Molecular studies on plants to enhance their stress tolerance [Elektronische Ressource] / by Alaa El-din A. Helaly

Molecular studies on plants to enhance their stress tolerance [Elektronische Ressource] / by Alaa El-din A. Helaly

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From the University of Potsdam and the Max-Planck Institute of Molecular Plant Physiology Molecular studies on plants to enhance their stress tolerance DISSERTATION A thesis submitted to the Institute of Biochemistry and Biology Faculty of Mathematics and Natural Sciences University of Potsdam For the degree of Doctor of natural sciences (doctor rerum naturalium) in molecular biology By Alaa El-din A. Helaly Potsdam 2004 Contents 1 Introductiuon 1 1.1 Transcription factors in eukaryotes 1 1.2 Transcription factor domain 2 1.3 Zinc finger proteins 3 1.4 AtSTO1 as a putative transcription factor 6 1.5 Regulatory role of transcription factor proteins under stress 7 1.6 Mechanism of salt stress and plant response 101.7 Proline biosynthesis in plants under stress 121.8Anthocyanin biosynthesis 14 2 Aim of this work 16 3Materials and methods 17 3.1 Chemicals and enzymes 173.2 Oligonucleotides 173.3 Vectors and plasmids 183.4 Bacteria 183.4.1 Escherichia coli 183.4.2 Agrobacterium tumefaciens 183.5 Plants 183.6Growth conditions 183.6.1 Bacteria 183.6.2 Tobacco BY2 cells 193.6.3 Plants 193.6.3.1 Transformation in A. thaliana 193.6.3.2 Seed sterilization193.6.3.2 Tissue culture 193.6.3.3 Phytotron 203.7 Molecular methods203.7.1 Cloning and sequence 203.7.2 Amplification of DNA fragments via polymerase chain reaction 21 i3.7.

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Publié le 01 janvier 2005
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From the University of Potsdam

and the

Max-Planck Institute of Molecular Plant Physiology






Molecular studies
on plants to enhance their
stress tolerance






DISSERTATION





A thesis submitted to the
Institute of Biochemistry and Biology
Faculty of Mathematics and Natural Sciences
University of Potsdam





For the degree of
Doctor of natural sciences (doctor rerum naturalium) in molecular biology








By

Alaa El-din A. Helaly

Potsdam 2004
Contents

1 Introductiuon 1
1.1 Transcription factors in eukaryotes 1
1.2 Transcription factor domain 2
1.3 Zinc finger proteins 3
1.4 AtSTO1 as a putative transcription factor 6
1.5 Regulatory role of transcription factor proteins under stress 7
1.6 Mechanism of salt stress and plant response 10
1.7 Proline biosynthesis in plants under stress 12
1.8Anthocyanin biosynthesis 14

2 Aim of this work 16

3Materials and methods 17

3.1 Chemicals and enzymes 17
3.2 Oligonucleotides 17
3.3 Vectors and plasmids 18
3.4 Bacteria 18
3.4.1 Escherichia coli 18
3.4.2 Agrobacterium tumefaciens 18
3.5 Plants 18
3.6Growth conditions 18
3.6.1 Bacteria 18
3.6.2 Tobacco BY2 cells 19
3.6.3 Plants 19
3.6.3.1 Transformation in A. thaliana 19
3.6.3.2 Seed sterilization19
3.6.3.2 Tissue culture 19
3.6.3.3 Phytotron 20
3.7 Molecular methods20
3.7.1 Cloning and sequence 20
3.7.2 Amplification of DNA fragments via polymerase chain reaction 21
i3.7.3 Cloning strategies 21
3.7.3.1 Isolation of the AtSTO1 cDNA 21
3.7.3.2 Isolation of the AtSTO1 promoter 21
3.7.3.3 Subcloning of AtSTO1 cDNA into pA7-GFP 22
3.7.3.4 Subcloning of AtSTO1 cDNA into pGreen0229 22
3.7.3.5 Generating an AtSTO1 RNAi construct 22
3.7.4 Isolation of DNA and RNA 22
3.7.4.1 Isolation of genomic DNA 22
3.7.4.2 Isolation of RNA 23
3.7.5 Northern blot hybridizations 23
3.7.6 3.7.6 T-DNA insertion line of AtSTO1 24
3.7.7 Stress experiments 24
3.7.7.1 Cold stress 24
3.7.7.2 Salt stress 25
3.7.7.3 Drought stress 26
3.7.7.4 Mannitol stress 27
3.7.7.5 Light stress 28
3.7.8 Histochemical localization of GUS activity 28
3.7.9 Microscopic analysis 28
3.7.10 Transformation of tobacco BY2 cells 29
3.7.11 Biochemical analysis 30
3.7.11.1 Determination of proline 30
3.7.11.2 Determination of soluble sugars 30
3.7.11.3 Determination of pigments 31
3.8 Statistical analysis 31

4Results 32

4.1Identification of the STO family 32
4.1.1 Alignment report of STO proteins in Arabidopsis, rice and other plants 32
4.1.2 Phylogenetic tree of the STO family 36
4.1.3 Sequence analysis of the intron/exon regions of AtSTO genes 37
4.2 Histochemical analysis of AtSTO1 promoter activity 38
4.2.1 Analysis of AtSTO1 promoter activity in different plant tissues 38
ii4.2.2 Stress-dependent regulation of AtSTO1 promoter activity 41
4.2.3 Cis-elements analysis of the AtSTO1 promoter 42
4.3 Intracellular localisation of AtSTO1 Protein 44
4.4 Effect of abiotic stress on AtSTO1 transcript level 47
4.4.1 Effect of salt stress on AtSTO1 transcript level 47
4.4.2 Effect of cold stress (4°C) on AtSTO1 transcript level 48
4.4.3 Effect of drought stress on AtSTO1 transcript level 48
4.4.4 Effect of light stress on AtSTO1 transcript level 49
4.4.5 Effect of osmotic stress on AtSTO1 transcript level 49
4.5 Overexpression of AtSTO1 in transgenic Arapidopsis plants 50
4.6 Generation of AtSTO1 RNAi transgenic lines 51
4.7 Identification of an AtSTO1 of T-DNA insertion line 52
4.8 Physiological analysis of AtSTO1 overexpression and RNAi plants 54
under different stress conditions
4.8.1 Analysis of AtSTO1 overexpression and RNAi plants under normal 54
growth conditions
4.8.2 Analysis of AtSTO1 overexpression and RNAi plants under salt stress 54
4.8.3 Analyssion and RNAi plants under cold stress 56
4.8.4 Analysis AtSTO1 overexpression and RNAi plants under drought 57
stress
4.8.5 Analysis AtSTO1 overexpressi osmotic 58
stress
4.9 Chemical analysis of AtSTO1 transgenic lines 59
4.9.1 The proline content of AtSTO1 transgenic lines 59
4.9.1.1 Proline content under normal growth conditions 59
4.9.1.2 Proline content under salt stress 59
4.9.1.3 Proline content under cold stress 61
4.9.1.4 Proline content under drought stress 62
4.9.2 Soluble sugars in AtSTO1 transgenic lines 62
4.9.2.1 Soluble sugar content under normal growth conditions 63
4.9.2.2 Soluble sugar content under salt stress 63
4.9.2.3 ontent under cold stress 66
4.9.2.4 Soluble sugar content under drought stress 68
4.9.3 Pigments content in AtSTO1 transgenic lines 70
iii4.9.3.1 Pigment content under normal growth condition 70
4.9.3.2 Pigment content under salt stress 70
4.9.3.3 Pigment content under cold stress 72
4.9.3.4 Pigment content under drought stress 75

5Discussion 76

5.1 Proteins of STO family are likely to be transcription factors 76
5.2 STO proteins and their phylogenetic relationship 77
5.3 Expression of AtSTO1 in response to abiotic stresses 79
5.3 Nuclear localization of AtSTO1 82
5.4 Phenotypes of AtSTO1 transgenic plants under abiotic stress 83
5.5 Effect of abiotic stress on the proline content in AtSTO1 transgenic 85
plants
5.6 Effect of abiotic stress on soluble sugars in AtSTO1 transgenic plants 86
5.7 Effect of abiotic stress on pigment contents in AtSTO1 transgenic 88
plants

6Summary 90

7References 92




















ivList of Tables

Table 1 Structural feature of conserved domains that are used to classify plant 3
transcription factors
Table 2 STO proteins in Arabidopsis thaliana, Oryza sativa and other plant 35
species
Table 3 Intron and exon size of the AtSTO genes family from Arabidopsis 38
thaliana
Table 4 Effect of abiotic and osmotic stress on root length (cm) of Arabidopsis 55
thaliana wild-type, overexpression and RNAi lines
Table 5 Effect of normal growth condition and abiotic stress on proline content 60
(µmol/100mg FW) in leaves of Arabidopsis thaliana wild type,
overexpression and RNAi lines after 1 day and 2 weeks of stress treated
Table 6 Effect of normal condition and abiotic stress on proline content 60
µmol/100mg FW in roots of Arabidopsis thaliana wild type,
overexpres1 day and 2 week of treated plants
Table 7 Soluble sugar contents in leaves and roots of Arabidopsis thaliana wild- 63
type, overexpression and RNAi lines grown under standard condition
Table 8 Effect of salt stress on soluble sugars in leaves of Arabidopsis thaliana 64
wild-type, overexpression and RNAi lines after 1 day and 2 weeks of
treatment
Table 9 Effect of salt stress on soluble sugars in roots of Arabidopsis thaliana 64
wild-type, overexpression and RNAi lines after 1 day and 2 weeks of
treatment
Table 10 Effect of cold stress on soluble sugars in leaves of Arabidopsis thaliana 67
wild-type, overexpression and RNAi
growth at 4°C
Table 11 Effect of cold stress on soluble sugars in roots of Arabidopsis thaliana 67
wild-type, overexpression and RNAi lines after 1 day and 2 week of
growth at 4°C
Table 12 Effect of drought stress on soluble sugars in leaves of Arabidopsis 68
thaliana wild-type, overexpression and RNAi lines after 4 day and 2
weeks of growth without watering
Table 13 Effect of drought stress on soluble sugar contents in roots of Arabidopsis 69
vthaliana wild-type, overexpression and RNAi lines after 4 day and 2 week
of grow without watering
Table14 Effect of normal growth condition on the content of chlorophyll a, b and 70
total chlorophyll (T. Chlorophyll), cartenoids and anthocyanins in leaves
of Arabidopsis thaliana wild-type, overexpression and RNAi lines (4-
week-old plants)
Table 15 Effect of salt stress on the content of chlorophyll a, b, total chlorophyll (T. 71
Chlorophyll), cartenoids and anthocyanins in leaves of wild-type and
transgenic plants (at 4-week-old)
Table 16 Effect of cold stress on the content of chlorophyll a, b and total 72
chlorophyll (T. Chlorophyll), cartenoids and anthocyanins in leaves of
Arabidopsis thaliana wild-type, overexpression and RNAi lines (4 week
old; 2 week of exposure to 4°C)
Table 17 Effect cold stress on the content of chlorophyll a, b and total chlorophyll 74
(T. chlorophyll), cartenoids and anthocyanin in leaves of Arabidopsis
thaliana wild- type, overexpression and RNAi lines (6 week old or 4
weeks of exposed to4°C)
Table 18 74
(T. Chlorophyll), cartenoids and anthoion and RNAi lines (8 week old or 6
weeks of exposed to4°C)
Table 19 Effect cold stress on the content of chlorophyll a, b and total chlorophyll 75
(T. chlorophyll), cartenoids and anthocyanin in flowers of Arabidopsis
thaliana wild- type, overexpression and RNAi lines (8 week old or 6
weeks of exposed to4°C)
Table 20 Effect of drought stress on the content of chlorophyll a, b and total 75
chlorophyll (T. chlorophyll), cartenoids and anthocyanins in leaves of
Arabidopsis thaliana wild-type, overexpression and RNAi lines (4 weeks
old; after 2 weeks of final watering)








viList of Figures

Figure 1 Cellular homeostasis established after salt (NaCl) adaptation 11
Figure 2 Alternative pathways of proline synthesis in higher plants 13
Figure 3 Alignment of 27 amino acid sequences of the STO family containing 33
zinc finger motifs and their flanking regions
Figure 4 The similarity percentage of 26 STO proteins 34
Figure 5 Phylogenetic tree of the STO-like family in Arabidopsis, rice and 36
other plant species
Figure 6 Schematic representation of the intron-exon structure of 8 genes 38
encoding AtSTO proteins
Figure 7 Histochemical analysis of GUS expression in leaves 39
Figure 8 is Of GUS activity in roots of plants carrying the 40
AtSTO1-GUS transgene
Figure 9 Histochemical analysis of GUS activity in leaves of plants carrying 40
the AtSTO1-GUS constructs
Figure 10 is of GUS activity in flowers carrying the 41
AtSTO1-GUS transgene
Figure 11 Histochemical analysis of AtSTO1 promoter activity under different 42
stress conditions
Figure 12 The structure of the promoter region of the AtSTO1 gene 43
Figure 13 pSTOA7-GFP fussion protein expressed transiently in tobacco BY2 45
protoplast
Figure 14 Effect of salt treatment (200 mM NaCl) on AtSTO1 transcript level 47
Figure 15 Effect of cold stress (4°C) on AtSTO1 transcript level 48
Figure 16 Effect of drought stress on AtSTO1 transcript level 49
Figure 17 Effect of light stress on AtSTO1 transcript level 49
Figure 18 Effect of osmotic stress on AtSTO1 transcript level 50
Figure 19 The AtSTO1 overexpression plants 50
Figure 20 Position of the RNA interference fragment in the AtSTO1 coding 51
region used to generate RNAi lines
Figure 21 Northern blot analysis of AtSTO1 RNAi transgenic lines 52
Figure 22 Analysis of AtSTO1 T-DNA insertion line 53
Figure 23 Analysis of AtSTO1 overexpression and RNAi plants grown for 4 54
weeks under normal condition
viiFigure 24 Analysis of AtSTO1 overexpression and RNAi plants grown for 4 55
weeks under salt stress
Figure 25 Analyspression and RNAi phenotype under cold 56
stress
Figure 26 Analysis of AtSTO1 overexpression and RNAi lines under drought 57
stress
Figure 27 Analysis of AtSTO1 overexpression and RNAi phenotype under 58
osmotic stress
Figure 28 Proline content in leaves and roots of 4-week-old Arabidopsis 61
thaliana wild-type (WT), AtSTO1 overexpression (O.x) and RNAi
lines (RNAi)
Figure 29 Effect of standard groth conditions on soluble sugar content 62
Figure 30 Effect of salt stress (200 mM NaCl) on soluble sugar content 65
Figure 31 Effect of cold stress (4°C) on soluble sugar content 66
Figure 32 Effect of drought stress on soluble sugar content 69
Figure 33 Effect of several stress conditions on chlorophyll a, b, total 71
chlorophyll (T. Chlorophyll) and cartenoids content
Figure 34. Effect long-term of cold and salt stress on pigment content 73
Figure 35 The CCT domain and COOH region peptides of Arabidopsis thaliana 76
proteins are included for comparison
Figure 36 Schematic representation of the induction of two rd29 genes and 80
their cís-acting elements involved in stress-responsive expression
Figure 37 Induction of AtSTO family members by different stresses 81

viii Introduction
1 Introduction

1.1 Transcription factors in eukaryotes

Detailed analyses of completely sequenced genomes reveal that a significant
percentage of the encoded proteins correspond to transcription factors. These can be
classified into several gene families according to the presence of particular DNA
binding domains (Boggon et al., 1999). However, the analysis of a particular
transcription factor should be done in the context of the family, to which it belongs,
taking into account that functional redundancy is a very frequent event within
eukaryotic TFs (Jakoby et al., 2002). Also the sequencing of the Arabidopsis genome
allows a comparative analysis of transcriptionl regulators among the three eukaryotic
kingdoms. Thereby the content of transcriptional regulators is compared with trans-
regulators in other eukaryotic organisms: the transcription regulators represent
approximately 3.5, 3.5, and 4.6 % of the genes in Saccharomyces cerevisiae,
Caenorhabditis elegans, and Drosophila melanogaster, respectively, and around 5%
in humans. Regulation of gene expression is associated with most biological
phenomena and is largely mediated through proteins that interact directly or indirectly
with specific DNA sequences (cis-elements) in the promoter region of genes.
Transcription factors play critical roles in all aspects of a higher plant’s life. It is the
programmed and regulated interactions between transcription factors and genomic
DNA that bring a genome to its life and define many of its functional features
(Grandori et al., 2000; Dimova et al., 2003; Kohler et al., 2003). An initial analysis
indicated that Arabidopsis thaliana has at least 1,572 transcription factors genes
(approximately 6% of the coding capacity of its genome) that belong to more than 45
different families, each possessing a highly conserved and characteristic region
recognized as the DNA-binding domain (Takatsuji 1998; Riechmann et al., 2000;
Ratcliffe and Riechmann, 2002). Transcription factors are usually classified as
proteins that show sequence-specific DNA binding and are capable of activating
and/or repressing transcription. Most of the known transcription factors can be
grouped into families according to their DNA binding domain. There is a large
variation in the transcription factor domains among different organisms, and the
Arabidopsis transcription factors that belong to families that are common to all
eukaryotes do not share significant similarity with those from the other kingdoms.
However, there are conserved DNA binding domains that define the respective
1