Received 24 Feb. 2004 Accepted 13 Jul. 2004Supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars ((2003)406), State Education Ministry andthe Science Foundation of Education Department of Shaanxi Province (03JC39).

* E-mail: <lwenzhe@nwu.edu.cn>.

http://www.chineseplantscience.com

Acta Botanica Sinica植 物 学 报 2004, 46 (10): 1242-1248

Secretory Structures and Their Relationship to Accumulation ofCamptothecin 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 CPTproduction strategies. The aim of this study was to anatomically and morphologically characterize thesecretory structure in leaves and stems of C. acuminata and determine their relationship to accumulationof CPT. Secretory canals and glandular trichomes were found in young stems and young leaves. Secretorycanals consisted of a sub-epidermal canal delimited by one to two layers of cells. Glandular trichomes wereunicellular. Fluorescence microscopy and CPT analysis showed that CPT was primarily accumulated insecretory canals and glandular trichomes of leaves and stems.Key words: Camptotheca acuminata ; secretory structures; camptothecin (CPT); fluorescence microscopy

Camptotheca acuminata (Nyssaceae) is a tree originallyfound in China. The various organs of the species containthe alkaloid camptothecin (CPT) and its derivatives withimportant biological activities. The CPT was isolated fromthe wood of C. acuminata and its structure was elucidatedby Wall et al. (1966). The CPT is a valuable compound as achemical precursor of topotecan and irinotecan which areused clinically as anticancer agents (Creemers et al., 1996).The antitumor activity of CPT is due to its ability to inhibitDNA topoisomeraseⅠ(Kjeldsen et al., 1992). The CPT alsoinhibits retroviruses such as the human immunodeficiencyvirus and the equine infectious anemia virus (Priel et al.,1991). The anti-HIV activity of CPT is due to the inhibitionof Tat-mediated transcription from the viral promoter (Li etal., 1994). Thus, CPT drugs will have broad uses and demandfor CPTs will dramatically increase in the future.

Secretory structures are known to be the primary sitesof production of bioactive secondary products which mayfunction as plant growth regulators and defend the plantagainst insects, other pathogens and, possibly, other plants(Wagner, 1991; Werker, 1993; Duke, 1994). CPT is a defencechemical (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 accumulatedin glandular trichomes of leaves and stems in C. acuminata.However, glandular trichomes are only distributed onsurface of young leaves and stems. The results do not

explain CPT accumulated in wood and bark. Other secretorystructures may accumulate CPT in leaves and stems. Littleinformation is available on secretory structures other thanglandular trichomes in C. acuminata.

In the present study, light and scanning electronmicroscopy were used to determine the distribution,morphology and structure of secretory structures, withemphasis on secretory canals, in C. acuminata .Fluorescence microscopy and CPT content analysis wereused to investigate the relationship between secretorystructure and the accumulation of CPT in C. acuminata.

1 Materials and Methods1.1 Plant materials

Two-year-old Camptotheca acuminata Decne.seedlings were grown from seed in the field at the BotanicalGarden of Northwest University, Xi’an. Seeds wereprovided by Botanical Garden of Xi’an. The samples of C.acuminata were identified by Prof. REN Yi of NorthwestUniversity.1.2 Light microscopy

Stems and leaves of plant were used as materials. Sec-tions (40 µm) of fresh material were cut using a Leica CM1850 cryostat at –19 ºC. Other sections (1–2 µm) were cutusing a Reichert-Jung ultramicrotome after the material hadbeen fixed in 3% glutaraldehyde for 12 h and post fixed in1% OsO4 for 2 h. The tissue was dehydrated in a gradedethanol 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 wereunstained for the observation of secretory canals by usingLeica 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 dryweight) of young stems. Five two-year-old plants were ran-domly selected. The internodes of stems and leaves werecollected from the top three juvenile shoots of five experi-mental plants in July. CPT content was analyzed. Secretorycanal density was measured by sectioning frozen. Secre-tory canal number per scope view (1 mm ×1 mm each) wascounted 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 LeicaDMLB fluorescence microscope.1.4 Scanning electron microscopy

The leaf segments of different developmental stageswere fixed for 24 h in FAA (formalin:acetic acid:alcohol) atroom temperature and subsequently dehydrated in a gradedethanol 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, themethanolic extract was evaporated to dryness at 40 ºC in vacuumusing 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 (AglientHP 1100), a reversed phase column (Sphermage-80, 4 mm× 250 mm) and a DAD (Diode Array detector, AgliengHP1100) for the detection of camptothecin at 254 nm. Theflow rate was 1 mL/min and a gradient of acetonitrile andwater 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 ResultsThe adaxial and abaxial surfaces of young leaves of C.

acuminata contained numerous non-glandular trichomesand glandular trichomes (Figs.1, 2). Large numbers of non-

glandular trichomes were found along the veins or at theleaf 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 abaxialsurface (Figs.2, 4). Trichomes were significantly denser onthe surfaces of young leaves than on that of old ones (Fig.5).

Secretory canals of C. acuminata consist of a canaldelimited by one or two layers of cells. The canals have avariable lumen diameter and/or a variable number of secre-tory cells (Fig.6). The ratio of length and width ranged from15 to 20 (Fig.7). Secretory canals were present in youngerstems and leaves. In young stems, secretory canals weredistributed in pith and cortex tissues near vascular tissue.The diameter of canals was larger in pith than those incortex issues by comparing cross sections of 10 differentsamples (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 thevascular tissue of veins and petioles (Figs.13, 14). Theyalso could be found around the vascular tissue and corticalparenchyma in veins of petioles (Fig.17). There were noanatomical differences in canals between the leaf and thestem.

There was a positive relationship between organs ageand the density of secretory canals. A high density of secre-tory canals associated with young stems (Figs.8, 9, 21) andleaves (Figs.10, 13, 22). There was a large decline in thedensity 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 canaldensity in stems of C. acuminata (Fig.23). Secretory canalscould not be found in the older tissues (Figs.9, 10).

Fluorescence microscopy was used to observe thesecretory structures. The secretory products in canals andglandular trichomes showed intense blue fluorescence un-der 360 nm UV light (Figs.16-20). It is the same color asCPT shows in the fluorescence micrographs (Fig.15).

The secretion product of the secretory canals appearedin gray-blank in section fixed by OsO4 (Figs.6-8). Glandulartrichomes also contained osmiophilic material (Fig.14). Inaddition, some parenchymatous cells of cortex and pith instems and leaves also contained osmiophilic material (Figs.8,13). However, they did not show intense blueautofluorescence 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 = 190µm. 2. The abaxial surfaces of young leaves. Bar = 190 µm. 3. An enlarged globoid glandular trichome. Bar = 6 µm. 4. An enlargedellipsoidal glandular trichome. Bar = 6 µm. 5. The adaxial surfaces of mature leaves. Bar = 190 µm. 6-10. Distribution and morphologyof secretory canals in C. acuminata. Bar = 10 µm. 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) andcortex (short arrow). 9. Cross section of internode 10, secretory canal were not found in older stems. 10. Cross section of main vein inmature 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 µm.12. Ellipsoidal unicellular glandular trichome. Bar = 5 µm. 13. The main vein of young leaves with secretory canals (arrow). Bar = 20 µm.14. Leaf primordium with unopened secretory canal appearing in the center of procambium (black arrow) and glandular trichomes withosmiophilic material (pink arrow). Bar = 20 µm. 15-20. Fluorescence micrographs of C. acuminata. V, vessel. Bar = 20 µm. 15.Fluorescence micrograph of camptothecin (CPT) standard. 16. Glandular trichomes with autofluorescence. 17. Cross of petiole showingautofluorescence of the secretory canals in cortex (white arrow) and associated with vascular bundles (pink arrow). 18. Cross section ofyoung stem showing autofluorescence of secretory canals in cortex (white arrow) and pith (pink arrow). 19. Longitudinal section ofyoung stem showing autofluorescence of secretory canals in cortex (arrow). 20. Cross section of main vein in young leaves showingautofluorescence of secretory canals (arrow).

Acta Botanica Sinica 植物学报 Vol.46 No.10 20041246

3 DiscussionThe 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 andleaves. 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 inolder tissues (López-Meyer et al., 1994; Liu et al., 1998).There was a positive relationship between the number ofsecretory canals and CPT concentration. High CPT con-centrations were associated with the high density of

secretory canals. No secretory structures were found inroots 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 genusCamptotheca by Li et al. (2002), who concluded thatcamptothecin was primarily accumulated in glandular tri-chomes of leaves.

CPT has absorption maxima at 256 and 363 nm (vanHengel et al., 1992; Liu, 2000), the peak of absorption at 360nm was chosen for detection of camptothecin in the fluo-rescent microscope analyses. Glandular trichomes and thecontents of secretory canals showed intense blueautofluorescence under the fluorescent microscope at 360nm 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 etal., 2002) and secretory canals. Trichomes and secretorycanals are significantly denser in the young tissues than inthe old ones, and CPT contents in young leaves and stemsare higher than those in old leaves and stems. It was sug-gested that glandular trichomes and secretory canals wereprimary 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 andstems were organs for camptothecin storage, and were notorgans for camptothecin biosynthesis. These results wereconsistent with the findings of López-Meyer et al. (1994),who rationalized that leaves are not the CPT producingorgans. Probably root tissues are the source of CPT and ayet unknown regulated transport system directs CPT toother plant organs. The observed discrepancy of plantsparts between strictosidine synthesis (STR) enzyme (a keyenzyme of camptothecin biosynthesis) activity andcamptothecin biosynthesis might occur in the limited partswhere expression of mRNA and enzyme activity took placeand then camptothecin was transported to other parts(Yamazaki et al., 2003).

Immature tissues serve as major sinks of photosynthatesand 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 andpathogenic attacks (Liu et al., 1998). Secretory structuresare known to be the primary sites of production of bioactivesecondary products which may function as plant growthregulators and defend the plant against insects, pathogensand, possibly, other plants (Wagner, 1991; Werker, 1993;Duke, 1994). Secretory canals which are associated withveins could transport photosynthates and protect the

Fig.21. The secretory canal density of internodes in youngstems from apex.

Fig.22. The secretory canal density in young leaves from apex.

Fig.23. Linear relationship between secretory canal density andcamptothecin (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 (Harborneand Grayer, 1993).

Higher CPT concentration in young C. acuminata leavesand stems than in old ones represents a chemical defensemechanism developed by young tissues to deter attacksby herbivores, micro-organisms, or both. This mechanismmight be a programmed defense strategy that has evolvedso that young tissues are protected by defense chemicalsduring 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 depositoriesplaces 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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