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Forschungszentrum Karlsruhe
in der Helmholtz-Gemeinschaft
Wissenschaftliche Berichte
FZKA 7472
Molecular Biological and
(Eco)physiological Studies
on Isoprene Emission in
Arabidopsis and Grey Poplar
M. Loivamäki
Institut für Meteorologie und Klimaforschung
Atmosphärische Umweltforschung
April 2009 Forschungszentrum Karlsruhe
in der Helmholtz-Gemeinschaft
Wissenschaftliche Berichte
FZKA 7472

Molecular biological and
(eco)physiological studies on isoprene
emission in Arabidopsis and Grey poplar

Maaria Loivamäki
Institut für Meteorologie und Klimaforschung
Atmosphärische Umweltforschung

von der Fakultät für Forst- und Umweltwissenschaften der Universität
Freiburg genehmigte Dissertation

Forschungszentrum Karlsruhe GmbH, Karlsruhe

Für diesen Bericht behalten wir uns alle Rechte vor
Forschungszentrum Karlsruhe GmbH
Postfach 3640, 76021 Karlsruhe
Mitglied der Hermann von Helmholtz-Gemeinschaft
Deutscher Forschungszentren (HGF)
ISSN 0947-8620

Molecular biological and (eco)physiological studies on
isoprene emission in Arabidopsis and Grey poplar

Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Forst-
und Umweltwissenschaften der Albert-Ludwigs-Universität Freiburg i. Brsg.

vorgelegt von
Maaria Loivamäki

Freiburg im Breisgau

Dekan: Prof. Dr. Heinz Rennenberg
Referent: Jörg-Peter Schnitzler
Koreferent: Dr. Heinz
Datum der mündlichen Prüfung: 09.07.2008


Plants interact with their environment with a great variety of volatile organic compounds (VOCs),
isoprenoids ( ≡ terpenes), i.e. isoprene, mono-, homo-, di- and sesquiterpenes, being the most
prominent group. Isoprene, a hemiterpene, is the simplest isoprenoid compound whose main
source, woody plant species, comprises 75% of the 500 Tg C isoprene emitted to the atmosphere
per year. Due to the significant influence of isoprene in atmospheric chemistry, growing research
interests have focused to investigate this C5 compound. However, physiological function(s) of
isoprene emission in planta is not elucidated to date. Actual studies indicate that isoprene can
enhance thermotolerance or quench oxidative stress, but the underlying mechanisms are widely
unknown. The work presented here significantly contributes to the understanding of physiological
function of isoprene and regulation of isoprene biosynthesis by exploiting transgenic Arabidopsis
thaliana and Populus x canescens as model systems.

The first part of the work aimed to elucidate whether isoprene biosynthesis in plants is triggered by
endogenous regulatory mechanisms like the circadian clock. Isoprene emission varies diurnally in
several species, also in the natural isoprene emitter Grey poplar (P. x canescens). Moreover it was
recently proved that the poplar isoprene synthase gene (PcISPS) displays diurnal variation in its
expression. Working on shoot cultures of Grey poplar, placed under different light regime in climate
chambers, it was possible to show that under continuous light PcISPS expression, measured by
quantitative reverse transcriptase PCR, oscillated with amplitude of approximately 24 hours
testifying for endogenous clock regulation. Furthermore, circadian rhythms were not only limited to
the level of gene expression. Isoprene emission rates also displayed circadian changes. In
contrast, on the protein level circadian changes could not be detected. It, however, appeared that
PcISPS activity and protein content became reduced under constant darkness, while under
constant light activity and protein content were higher than under day/night regime. Measurement
of additional selected isoprenoid genes revealed that phytoene synthase (PcPSY; carotenoids
pathway) also displays circadian fluctuations of gene expression whereas 1-deoxy-D-xylulose 5-
phosphate reductoisomerase (PcDXR), the first committed enzyme of the methylerythritol
phosphate (MEP)-pathway only shows a light regulation of its expression.

In the second part of the work Arabidopsis thaliana (ecotype Columbia-0), a natural non-emitter of
isoprene, has been constitutively transformed with PcISPS from Grey poplar. Over-expression of
poplar ISPS in Arabidopsis resulted in isoprene emitting rosettes that showed enhanced growth
rates compared to wild type under moderate thermal stress. The fact that highest growth rates,
higher DMADP levels and ISPS enzyme activities were detected in young developing plants
indicates that enhanced growth of the transgenic plants under thermal stress is due to the
introduced PcISPS gene. However, the emission rates did not reach the level of natural isoprene
emitters, like poplars, suggesting possible different regulation of the isoprene biosynthesis and/or
lack of substrate for that. To study the physiology of these plants a dynamic gas exchange system
was developed allowing miming the natural rapid fluctuations of leaf temperature and light intensity
(‘sun- or lightflecks’) in order to study isoprene emission. The results showed that wild type
Arabidopsis is already well enough thermotolerant against transient and moderate light and/or heat
stress. In contrast, when the same conditions, combining light and heat flecks, were applied to wild
type and transgenic Grey poplar lines in which gene-expression of PcISPS was knock-down by
RNA interference technology, assimilation was impaired in the non-isoprene emitting lines
compared to wild type. Thus, for poplar the ability to emit isoprene can be detrimental.

Transgenic Arabidopsis lines were further applied in ecophysiological studies to investigate the role
of isoprene in plant-insect interactions. Feeding of herbivores on plants makes plants release
volatile compounds that attract herbivore enemies. In all the performed studies the parasitic wasp
Diadegma semiclausum searching for its host Plutella xylostella (Diamondback moth) preferred the
volatiles emitted by wild type Arabidopsis to those from transgenic, isoprene emitting Arabidopsis
plants. Furthermore low external isoprene concentration in the volatile blend of either Arabidopsis
or herbivore-infested Brassica oleracea, the natural host of the Diamondback moth, repelled the
parasitic wasps. The behaviors of the two examined herbivores (Pl. xylostella and Pieris rapae
(Small White Cabbage butterfly)) were not affected by isoprene emission, and GC-MS detection
showed despite of isoprene no other differences in the VOC blends of wild type and transgenic
plants. These findings suggest that isoprene emission of plants plays a complex ecophysiological
role, influencing biotic interactions between plants and insects.