15th International Conference on Arabidopsis Research - TAIR

T07-079
Biochemical and Molecular Analysis of Constitutive
and Inducible Terpene Volatile Emission from
Arabidopsis thaliana
Dorothea Tholl(1, 2), Feng Chen(2), Christian Abel(1), Jana Petri(1), Eran
Pichersky(2), Jonathan Gershenzon(1)
1-Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany
2-Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor
MI 48109, USA
Terpene secondary metabolites have been described to exhibit important
functions in direct plant defenses against herbivores and pathogens and as
volatile signaling compounds in indirect defense or the attraction of pollinators.
Recent results have led to exciting new insights into the potential role
of volatile terpenes in thermotolerance and the protection of plants against
ozone and oxidative stress.
We are using Arabidopsis as model system to gain deeper insights into
the biochemistry, regulation and biological functions of terpene volatile
biosynthesis. The Arabidopsis genome contains a large family of over thirty
terpene synthase (AtTPS) genes. Terpene synthases catalyze the formation of
monoterpenes (C10), sesquiterpenes (C15) or diterpenes (C20) from geranyl
diphosphate (GPP), farnesyl diphosphate (FPP) or geranylgeranyl diphosphate
(GGPP), which are central intermediates of the isoprenoid pathway. Applying a
functional genomics approach, we have identified several terpene synthases
which are constitutively expressed in Arabidopsis roots and flowers and are
responsible for the emission of a complex blend of mono- and sesquiterpenes
volatiles (Chen, Tholl et al., 2003). Promoter-GUS analyses revealed that
floral AtTPS genes are expressed in several different organs including sepals,
anther filaments, ovules, stigma and nectaries. A detailed investigation of
several Arabidopsis ecotypes showed variation and differential regulation of
floral volatile emission. We are currently investigating particular ecotypes and
floral TPS gene knock out and overexpression lines to explore the potential
role of floral sesquiterpenes in protection of the reproductive tissues against
oxidative stress conditions or microbial attack. Similar molecular approaches
are applied to analyze antioxidant, signaling or defense roles of stress inducible
terpene volatiles released from Arabidopsis rosette leaves. Moreover, we
are undertaking comparative biochemical and molecular analyses of terpene
biosynthesis and emission from A. thaliana and its close relatives including A.
lyrata and Boechera drummondii. Overall, the results should lead to new insights
and a more precise understanding of the ecophysiological significance,
regulation and evolution of plant terpene volatile biosynthesis in response to
abiotic and biotic stress factors.
Chen, F.*, Tholl, D.*, D’Auria, J., Farooq, A., Pichersky, E., and Gershenzon, J., 2003, Plant Cell,
15, 481-494
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)
T07-080
Uniform stable isotope labeling of Arabidopsis
thaliana opens hetero-nuclear multi-dimensional
NMR-based metabolomics
Jun Kikuchi(1, 2), Kazuo Shinozaki(3, 4), Takashi Hirayama(2, 3)
1-Protein Research Gr., GSC, RIKEN Yokohama Inst.
2-Grad. Sch. Integr. Sci., Yokohama City Univ.
3-Plant Funct. Gr., GSC, RIKEN Yokohama Inst.
4-Lab. Plant Mol. Biol., RIKEN Tukuba Inst.
As any plant scientist does not doubt importance of plant metabolomics in
post-genomics-proteomics era, recent methodological advances of FT-MS
analysis lead in this field. However, the MS technology has potentially
disadvantages to differentiate identical molecular mass isomers and low
reproducibility due to ion-suppression effect. Therefore, NMR will become a
key technology in plant metabolomics with the use of stable isotope labeling
and advanced hetero-nuclear NMR methodologies. Since the NMR-based
approach has an advantage in comparison with different samples, spectral
subtraction between different mutants or stimuli enable quantification of increased
or decreased metabolites among those samples. To demonstrate the
power of this approach, we performed multi-dimensional hetero-nuclear NMR
analysis of metabolic movement of carbon and nitrogen nuclei in Arabidopsis
thaliana [1]. First, distinct ethanol-stress response was clearly investigated
by the uniformly 13C-Glc-labeled plants in the wild type and an ethanol-hypersensitive
mutant [2]. Furthermore, inexpensive, and non-harmful uniform
13C labeling was achieved by photosynthetic incorporation of 13CO2. We
monitored time-dependence and tissues dependent changes of 13C incorporation
by use of multi-dimensional NMR. Over two weeks growth in 13CO2
atmosphere, 1H-detected multi-dimensional NMR measurements such as
2D-1H-13C HSQC, 3D-1H-13C-HCCH-COSY exhibit to detect reasonable
number of cross-peaks to perform non-targeting metabolomics analysis. In
addition to above extracted, in vitro sampling, the NMR-based methods can
be used non-invasively and can yield (albeit limited) spatial information about
gradients of solute concentrations. Therefore, we followed nitrogen fluxes
in 15N-labeled seeds during the initiation of germination in vivo. Although
above advantages in NMR-based methodologies, their low sensitivity offers
to restrict measurements of relatively high concentration compounds. This
disadvantage is being overcome due to progress in NMR technology, like as
the world highest magnetic-field (920 MHz) NMR spectrometer [3] and the
achievement of the coolest (4.5 K) high-sensitive cryogenically cooled probe
[4], both developed by our groups.
[1]Kikuchi et al(2004)P.C.P. [2]Hirayama et al(2004)P.C.P. [3]Kiyoshi et al(2004)I.T.A.S. [4]Yokota
et al(2004)A.C.E.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin