Sinapate ester metabolism in Brassica and Arabidopsis [Elektronische Ressource] / von Carsten Milkowski

Milkowski

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2009
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 11 Mo

Extrait

Sinapate ester metabolism in Brassica
and Arabidopsis

HABILITATIONSSCHRIFT


zur Erlangung des akademischen Grades

doctor rerum naturalium habilitatus (Dr. rer. nat. habil.)


vorgelegt der


Biologisch-Pharmazeutischen Fakultät

der Friedrich-Schiller-Universität Jena

von


Dr. rer. nat. Carsten Milkowski

geb. am 30.07.1963 in Lübz
(Mecklenburg-Vorpommern)


Jena, den 9.April 2009






Gutachter

Prof. Dr. Jonathan Gershenzon
Max-Planck-Institut für Chemische Ökologie
Abteilung Biochemie
Hans-Knöll-Straße 8
07745 Jena




Prof. Dr. Birger Lindberg Møller
Department of Plant Biology
The Royal Veterinary and Agricultural University
Thorvaldsensvej 40
DK-1871 Frederiksberg C, Copenhagen
Denmark




Prof. em. Dr. Thomas Hartmann
Technische Universität Carolo-Wilhelmina zu Braunschweig
Institut für Pharmazeutische Biologie
Mendelssohnstraße 1
38106 Braunschweig







Erteilung der Lehrbefähigung für das Fach „Allgemeine Botanik“

14. Dezember 2009 TABLE OF CONTENTS

1 Introduction……………………………………………………….3

2 Genes and Enzymes of Sinapate Ester Metabolism…………….5

2.1 Glucosyltransferases……………………………………………...7
2.2 Acyltransferases…………………………………………………..9
2.3 Sinapine esterase………………………………………………...11

3 Evolution………………………………………………………...12

4 Metabolic engineering…………………………………………..24

4.1 Suppression of biosynthesis…………………………………….25
4.2 Metabolic diversion……………………………………………..28
4.3 Induced degradation……………………………………………29
4.4 Economical relevance…………………………………………..31

5 Summary and outlook………………………………………….32

6 References………………………………………………………34

7 List of included publications…………………………………..42

8 Statement on personal contributions ………………………….43


1 Introduction

Higher plants display a tremendous metabolic plasticity. This is illustrated by their
ability to synthesize myriads of so-called secondary organic compounds (‘natural products’)
that seem to be dispensable for growth and development and are often differentially
distributed among limited taxonomic groups (Harborne and Turner 1984). Investigation of
these plant products was initiated about 200 years ago by Friedrich Wilhelm Sertürner, who
confined the active principle of opium poppy to a single organic substance, known as
morphine. Since then, the structures of more than 200,000 plant natural products have been
elucidated and assigned to the groups of terpenoids, alkaloids, polyketides or
phenylpropanoids and various derived phenolic compounds (Wink 1988; Hartmann 2007).
As functional aspects of plant secondary metabolism were hardly apparent, its
products have long been considered to be inert metabolic waste or detoxification products
(Peach 1950). This view changed when entomologists recognized secondary metabolites as
chemical mediators between plants and insects and established the concept of coevolution
(Fraenkel 1959). Eventually, this line of research unravelled the enormous ecological
importance of plant secondary compounds.
On the other hand, products of secondary metabolism pathways have also proven
functional in internal plant processes. This is ideally illustrated by some terpenoids acting as
plant hormones like abscisic acid or gibberellins. Lignin is the second most abundant
biopolymer on earth (Buchanan et al. 2000). It provides rigidity and impermeability to
secondary thickened cell walls, thus being indispensable for plant structure and defense as
well as for the development of xylem elements as an efficient long-distance water transport
system. Accordingly, in evolutionary terms, the establishment of the phenylpropanoid
metabolic pathway toward lignin was a basic requirement for plants to conquer terrestrial
biotopes (Boudet 2007). As products of another branch of the phenylpropanoid pathway,
flavonoids are ubiquitously distributed among higher plants and serve a wide array of
fundamental functions as revealed by their involvement in attraction of pollinators and seed
dispersers, protection against UV-B radiation or the establishment of plant-rhizobium bacteria
interactions for nitrogen fixation in legumes. In Arabidopsis, flavonoids have been shown to
be involved in auxin transport (Brown et al. 2001). These are only few prominent examples,
which indicate that a strict line between primary and secondary metabolism is difficult to be
drawn. Hence, in contrast to the traditional meaning of ‘less important’, the term ‘secondary’
is now interpreted as an indispensable layer of functionality that is inherent to plant metabolic
3networks and contributes significantly to the plasticity of plant metabolism, which is required
to afford the sessile life style of a land plant under changing environmental conditions
(Hartmann 2007).
Only in recent years, the combined application of the upcoming Omics technologies
for gene, protein and metabolite analyses begins to discern the many interactions within the
network of secondary metabolism and between secondary and primary metabolism (Böttcher
et al. 2008). This paves the way to a better understanding of plant metabolism in its
outstanding complexity and will support targeted metabolic engineering approaches to
generate plants with altered metabolite contents for food industry or pharmaceutical use
(Dixon 2005). The complete transfer of the biosynthetic pathway of the cyanogenic glucoside
dhurrin from Sorghum bicolor to Arabidopsis making this plant cyanogenic demonstrated
both the feasibility and the power of engineering secondary plant metabolism (Tattersall et al.
2001). The most prominent example of such a combinatorial biosynthesis in plants is the so-
called ‘Golden Rice’ (Ye et al. 2000). It was generated by simultaneous expression of a
phytoene synthase and a lycopene β-cyclase from Narcissus pseudonarcissus as well as a
bacterial phytoene desaturase from Erwinia uredovora in the rice endosperm to produce β-
carotene (provitamin A). Concerning phenylpropanoid metabolism, a fundamental interest in
improving the processing efficiency of plant biomass for pulping, forage digestibility and
biofuels has produced a wealth of approaches to engineer the amount and composition of
lignin in several plant species (reviewed by Vanholme et al. 2008).
The inherent flexibility of plant secondary metabolism is reflected by a remarkable
plasticity of plant genomes. Within the Arabidopsis genome 15-20% of genes are predicted to
be involved in secondary metabolism (D’Auria and Gershenzon 2005). Accordingly, many
enzymes of plant secondary metabolism are organized in gene families that have developed
from single or few hypothetical ancestors (Moore and Purugganan 2005). In the last decade,
of several enzymes involved in plant secondary metabolism unexpected homologs with
functions in primary metabolism have been detected (Ober and Hartmann 1999; Steffens
2000). This has encouraged novel research strategies aimed at understanding the evolution of
metabolic diversity.
Our research is focused on the metabolism of soluble sinapate esters. These
compounds are a hallmark of Brassicaceae plants (Bouchereau et al. 1991) that enabled us to
work with the model plant Arabidopsis. For a long time it has been known that seeds of many
Brassicaceae species accumulate considerably high amounts of sinapine, the choline ester of
sinapate, as the predominating phenolic compound (Gadamer 1897). Given its antinutritive
4impact on the seed protein fraction of the major crop plant Brassica napus (oilseed rape;
Canola), a low sinapine content has become a major aim of conventional and molecular
breeding programmes designed to increase the nutritional value of seeds.
The subsequent chapters describe the progress in gene identification, characterization
of enzymes and targeted engineering of sinapate ester metabolism made by our and other
laboratories throughout the last decade. With emphasis on B. napus and Arabidopsis, the
structure and function of the relevant proteins and genes will be discussed and aspects of
evolutionary enzyme recruitment will be surveyed.


2 Genes and Enzymes of Sinapate Ester Metabolism

Sinapate (3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-enoate), the phenolic component
of sinapate esters, is produced by the phenylpropanoid pathway (Figure 1). The general part
of this metabolism converts the aromatic amino acid L-phenylalanine, which is provided by
the plastid-located shikimate pathway, to 4-coumaroyl-CoA. This energy-rich thioester marks
an important branchpoint of phenylpropanoid metabolism since it feeds into different types of
hydroxycinnamate side-chain reactions (Barz et al. 1985), i.e. extension with formation of
additional ring systems (e.g. flavonoids or stilbenes), degradation (e.g. hydroxybenzoates),
reduction (e.g. hydroxycinnamyl alcohols fueling lignin biosynthesis), oxidation and
lactonization (e.g. coumarins) o