Functional analysis of the sucrose synthase gene family in Arabidopsis thaliana [Elektronische Ressource] / von Zuzanna Bieniawska

Zuzanna Bieniawska

Sujets

• Biologie

Informations

• Publié par: universitat_potsdam
• Publié le: 01 janvier 2006
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Dissertation



Functional analysis of the sucrose synthase gene family
in Arabidopsis thaliana






vorgelegt an der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Potsdam




von Zuzanna Bieniawska


geb. am 12.04.1978 in Wroclaw (Polen)




zur Erlangung des akademischen Grades


Dr. rer. nat.








Wissenschaftsdisziplin: Molekularbiologie der Pflanzen



6. März 2006













































Cz łowiek mo że znieść bardzo du żo,
lecz pe łnia błąd s ądz ąc,
że potrafi znieść wszystko

Fiodor Dostojewski



Abstract

Abstract

Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the
reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its
activity, localization and function have been studied in various plant species. It has been
shown that Susy can play a role in supplying energy in companion cells for phloem loading
(Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and
supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001).
Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of
these isoforms was investigated using promoter-reporter gene constructs (GUS) and real
time RT-PCR. Although these isoforms are closely related at the protein level they have
radically different spatial and temporal patterns of expression in the plant with no two
isoforms showing the same distribution. More than one isoform is expressed in all organs
examined. Some of them have high but specific expression in particular organs or
developmental stages whilst others are constantly expressed throughout the whole plant and
across various stages of development.
The in planta function of the six Susy isoforms were explored through analysis of
T-DNA insertion mutants and RNA lines. Plants without the expression of individual i
isoforms show no differences in growth and development, and are not significantly
different from wild type plants in soluble sugars, starch and cellulose contents under all
growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform
that was exclusively expressed in stomata cells only had a minor influence on guard cell
osmoregulation and/or bioenergetics.
Although none of the sucrose synthases appear to be essential for normal growth
under our standard growth conditions, they may be necessary for growth under stress
conditions. Different isoforms of sucrose synthase respond differently to various abiotic
stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and
increases total Susy activity. However, the analysis of the plants with reduced expression of
both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen
deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has
no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data
provide a comprehensive overview of the expression of this gene family which supports
some of the previously reported roles for Susy and indicates the involvement of specific
isoforms in metabolism and/or signalling.
3 Index

Index

Abstract .................................................................................................................................3

Index.......4

Abbreviations........................................................................................................................7


1 Introduction ........................................................................................................... 9
1.1 Sucrose synthesis ................................................................................................................ 9
1.2 Transport of sucrose.......................................................................................................... 10
1.3 Sucrose breakdown 11
1.3.1 Sucrose synthase ............................................................................................................... 12
1.3.1.1 Cell and tissue specific expression ................................................................................... 12
1.3.1.2 Environmental influence on expression............................................................................ 13
1.3.1.3 Protein modification 14
1.3.1.4 Subcellular distribution..................................................................................................... 15
1.3.2 Invertases .......................................................................................................................... 16
1.3.2.1 Neutral/Alkaline invertases............................................................................................... 16
1.3.2.2 Acid invertases.................................................................................................................. 16
1.4 UGPase, important crossroad of sucrose biosynthesis and breakdown ............................ 18
1.5 Importance of UDPG ........................................................................................................ 19
1.6 Aims and objectives of the present work.......................................................................... 22


2 Materials and Methods ....................................................................................... 23
2.1 Chemicals 23
2.2 Plant material .................................................................................................................... 23
2.3 Seed sterilization and tissue culture.................................................................................. 24
2.4 Plant growth and harvest................................................................................................... 24
2.5 Plasmids and cloning work ............................................................................................... 26
2.6 Transformation of bacteria................................................................................................ 27
2.7 Plant transformation.......................................................................................................... 27
2.8 GUS staining..................................................................................................................... 27
2.9 RNA extraction and cDNA synthesis ............................................................................... 27
2.10 Primer designing and real-time RT-PCR reaction ............................................................ 28
2.11 Mutant screening............................................................................................................... 29
2.12 Extraction of soluble sugars and cell wall components .................................................... 30
2.13 Assay of reducing sugars, sucrose and starch ................................................................... 31
2.14 Anthron assay for cellulose determination........................................................................ 31
2.15 Extraction and assay of Susy ............................................................................................ 32
2.16 of UGPase ....................................................................................... 32
2.17 Gas exchange measurements 32
2.18 Chlorophyll fluorescence.................................................................................................. 33
2.19 Extraction of Susy for protein gels and immunoblotting.................................................. 33
2.20 Freezing experiments........................................................................................................ 34
2.21 Data calculation for array hybridisation expression analysis............................................ 34
2.22 Construction of phylogenetic tree..................................................................................... 35



4 Index

3 Results................................................................................................................... 36
3.1 Phylogenetic analysis of sucrose synthases from Arabidopsis and other plant species.... 36
3.2 Gene expression studies of the sucrose synthase gene family in Arabidopsis.................. 38
3.2.1 Quantitative expression analysis of sucrose synthase gene family using real-time
RT-PCR ............................................................................................................................ 38
3.2.2 Expression analysis using publicly available data of array hybridisations ....................... 39
3.2.2.1 Expression of sucrose synthase isoforms in different organs and in different
developmental stages ............................................................................