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

Sujets

• Biologie

Informations

• Publié par: universitat_potsdam
• Publié le: 01 janvier 2005
• Nombre de lectures: 65
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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