SNARE-RNAi results in higher terpene emission from ectopically expressed caryophyllene synthase in nicotiana benthamiana
Ting, Jimmy ; Delatte, Thierry L. ; Kolkman, P. ; Misas-Villamil, Johana C. ; Hoorn, Renier A.L. Van Der; Bouwmeester, Harro J. ; Krol, Sander van der - \ 2015
Molecular Plant 8 (2015)3. - ISSN 1674-2052 - p. 454 - 466.
caryophyllene synthase - linalool synthase - Nicotiana benthamiana - proteasome - terpene transport - vesicle-associated membrane proteins (VAMP72)
Plants produce numerous terpenes and much effort has been dedicated to the identification and characterization of the terpene biosynthetic genes. However, little is known about how terpenes are transported within the cell and from the cell into the apoplast. To investigate a putative role of vesicle fusion in this process, we used Agrobacterium tumefaciens-mediated transient coexpression in Nicotiana benthamiana of an MtVAMP721e-RNAi construct (Vi) with either a caryophyllene synthase or a linalool synthase, respectively. Headspace analysis of the leaves showed that caryophyllene or linalool emission increased about five-fold when N. benthamiana VAMP72 function was blocked. RNA sequencing and protein ubiquitination analysis of the agroinfiltrated N. benthamiana leaf extracts suggested that increased terpene emissions may be attributed to proteasome malfunction based on three observations: leaves with TPS+Vi showed (1) a higher level of a DsRed marker protein, (2) a higher level of ubiquitinated proteins, and (3) coordinated induced expression of multiple proteasome genes, presumably caused by the lack of proteasome-mediated feedback regulation. However, caryophyllene or linalool did not inhibit proteasome-related protease activity in the in vitro assays. While the results are not conclusive for a role of vesicle fusion in terpene transport, they do show a strong interaction between inhibition of vesicle fusion and ectopic expression of certain terpenes. The results have potential applications in metabolic engineering.
Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns
Yang, T. ; Stoopen, G. ; Yalpani, N. ; Vervoort, J.J.M. ; Vos, R. de; Voster, A. ; Verstappen, F.W.A. ; Bouwmeester, H.J. ; Jongsma, M.A. - \ 2011
Metabolic Engineering 13 (2011)4. - ISSN 1096-7176 - p. 414 - 425.
altered monoterpene composition - seed-borne fungi - essential oils - fusarium-graminearum - terpenoid metabolism - linalool synthase - limonene synthase - mass-spectrometry - beta-glucosidase - tomato fruits
Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC
Functional characterization of enzymes forming volatile esters from strawberry and banana
Beekwilder, M.J. ; Alvarez-Huerta, M. ; Neef, E.J. ; Verstappen, F.W.A. ; Bouwmeester, H.J. ; Aharoni, A. - \ 2004
Plant Physiology 135 (2004)4. - ISSN 0032-0889 - p. 1865 - 1878.
floral scent production - clarkia-breweri - alcohol acyltransferase - molecular-cloning - linalool synthase - escherichia-coli - transferase cdna - gene-expression - messenger-rnas - acetyl-coa
Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria x ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess the potential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants overexpressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is an important parameter to consider when engineering volatile ester formation in plants
Metabolic engineering of monoterpende biosysnthesis: two step production of (+)-trans-Isopiperitenol by tobacco
Lücker, J. ; Schwab, W. ; Franssen, M.C.R. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
The Plant Journal 39 (2004)1. - ISSN 0960-7412 - p. 135 - 145.
peppermint mentha-piperita - functional expression - limonene enantiomers - linalool synthase - cdna isolation - s-linalool - plants - mint - (-)-limonene - cloning
Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.
The influence of monoterpene synthase transformation on the odour of tabacco.
Tamer, M.K. el; Smeets, M.A.M. ; Holthuysen, N.T.E. ; Lucker, J. ; Tang, A. ; Roozen, J.P. ; Bouwmeester, H.J. ; Voragen, A.G.J. - \ 2003
Journal of Biotechnology 106 (2003)1. - ISSN 0168-1656 - p. 15 - 21.
linalool synthase - gene-expression - s-linalool - biosynthesis - plants - cancer
Monoterpenes are an important class of terpenoids that are commonly present in plant essential oils. These can be extracted from plants and are used in the flavouring and perfumery industry. Monoterpene synthases are the key enzymes in monoterpene biosynthesis, as they catalyse the cyclisation of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. Tobacco is one of the most studied model plants, it can easily and efficiently be transformed, and is a suitable model to study the release of plant volatiles. Thus, we have isolated monoterpene synthases from lemon, transformed tobacco with these cDNAs and have used human panelists to study the change in fragrance of the transgenic in comparison to the wild type plants. In a triangle test, we found that subjects were capable of smelling significant differences between leaf samples. However, as a result of variability in panel ratings, no significant difference between two sets of transgenic flowers and the wild type tobacco flowers was found for the generated attributes in a descriptive test.
Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.
Tamer, M.K. el; Lucker, J. ; Bosch, D. ; Verhoeven, H.A. ; Verstappen, F.W.A. ; Schwab, W. ; Tunen, A.J. van; Voragen, A.G.J. ; Maagd, R.A. de; Bouwmeester, H.J. - \ 2003
Archives of Biochemistry and Biophysics 411 (2003). - ISSN 0003-9861 - p. 196 - 203.
site-directed mutagenesis - 5-epi-aristolochene synthase - trichodiene synthase - linalool synthase - germacrene-a - biosynthesis - expression - biology
Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lucker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClyTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of P-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClyTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClyTS and Cl(-)betaPINS. (C) 2003 Elsevier Science (USA). All rights reserved.
Regulation of floral scent production in petunia revealed by targeted metabolomics
Verdonk, J.C. ; Vos, C.H. de; Verhoeven, H.A. ; Haring, M.A. ; Tunen, A.J. van; Schuurink, R.C. - \ 2003
Phytochemistry 62 (2003). - ISSN 0031-9422 - p. 997 - 1008.
solid-phase microextraction - snapdragon flowers - linalool synthase - rosa-hybrida - in-situ - emission - volatiles - biosynthesis - expression - gene
Petunia hybrida line W115 (Mitchell) has large white flowers that produce a pleasant fragrance. By applying solid phase micro extraction (SPME) techniques coupled to GC-MS analysis, volatile emission was monitored in vivo using a targeted metabolomics approach. Mature flowers released predominantly benzenoid compounds of which benzaldehyde, phenylacetaldehyde, methylbenzoate, phenylethylalcohol, iso-eugenol and benzylbenzoate were most abundant. This emission had a circadian rhythm reaching its maximum at dusk. During petal limb expansion two sesquiterpenes were emitted by the petunia flowers, tentatively identified as germacrene D and cadina-3,9-diene. In vitro analysis showed that the petal limbs and stigma were the main producers of the benzenoids and sesquiterpenes, respectively. Moreover, comparison of in vivo and in vitro analysis indicated that volatiles were not stored during periods of low emission but rather were synthesized de novo. DNA-microarray analysis revealed that genes of the pathways leading to the production of volatile benzenoids were upregulated late during the day, preceding the increase of volatile emission. RNA-gel blot analyses confirmed that the levels of phenylalanine ammonia lyase (PAL) and S-adenosyl methionine (SAM) synthase transcripts increased towards the evening. Our results suggest that the circadian production of volatile benzenoids in petunia W115 is, at least partly, regulated at the transcript level.