Leaf Peltate Glandular Trichomes of Vernonia Galamensis Ssp
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I Int. J. Plant Sd. 169(5):605-614. 2008. © 2008 by The University of Chicago. All rights reserved. 1058-5893/2008/16905-0002$15.00 DOt: 10.1086/533598 LEAF PELTATE GLANDULAR TRICHOMES OF VERNONIA GALAMENSIS SSP. GALAMENSIS VAR. ETHIOPICA GILBERT: DEVELOPMENT, ULTRASTRUCTURE, AND CHEMICAL COMPOSITION Françoise Favi,l Charles L. Cantrell,t Tadesse Mebrahtu, and Mark E. Kraemer *Agricultural Research Station, Virginia State University, Petersburg, Virginia 23805, U.S.A.; and tUSDA-ARS, Natural Products Utilization Research Unit, University, Mississippi 38677, U.S.A. Plants from the genus Vernonia produce a variety of flavonoids and bitter sesquiterpene tactones important for agriculture and human health. Leaf glandular trichornes of Vernonia galainensis ssp. galamensis var. ethiopica Gilbert (VGAE) were investigated for ultrastructural development and content composition because sesquiter- pene lactones that impart a bitter taste to the leaves have been associated with the presence of these glands. Trichome ultrastructure was examined using LM, SEM, and TEM. Glands were removed from the leaf surface, and the chemical composition of gland contents was determined using HPLC and high-resolution mass spec- trometry. Immature and mature 10-celled peltatc hiseriate glandular trichomes were present only at the abaxial side of the leaf. A large subcuticular space (head) developed from the most distal cell pair of the mature trichome and gradually filled with an osmiophillic substance. Mass spectrometry analysis revealed that the peltate trichome is a major source of prevernocistifolide-8-O-isobutyrate. This glaucolide-type sesquiterpene lactone was previously identified as a major constituent of the aerial parts of VGAL. Keywords: glaucolides, sesquiterpene lactones, Vernonia galanzensis, trichomes. Introduction believed to be responsible for the production of these com- pounds. The genus Vernonia (Asteraceae) contains more than 500 Fine study of the glandular structure of the peltate glands of species distributed in Africa and the Americas, most of which VGAE will advance our knowledge of the production mecha- have bitter-tasting leaves. Vernonia galamensis (Cass.) Less is nism for an important group of biologically active compounds divided into several subspecies and varieties. Gilbert (1986) re- and will support future efforts toward the manipulation of defined this widely distributed species from East Africa into this production through genetic engineering. This study is a six subspecies, one of which has four varieties, among them contribution toward the understanding of trichome structure, Vernonia galarnensis ssp. galarnensis var. ethiopica, formally development, and glandular content. Three types of trichomes known as Vernonia pauciflora (Willd.). Our Agricultural Re- were observed on the foliage of VGAE: (1) peltate biseriate search Station is interested in V. galainensis ssp. galaniensis glandular, (2) awl-shaped glandular, and (3) nonglandular var. ethiopica Gilbert (VGAE), an herbaceous plant from Eri- (hair). Here we report the development and chemical content trea in northeast Africa, as a potential new oilseed crop. The of the peltate hiseriate glandular trichome. seed is rich in vernolic acid, a unique epoxy oil characterized by unusually low viscosity and thus valuable in paints, plasti- Material and Methods cizers, and flexible resins (Mohamed et al. 1999; King et al. 2000). Leaves from plants in the Vernonia genus contain fla- Plants vonoids, sesquiterpene lactones (Mabry et al. 1975; El-Sayed Seeds of Vernonia galamensis van. ethiopica were planted shal- et al. 1999; Miserez 1999), and other biologically active com- 3) pounds that have been used for centuries in traditional African lowly in a 78-hole (40 cc seedbed in prewetted mix (1: 1) of medicine (Tadesse et al. 1993; Igile et al. 1994) and even by Baccto potting soil (Michigan Peat) and Metro-Mix 360 (Sun chimpanzees to cure ailments (Huffman and Seifu 1989; Jisaka Gro I lorticulture) and kept moist. Seedlings were grown in a et al. 1992, 1993). They have also been used by Africans to glass greenhouse with supplemental lighting (1000-W metal ha- control and repel insects (Ganjian et at. 1983; El-Sayed et al. lide) to achieve a 13L : 11 photoperiod. Day/night temperature 1999). Leaf extracts from Vernonia have shown potential for was Ca. 25°/20°C. After 30 d, the seedlings were transplanted drug (Miserez 1999) and pesticide development (Fl-Sayed into 20-cm-diameter pots. Plants were watered as needed and et at. 1999), but nothing has been published on the ultrastruc- fertilized twice per week using Peters 20-20-20 all-purpose ture and chemical composition of the glandular trichomes fertilizer (25 gIL) to soil wetness. Both developing and fully elongated leaves from 60-d-old plants were sampled for mi- croscopy studies. The fully expanded leaves used in this study Author for correspondence; e-mail: [email protected]. were from the main stem and had axillary buds beginning de- Manuscript rcccwed June 2007; revised manuscript rcuzved October 2007 velopment into secondary vegetative branching. Glands used 606 INTERNATIONAL JOURNAL OF PLANT SCIENCES for content analysis were isolated from fully expanded leaves whole apparatus was tightly sealed and covered with ice. A up to 4 wk old. rheostat was used to supply the low voltage required for gen- tle tissue disruption. Three pulses of I -min duration with rheo- stat set at between 40 and 45 V were used, with a 1 -min rest SEM between pulses. Glands were then separated from leaf mate- Two types of SEM were used. Sections (2 cm 2 ) of freshly rial by passing the resulting mixture through a series of nylon harvested leaves from greenhouse-grown plants were either cloths with mesh sizes of 350, 105, 60, and 40 pm (Small Parts, freeze-dried or fixed in Trumps fixative (formalin, glutaralde- Miami Lakes, FL) to remove large debris and glass beads, leaf hyde in 0.2 M sodium cacodylate buffer with glucose) for debris and nonglandular trichomes, and awl-shaped glands, re- 48 h, washed three times with cacodylate buffer, serially dehy- spectively. The peltate glands were retained by the 40- Am mesh drated in ethanol, critical-point dried, and sputter-coated with cloth. Up to 600 mL of storage buffer (isolation buffer without gold. Digital images were made with a Hitachi S-4500 SEM at f3-mercaptoethanol, methylcellulose, and polyvinyl pyrrol idone) 20 kV accelerating voltage. An environmental SEM was used was used to facilitate flow through the mesh cloths. Peltate to obtain images of untreated leaves. Sections (4 cm x 2.2 cm) glands were washed at least eight times with storage buffer, of freshly harvested leaves from greenhouse-grown plants transferred to a 1.5-mL microfuge tube, and placed on ice. were mounted on stubs and scanned with a Hitachi tabletop Yield was ca. 30 AL of peltate glands (packed volume) per 12 g microscope (TM- 1000)at low vacuum. All SEM images were of leaf sample. Digital pictures of peltate glands were obtained digital (.tiff or .bmp) and made using software programs asso- using a digital camera (Nikon Coolpix 8400) attached to a ciated with these microscopes. phase contrast/epifluorescent microscope (VanGuard 1486 FL). Peltate glands were extracted with 5 mL of distilled ethyl alcohol (HPLC grade) and filtered (Whatman no. 2 filter pa- TEM per), and the extract was concentrated to 100 AL under pun- Leaves harvested from 2-mo-old plants were sectioned 0 fled nitrogen gas for gland content analysis. Dry weight was ca. lOtg. mm2 ) and fixed for 48 h at 4°C in Trumps fixative, washed with cacodylate buffer, postfixed and stained with osmium This technique yielded only small quantities of gland con- tetroxide, serially dehydrated in ethanol, and embedded in tent that were used to identify the major compound. Thereaf- Spurrs resin. Thick (100-150 nm) sections were stained with ter, leaf wash extract was used to obtain larger quantities of toluidine blue and visualized using a phase contrast/epifluo- glandular material that were used for mass spectra analysis. rescent microscope (VanGuard 1482 FL) to determine orienta- Fresh mature leaves (600 g) were placed in 3 L of distilled tion of the trichornes. Blocks whose thick sections displayed ethyl alcohol for i mm. Leaves were removed and the solvent trichomes with biseriate cells were retained for thin section- filtered under vacuum with Whatman no. 2 paper. The solu- ing appropriate for TEM. Slides containing selected thick sec- tion was concentrated to 50 mL using a rotary evaporator and tions were digitally imaged with a Nikon Eclipse ME-600 was partitioned. A 200-mL mixture of chloroform and water microscope equipped with a Digital Sight (DS-SM-L1) imag- (1 : 1) was added to concentrated leaf wash and partitioned in a separatory funnel. The upper chloroform fraction was col- ing system. Thin (40-60 nm) sections were stained for S mm using alcohol saturated with uranyl acetate and were coun- lected, concentrated to 20 mL, and further partitioned with terstained for 5 min with lead citrate. All specimens were ob- 50 mL each of hexane and methanol. The methanol fraction served and photographed using a JEOL JSM-1210 TEM at was used to identify the major component found in the gland 60 kV accelerating voltage. content extract. Instrumentation and Gland Content Analysis Gland Isolation Instrumentation. HPLC method development and analyti- Peltate glandular trichomes were isolated from fully expanded cal and semipreparative purifications were performed using leaves using a method modified from that of Gershenzon et al. an Agilent 1100 (Palo Alto, CA) HPLC system equipped with (1992) and Gang et al. (2001). All buffers and extracts were a quaternary pump, autosampler, diode-array detector, and maintained near freezing with ice baths. Fully expanded leaves vacuum degasser. High-resolution mass spectra were obtained (12 g) were harvested from main stems, placed in a 300-mL using an Agilent 1100 HPLC coupled to a JEOL AccuTOF beaker, washed with buffer (0.5 mM Tris-HCI, pH 7.5) for (JMS-TIOOLC).