Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis-enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-longchain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

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SummaryThe Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ß-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cellspecific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.catharanthine | Catharanthus roseus | surface secretion | vindoline

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as b-primeverosides (6-O-b-D-xylopyranosyl-b-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into b-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 69-hydroxy group of the sugar moiety of geranyl b-D-glucopyranoside, producing geranyl b-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with b-PRIMEVEROSIDASE encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma b-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.

SummaryThe Madagascar periwinkle (Catharanthus roseus) produces the well-known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine that are derived from the coupling of vindoline and catharanthine monomers. This study describes the novel application of a carborundum abrasion (CA) technique for large-scale isolation of leaf epidermis-enriched proteins in order to purify to apparent homogeneity 16-hydroxytabersonine-16-O-methyltransferase (16OMT), which catalyses the second of six steps in the conversion of tabersonine into vindoline, and to clone the gene. Functional expression and biochemical characterization of recombinant 16OMT demonstrated its very narrow substrate specificity and high affinity for 16-hydroxytabersonine. In addition to allowing the cloning of this gene, the CA technique clearly showed that 16OMT is predominantly expressed in Catharanthus leaf epidermis. The results provide compelling evidence that most of the pathway for vindoline biosynthesis, including the O-methylation of 16-hydroxytabersonine, occurs exclusively in the leaf epidermis, with subsequent steps occurring in other leaf cell types.

Komine, Hidehiko, Kanji Matsukawa, Hirotsugu Tsuchimochi, and Jun Murata. Central command blunts the baroreflex bradycardia to aortic nerve stimulation at the onset of voluntary static exercise in cats. Am J Physiol Heart Circ Physiol 285: H516-H526, 2003. First published May 1, 2003 10.1152/ajpheart.00013.2003.-To examine whether the central characteristics of the aortic baroreflex alter from moment to moment during static exercise, we identified the dynamic changes in the sizes of the bradycardia and depressor response evoked by stimulation of the aortic depressor nerve (ADN). Three conscious cats were trained to voluntarily extend the right forelimb and press a bar for 31 Ϯ 1 s with a peak force of 337 Ϯ 22 g while maintaining a sitting posture. The ADN stimulation-induced bradycardia was attenuated at the initial period of exercise (up to 8 s from the exercise onset) to 62 Ϯ 5% of the preexercise bradycardia and remained blunted until the end of exercise. The most blunted bradycardia was observed immediately before or when the forelimb was extended before force development. The baroreflex-induced bradycardia was suppressed again at cessation of exercise when the forelimb was retracted and recovered within a few seconds. In contrast, static exercise did not affect the ADN stimulation-induced depressor response. The ADN stimulation-induced bradycardia was also blunted at the beginning of naturally occurring body movement such as spontaneous postural change or grooming behavior. Thus it is likely that the central characteristics of the aortic baroreflex dynamically change from moment to moment during voluntary static exercise and during natural body movement and that particularly a central inhibition of the cardiac component of the aortic baroreflex is induced by central command at the onset of static exercise, whereas the central property of the vasomotor component of the baroreflex is preserved.aortic depressor nerve; aortic baroreceptors; baroreflex sensitivity; baroreflex depressor response; central modulation of the arterial baroreflex ARTERIAL BLOOD PRESSURE (AP) is sensed by arterial baroreceptors in the carotid sinus and aortic arch regions, whose activities transmit the beat-to-beat changes in AP to the central nervous system and evoke the baroreflex responses of autonomic efferent nerve activities to the heart and blood vessels. For example, if AP increases during resting, arterial baroreceptors are stimulated in proportion to the rise in AP, which elicits reflex bradycardia and depressor response and thereby restores AP to the control level. However, it is well known that heart rate (HR) and AP increase simultaneously during static or dynamic exercise in humans and conscious animals, indicating that arterial baroreflex function is modulated by exercise.The effect of exercise on the arterial baroreflexes has been extensively studied in humans and animals. When baroreflex sensitivity was assessed as a ratio of the change in R-R interval or HR in response to an alteration in AP, it was reduced during d...

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