order to improve CPT
production strategies. The aim of this study was to anatomically and morphologically characterize the
secretory structure in leaves and stems of C. acuminata and determine their relationship to accumulation
of CPT. Secretory canals and glandular trichomes were found in young stems and young leaves. Secretory
canals consisted of a sub-epidermal canal delimited by one to two layers of cells. Glandular trichomes were
unicellular. Fluorescence microscopy and CPT analysis showed that CPT was primarily accumulated in
secretory canals and glandular trichomes of leaves and stems.
Key words: Camptotheca acuminata ; secretory structures; camptothecin (CPT); fluorescence microscopy
Camptotheca acuminata (Nyssaceae) is a tree originally
found in China. The various organs of the species contain
the alkaloid camptothecin (CPT) and its derivatives with
important biological activities. The CPT was isolated from
the wood of C. acuminata and its structure was elucidated
by Wall et al. (1966). The CPT is a valuable compound as a
chemical precursor of topotecan and irinotecan which are
used clinically as anticancer agents (Creemers et al., 1996).
The antitumor activity of CPT is due to its ability to inhibit
DNA topoisomeraseⅠ(Kjeldsen et al., 1992). The CPT also
inhibits retroviruses such as the human immunodeficiency
virus and the equine infectious anemia virus (Priel et al.,
1991). The anti-HIV activity of CPT is due to the inhibition
of Tat-mediated transcription from the viral promoter (Li et
al., 1994). Thus, CPT drugs will have broad uses and demand
for CPTs will dramatically increase in the future.
Secretory structures are known to be the primary sites
of production of bioactive secondary products which may
function as plant growth regulators and defend the plant
against insects, other pathogens and, possibly, other plants
(Wagner, 1991; Werker, 1993; Duke, 1994). CPT is a defence
chemical (Liu et al., 1998) and is found in leaves, bark, wood,
roots and fruits of C. acuminata (López-Meyer et al., 1994;
Liu and Adams, 1996; Vincent et al., 1997; Liu et al., 1998).
Li et al. (2002) suggested that CPT is primarily accumulated
in glandular trichomes of leaves and stems in C. acuminata.
However, glandular trichomes are only distributed on
surface of young leaves and stems. The results do not
explain CPT accumulated in wood and bark. Other secretory
structures may accumulate CPT in leaves and stems. Little
information is available on secretory structures other than
glandular trichomes in C. acuminata.
In the present study, light and scanning electron
microscopy were used to determine the distribution,
morphology and structure of secretory structures, with
emphasis on secretory canals, in C. acuminata .
Fluorescence microscopy and CPT content analysis were
used to investigate the relationship between secretory
structure and the accumulation of CPT in C. acuminata.
1 Materials and Methods
1.1 Plant materials
Two-year-old Camptotheca acuminata Decne.
seedlings were grown from seed in the field at the Botanical
Garden of Northwest University, Xi’an. Seeds were
provided by Botanical Garden of Xi’an. The samples of C.
acuminata were identified by Prof. REN Yi of Northwest
University.
1.2 Light microscopy
Stems and leaves of plant were used as materials. Sec-
tions (40 mm) of fresh material were cut using a Leica CM
1850 cryostat at –19 ºC. Other sections (1–2 mm) were cut
using a Reichert-Jung ultramicrotome after the material had
been fixed in 3% glutaraldehyde for 12 h and post fixed in
1% OsO4 for 2 h. The tissue was dehydrated in a graded
ethanol series and embedded in Epon 812. Semi-thin
LIU Wen-Zhe: Secretory Structures and Their Relationship to Accumulation of Camptothecin in Camptotheca acuminata
(Nyssaceae) 1243
sections were stained with Toluidine Blue O for the obser-
vation of tissue structure. Sections from fresh material were
unstained for the observation of secretory canals by using
Leica DMLB light microscopy.
The relationship between secretory canal and CPT con-
tent was examined by secretory canal density (number/
section) and CPT content (% (W/W), on the basis of dry
weight) of young stems. Five two-year-old plants were ran-
domly selected. The internodes of stems and leaves were
collected from the top three juvenile shoots of five experi-
mental plants in July. CPT content was analyzed. Secretory
canal density was measured by sectioning frozen. Secre-
tory canal number per scope view (1 mm ×1 mm each) was
counted with 20 views per internode or leaf.
1.3 Fluorescence microscopy
For fluorescence microscopy, sections of fresh materi-
als were cut using a Leica CM1850 cryostat microtome at
–19 ºC. Unstained sections were observed under Leica
DMLB fluorescence microscope.
1.4 Scanning electron microscopy
The leaf segments of different developmental stages
were fixed for 24 h in FAA (formalin:acetic acid:alcohol) at
room temperature and subsequently dehydrated in a graded
ethanol series. The specimens underwent critical point dry-
ing with liquid CO2 were spurted with gold, and finally ex-
amined with a Hitachi S-570 SEM.
1.5 CPT content analysis
Plant samples were dried in an oven at 60 ºC for 24 h.
Dried powdered material (100 mg) was extracted with sonica-
tion for 30 min at room temperature. After sonication, the
methanolic extract was evaporated to dryness at 40 ºC in vacuum
using a rotavapor. For the analysis of CPT content, the con-
centrate was re-dissolved in HPLC-grade methanol (1 mL).
The HPLC system consisted of a HPLC pump (Aglient
HP 1100), a reversed phase column (Sphermage-80, 4 mm
× 250 mm) and a DAD (Diode Array detector, Aglieng
HP1100) for the detection of camptothecin at 254 nm. The
flow rate was 1 mL/min and a gradient of acetonitrile and
water was used: 10% actonitrile for 5 min, 10% to 40% ac-
etonitrile for 15 min, 40% acetonitrile for 10 min (Liu, 2003).
Concentrations were calculated with camptothecin refer-
ence solution (CPT standard was kindly supplied by Dr. H.
Bischoff, Boehringer lngelheim Pharma KG, Germany). Ev-
ery sample was analyzed in duplicate.
2 Results
The adaxial and abaxial surfaces of young leaves of C.
acuminata contained numerous non-glandular trichomes
and glandular trichomes (Figs.1, 2). Large numbers of non-
glandular trichomes were found along the veins or at the
leaf margins on the abaxial surface (Fig.2). Mature, unicel-
lular glandular trichomes were situated on both leaf surfaces
（Figs.11, 12）. They can be divided into two types ac-
cording to the morphology of glandular head: globoid glan-
dular trichomes were found on the adaxial surface (Figs.1,
3); ellipsoidal glandular trichomes were found on the abaxial
surface (Figs.2, 4). Trichomes were significantly denser on
the surfaces of young leaves than on that of old ones (Fig.
5).
Secretory canals of C. acuminata consist of a canal
delimited by one or two layers of cells. The canals have a
variable lumen diameter and/or a variable number of secre-
tory cells (Fig.6). The ratio of length and width ranged from
15 to 20 (Fig.7). Secretory canals were present in younger
stems and leaves. In young stems, secretory canals were
distributed in pith and cortex tissues near vascular tissue.
The diameter of canals was larger in pith than those in
cortex issues by comparing cross sections of 10 different
samples (Fig.8). Sometimes they could be found in the pa-
renchyma below the epidermis (data not shown). In leaves,
these structures could be found in spaces delimited by the
vascular tissue of veins and petioles (Figs.13, 14). They
also could be found around the vascular tissue and cortical
parenchyma in veins of petioles (Fig.17). There were no
anatomical differences in canals between the leaf and the
stem.
There was a positive relationship between organs age
and the density of secretory canals. A high density of secre-
tory canals associated with young stems (Figs.8, 9, 21) and
leaves (Figs.10, 13, 22). There was a large decline in the
density of secretory canal from internode/leaf 5 to intern-
ode/leaf 10 (Figs.21, 22). Further, there was a linear relation-
ship (r = 0.923 9) between CPT content and secretory canal
density in stems of C. acuminata (Fig.23). Secretory canals
could not be found in the older tissues (Figs.9, 10).
Fluorescence microscopy was used to observe the
secretory structures. The secretory products in canals and
glandular trichomes showed intense blue fluorescence un-
der 360 nm UV light (Figs.16-20). It is the same color as
CPT shows in the fluorescence micrographs (Fig.15).
The secretion product of the secretory canals appeared
in gray-blank in section fixed by OsO4 (Figs.6-8). Glandular
trichomes also contained osmiophilic material (Fig.14). In
addition, some parenchymatous cells of cortex and pith in
stems and leaves also contained osmiophilic material (Figs.
8,13). However, they did not show intense blue
autofluorescence under UV light.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041244
Figs.1-10. 1-5. Scanning electron micrographs of leaves in Camptotheca acuminata. 1. The adaxial surfaces of young leaves. Bar = 190
mm. 2. The abaxial surfaces of young leaves. Bar = 190 mm. 3. An enlarged globoid glandular trichome. Bar = 6 mm. 4. An enlarged
ellipsoidal glandular trichome. Bar = 6 mm. 5. The adaxial surfaces of mature leaves. Bar = 190 mm. 6-10. Distribution and morphology
of secretory canals in C. acuminata. Bar = 10 mm. 6. Cross section of young stem, showing the structure of secretory canal in pith. 7.
Longitudinal section of secretory canal (arrow) in stem. 8. Cross section of internode 3, showing secretory canals in pith (long arrow) and
cortex (short arrow). 9. Cross section of internode 10, secretory canal were not found in older stems. 10. Cross section of main vein in
mature leaves, secretory canal were not found in older leaves.
LIU Wen-Zhe: Secretory Structures and Their Relationship to Accumulation of Camptothecin in Camptotheca acuminata
(Nyssaceae) 1245
Figs. 11-20. 11-14. Cross section of young leaves in Camptotheca acuminata. 11. Globoid unicellular glandular trichomes. Bar = 5 mm.
12. Ellipsoidal unicellular glandular trichome. Bar = 5 mm. 13. The main vein of young leaves with secretory canals (arrow). Bar = 20 mm.
14. Leaf primordium with unopened secretory canal appearing in the center of procambium (black arrow) and glandular trichomes with
osmiophilic material (pink arrow). Bar = 20 mm. 15-20. Fluorescence micrographs of C. acuminata. V, vessel. Bar = 20 mm. 15.
Fluorescence micrograph of camptothecin (CPT) standard. 16. Glandular trichomes with autofluorescence. 17. Cross of petiole showing
autofluorescence of the secretory canals in cortex (white arrow) and associated with vascular bundles (pink arrow). 18. Cross section of
young stem showing autofluorescence of secretory canals in cortex (white arrow) and pith (pink arrow). 19. Longitudinal section of
young stem showing autofluorescence of secretory canals in cortex (arrow). 20. Cross section of main vein in young leaves showing
autofluorescence of secretory canals (arrow).
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041246
3 Discussion
The findings on the secretory canals in C. acuminata
young stems and leaves in the present study were novel.
The secretory canals were located in young stems and
leaves. Secretory structures decreased in density with de-
velopment and growth of stems and leaves. CPT concen-
tration was higher in young C. acuminata tissues than in
older tissues (López-Meyer et al., 1994; Liu et al., 1998).
There was a positive relationship between the number of
secretory canals and CPT concentration. High CPT con-
centrations were associated with the high density of
secretory canals. No secretory structures were found in
roots of C. acuminata (data not shown). A linear relation-
ship between CPT content and glandular trichomes den-
sity on leaf surfaces was also observed in genus
Camptotheca by Li et al. (2002), who concluded that
camptothecin was primarily accumulated in glandular tri-
chomes of leaves.
CPT has absorption maxima at 256 and 363 nm (van
Hengel et al., 1992; Liu, 2000), the peak of absorption at 360
nm was chosen for detection of camptothecin in the fluo-
rescent microscope analyses. Glandular trichomes and the
contents of secretory canals showed intense blue
autofluorescence under the fluorescent microscope at 360
nm UV light. It has a similar color to CPT under the fluores-
cent microscope. This indicated that there are highly con-
centrated alkaloids (CPTs) in the glandular trichomes (Li et
al., 2002) and secretory canals. Trichomes and secretory
canals are significantly denser in the young tissues than in
the old ones, and CPT contents in young leaves and stems
are higher than those in old leaves and stems. It was sug-
gested that glandular trichomes and secretory canals were
primary sites of accumulation of CPTs.
No secretory structures were found in roots. However,
camptothecin was detected in roots (Liu and Adams, 1996).
This suggested that secretory structures in leaves and
stems were organs for camptothecin storage, and were not
organs for camptothecin biosynthesis. These results were
consistent with the findings of López-Meyer et al. (1994),
who rationalized that leaves are not the CPT producing
organs. Probably root tissues are the source of CPT and a
yet unknown regulated transport system directs CPT to
other plant organs. The observed discrepancy of plants
parts between strictosidine synthesis (STR) enzyme (a key
enzyme of camptothecin biosynthesis) activity and
camptothecin biosynthesis might occur in the limited parts
where expression of mRNA and enzyme activity took place
and then camptothecin was transported to other parts
(Yamazaki et al., 2003).
Immature tissues serve as major sinks of photosynthates
and sites of production for some phytohormones. Imma-
ture tissues are nutrient-rich and tender in physical structure.
These features may make them attractive to herbivory and
pathogenic attacks (Liu et al., 1998). Secretory structures
are known to be the primary sites of production of bioactive
secondary products which may function as plant growth
regulators and defend the plant against insects, pathogens
and, possibly, other plants (Wagner, 1991; Werker, 1993;
Duke, 1994). Secretory canals which are associated with
veins could transport photosynthates and protect the
Fig.21. The secretory canal density of internodes in young
stems from apex.
Fig.22. The secretory canal density in young leaves from apex.
Fig.23. Linear relationship between secretory canal density and
camptothecin (CPT) content in young stems.
LIU Wen-Zhe: Secretory Structures and Their Relationship to Accumulation of Camptothecin in Camptotheca acuminata
(Nyssaceae) 1247
(Managing editor: WANG Wei)
phloem (Williams, 1954). Glandular trichomes produce bio-
logically active metabolites (e.g., alkaloids), which may pro-
tect the plant against herbivores and parasites (Harborne
and Grayer, 1993).
Higher CPT concentration in young C. acuminata leaves
and stems than in old ones represents a chemical defense
mechanism developed by young tissues to deter attacks
by herbivores, micro-organisms, or both. This mechanism
might be a programmed defense strategy that has evolved
so that young tissues are protected by defense chemicals
during their normal ontogenic development (Zu et al.,
2003b). The distribution of CPTs in naturally developed C.
acuminata are under rigid tissue control, and young leaves,
young stems, fruits and seeds are the main depositories
places of alkaloids (Zu et al., 2003a).
Acknowledgements: We are grateful for Prof. HU Zheng-
Hai of Northwest University for reviewing the manuscript;
Dr. ZHENG Hong-Chun of Northwest University for Scan-
ning electron microscopy and Dr. SUE Zi-Rong of North-
west University for the CPT content analysis.
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