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Geranyl Acetate Esterase Controls and Regulates the Level of Geraniol in Lemongrass (Cymbopogon fl Exuosus Nees Ex Steud.) Mutant Cv

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Geranyl Acetate Esterase Controls and Regulates the Level of Geraniol in Lemongrass (Cymbopogon fl Exuosus Nees Ex Steud.) Mutant Cv Geranyl Acetate Esterase Controls and Regulates the Level of Geraniol in Lemongrass (Cymbopogon fl exuosus Nees ex Steud.) Mutant cv. GRL-1 Leaves Deepak Ganjewalaa,b,* and Rajesh Luthraa,c a Central Institute of Medicinal and Aromatic Plants (CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow-226 105, U.P., India b Present address: School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore-632 014, Tamil Nadu, India. E-mail: [email protected] c Present address: Human Resource and Development Group, CSIR Complex, Library Avenue, Pusa, New Delhi-110 012, India * Author for correspondence and reprint requests Z. Naturforsch. 64 c, 251 – 259 (2009); received August 25/September 21, 2008 Essential oil isolated from lemongrass (Cymbopogon fl exuosus) mutant cv. GRL-1 leaves is mainly composed of geraniol (G) and geranyl acetate (GA). The proportion of G and GA markedly fl uctuates during leaf development. The proportions of GA and G in the essential oil recorded at day 10 after leaf emergence were ~59% and ~33% respectively. However, the level of GA went down from ~59 to ~3% whereas the level of G rose from ~33 to ~91% during the leaf growth period from day 10 to day 50. However, the decline in the level of GA was most pronounced in the early (day 10 to day 30) stage of leaf growth. The trend of changes in the proportion of GA and G has clearly indicated the role of an esterase that must be involved in the conversion of GA to G during leaf development. We isolated an esterase from leaves of different ages that converts GA into G and has been given the name geranyl acetate esterase (GAE). The GAE activity markedly varied during the leaf development cycle; it was closely correlated with the monoterpene (GA and G) composition throughout leaf development. GAE appeared as several isoenzymes but only three (GAE-I, GAE-II, and GAE-III) of them had signifi cant GA cleaving activity. The GAE isoenzymes pattern was greatly infl uenced by the leaf developmental stages and so their GA cleaving activities. Like the GAE activity, GAE isoenzyme patterns were also found to be consistent with the monoterpene (GA and G) composition. GAE had an optimum pH at 8.5 and tem- perature at 30 ºC. Besides GAE, a compound with phosphatase activity capable of hydrolyz- ing geranyl diphosphate (GPP) to produce geraniol has also been isolated. Key words: Geraniol-Rich Lemongrass (GRL-1), Geraniol, Geranyl Acetate, Geranyl Acetate Esterase Introduction et al., 2004), and it is a major constituent of palma- rosa (C. martinii) essential oil (Dubey et al., 2000). The geraniol-rich lemongrass (Cymbopogon It is also present in vegetative tissues of many fl exuosus Nees ex Steud.) Wats mutant cv. GRL- herbs (Charles and Simon, 1992; Patra et al., 1997; 1 was isolated from normal citral-rich cv. OD-19 Mallavarapu et al., 1998; Mockute and Bernotiene, during a chemo-genetical improvement program. 1999; Vieira et al., 2001; Sidibe et al., 2001). GRL-1 is characterized by a high geraniol (G) In general, G is synthesized from geranyl di- content (~91%) compared to the normal citral- rich cv. OD-19 and other cultivars (Patra et al., phosphate (GPP), the universal precursor of all 1997). The high level of G in mutant cv. GRL- monoterpenes (Croteau, 1987; Gershenzon and 1 might be due to the blockage of the NADP- Croteau, 1993; McGarvey and Croteau, 1995; geraniol dehydrogenase activity which otherwise Wise and Croteau, 1999). However, in lemon- in parent cv. OD-19 oxidizes G into geranial (ci- grass mutant cv. GRL-1 we suggest at least three tral) (Fig. 1). G is an acyclic monoterpene alco- enzymatic activities to be involved in regulating hol which is frequently used in perfumery, soaps, the level of G. detergents and cosmetics. G is also emitted from (1) Either a phosphatase- or monoterpene syn- the fl owers of many species, notably roses (Iijima thase-based catalysis of GPP leading to G. Perez 0939 – 5075/2009/0300 – 0251 $ 06.00 © 2009 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D NNC_3_4_2009.indbC_3_4_2009.indb 251251 004.05.20094.05.2009 111:08:201:08:20 252 D. Ganjewala and R. Luthra · Control of Geraniol Level in Lemongrass Geranyl diphosphate (GPP) velopment in lemongrass mutant cv. GRL-1. The study revealed that the monoterpene (GA and G) Phosphatase (2Pi/PPi) content and composition markedly fl uctuate dur- Dehydrogenase ing leaf development. In fact, the amount of GA in the immature (10 d) got completely converted Geraniol (G) Geranial (citral a) into G; by the time all leaves reached maturation Isomerase Transacetylase GAE (50 d). Here we report as the enzyme GAE which is mainly involved in the conversion of GA to G. Geranyl acetate (GA) Neral (citral b) GAE is a developmentally regulated enzyme. Fig. 1. Geraniol biosynthetic pathway in lemongrass During leaf development, the GAE activity has (Cymbopogon fl exuosus) mutant cv. GRL-1 depicting been found to be consistent with the monoter- the metabolic block by the cross. pene (GA and G) compositions substantiating its role in controlling and regulating the level of G. Moreover, the GAE isoenzyme patterns have also been in full agreement with the monoterpene et al. (1980) have reported phosphatases which composition during leaf development. Besides the hydrolyze prenyldiphosphates (GPP; farnesyl di- role of GAE, the role of phosphatases in the for- phosphate, FPP, and neryl diphosphate, NPP) to mation of G from GPP has also been investigated their corresponding alcohols such as geraniol, at a preliminary level. For the present study, we farnesol and nerol in citrus. In contrast, more re- choose lemongrass mutant cv. GRL-1 as it offers cently geraniol synthase was reported to be in- an excellent system to study the metabolism of volved in geraniol formation from GPP in basil, G because in this mutant G does not further oxi- Cinnamomum and Perilla (Iijima et al., 2004; Yang dize to citral. et al., 2005; Michiho and Honda, 2007). (2) Acetylation of G to geranyl acetate (GA) by a very active transacetylase. Shalit et al. (2003) Material and Methods have cloned and characterized the fi rst acetyl- Plant material CoA acetyl transferase from rose petals which Cymbopogon fl exuosus (Ness ex Steud.) Wats converts G into GA. However, transacetylase mutant GRL-1 plants were grown in the Ex- from Cymbopogon sp. could not be isolated. perimental Farm, Central Institute of Medicinal (3) Finally, GA slowly hydrolyzes into G during and Aromatic Plants (CIMAP), Lucknow, India. leaf development. This reaction is catalyzed by Leaves were harvested after 10 d of emergence an esterase referred to a geranyl acetate esterase and consequently at regular intervals of 10 d to (GAE). One such GAE catalyzing the biotrans- study the monoterpene content and composition formation of GA into G has been reported previ- of the essential oils. In parallel, essential oils were ously from palamarosa (C. martinii) infl orescence also isolated to study the monoterpene content (Dubey and Luthra, 2001; Dubey et al., 2003). and composition of leaves at different positions Though, the level of G in the lemongrass mutant in 50-day-old (fully expanded) tillers as the leaves cv. GRL-1 is regulated by these three enzymatic from inside (1st) to outside (6th) of a tiller rep- steps, but GAE plays a crucial role in the forma- resent a gradient increase in leaf age. Moreover, tion of G during leaf development. In addition, to augment the developmental changes in the different regulatory controls operating at organ, monoterpene compositions, 15-day-old leaves cellular, subcellular and enzyme/isoenzyme levels were cut into three equal parts, viz. basal (proxi- also play a role in controlling the level of GA and mal), middle and apical (distal), and subjected to G (Dubey and Luthra, 2001; Dubey et al., 2003). essential oil extraction. Being a monocot in na- Therefore, investigating these enzymatic steps is ture, these three regions within the leaf represent crucial to obtain a deeper understanding of the the gradient increase in leaf age. formation of G in lemongrass. The aim of the present study was to examine the changes in the monoterpene (GA and G) Essential oil extraction content and composition and the enzymatic proc- For the isolation of essential oils, fresh leaf sam- esses underlying these changes during leaf de- ples (~100 g) were collected, thoroughly washed NNC_3_4_2009.indbC_3_4_2009.indb 252252 004.05.20094.05.2009 111:08:201:08:20 D. Ganjewala and R. Luthra · Control of Geraniol Level in Lemongrass 253 under the tap, and cut into small pieces. Essential assay buffer and incubated with GA. G produced oil was isolated by steam-distillation technique and left over GA after the incubation period was using a mini-Clevenger apparatus. The volume of extracted twice with diethyl ether and subjected oil was measured and collected in small stopper to GC analysis. A blank with the boiled enzyme tubes containing anhydrous sodium sulfate to re- was also run simultaneously. The appearance of move traces of moisture. the G peak indicated the presence of GAE ac- tivity. GC analysis The monoterpene compositions of the essen- Native-PAGE tial oils were analyzed by a Perkin Elmer 8500 Native-PAGE was performed as per Laemmli gas chromatograph equipped with a fl ame ioniza- (1970). The separating (1.0 mm thick) and stack- tion detector and a BP-1 (diethyl polysiloxane) ing gels (1.0 mm thick) were 13 and 5%, re- column (30 m × 0.32 mm i.d., 0.25 μm fi lm thick- spectively. As electrophoresis buffer, 0.25 M Tris nesses).
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