Selection and characterisation of the awake mutants with altered seed dormancy in response to ...

166 Pages·2016·5.63 MB·English

Selection and characterisation of the awake mutants
with altered seed dormancy in response to
temperature in Arabidopsis thaliana (L.) Heyn.

Submitted by FABIO FEDI to the University of Exeter as a thesis for the degree
of Doctor of Philosophy in Biological Sciences
In August 2015

This thesis is available for Library use on the understanding that it is copyright
material and that no quotation from the thesis may be published without proper
acknowledgement.

I certify that all material in this thesis which is not my own work has been
identified and that no material has previously been submitted and approved for
the award of a degree by this or any other University.

(Signature) ………………………………………………………………………………
Abstract
Abstract

Seed dormancy is a mechanism with great importance in plant fitness and it
inhibits seed germination until is broken and seeds can germinate under optimal
environmental conditions favorable for successful reproduction. Primary
dormancy is contingent to the environment that seeds and the mother plant
experience. Temperature is a major factor participating in the regulation of this
complex trait. High and low levels of dormancy are induced during seed
maturation by cold and warm temperatures respectively but the mechanism at
the basis of temperature signaling in seeds is not well understood. Climate
change and increased weather variability threaten the constant supply of high
quality seeds into the market hence agriculture productivity. Therefore,
understanding and taking control of the molecular mechanism behind the
regulation of seed dormancy and germination will help to control and predict
seed behavior in the field.

Here I describe and discuss a forward genetic screen for the selection of mutant
seed lines with altered seed dormancy in response to cool temperature during
seed set. Putative mutant seed lines designated awake1 to awake52, were
preliminarily characterized. Eleven awake lines were selected for further
analysis and one was investigated in more detail. It was revealed that awake1
seeds shares common phenotype with seeds of a suberin deficient mutant
which were previously reported to display increased dormancy but, here, I show
they also display a reduction of seed dormancy. Segregation analysis suggests
that the reduced dormancy phenotype is maternally inherited as the suberin
deficient mutants. Also, transcriptomic analysis shows that many suberin
associated genes are temperature-regulated. I conclude that control of suberin
deposition may play a role in the regulation of dormancy in response to cool
temperature.

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Table of contents
Table of contents
Abstract ............................................................................................................. ii
List of figures .................................................................................................... ii
List of tables ..................................................................................................... v
Publications ..................................................................................................... vi
Acknowledgments .......................................................................................... vii
Abbreviations ................................................................................................ viii
1. Introduction ................................................................................................ 1
1.1. Seed dormancy and germination .............................................................. 1
1.1.1. Agronomical and economical importance of environmental fluctuation
on dormancy and germination ......................................................................... 2
1.2. Hormonal regulation of seed dormancy and germination ......................... 3
1.2.1. Abscisic acid ...................................................................................... 4
1.2.2. Gibberellins ........................................................................................ 9
1.2.3. ABA and GA crosstalk...................................................................... 11
1.2.4. Ethylene, Brassinosteroids and Auxin .............................................. 11
1.3. Seed maturation and dormancy imposition ............................................ 12
1.3.1. Molecular regulation of seed maturation .......................................... 12
1.3.2. The role of DOG1 in seed dormancy ............................................... 14
1.3.3. The role of MFT in seed dormancy .................................................. 15
1.3.4. Influence of chromatin factors in seed dormancy ............................. 16
1.4. Influence of maternal environment on dormancy during seed set .......... 17
1.4.1. Maternal control of seed dormancy ............................................... 17
1.4.1.1. Seed coat and endosperm influence on seed dormancy and
germination ................................................................................................ 17
1.4.2. Temperature ................................................................................. 18
1.4.3. Photoperiod and light quality ........................................................ 21
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Table of contents
1.4.4. Nitrate ........................................................................................... 22
1.4.5. Water deficit stress ....................................................................... 23
1.5. Environmental control of germination ..................................................... 24
1.5.1. Temperature regulation of germination ............................................ 24
1.5.2. Light regulation of germination ......................................................... 26
1.5.3. Nitrate stimulation of germination ..................................................... 27
1.5.4. Smoke stimulation of germination .................................................... 29
1.5.5. After-ripening ................................................................................... 30
1.6. Secondary dormancy .............................................................................. 31
1.7. Thesis aim .............................................................................................. 32
2. Material and Methods .............................................................................. 34
2.1. Seeds and genotypes ............................................................................. 34
2.2. Seed coat surface sterilisation ................................................................ 34
2.3. Plant growth............................................................................................ 34
2.4. Crossing ................................................................................................. 35
2.5. Dormancy assays ................................................................................... 35
2.6. Germination on ABA, PAC, PEG and salts ............................................. 35
2.7. Seedling establishment on glucose and salts ......................................... 36
2.8. Red/Far-red germination reversibility ...................................................... 36
2.9. Flowering time analysis .......................................................................... 36
2.10. β-glucuronidase (GUS) assay ............................................................. 37
2.11. Seed clearing and DIC microscopy ..................................................... 37
2.12. Suberin detection ................................................................................ 37
2.13. Mucilage detection .............................................................................. 38
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Table of contents
2.14. Seed coat permeability to tetrazolium ................................................. 38
2.15. Lignin staining ..................................................................................... 38
2.16. ABA analysis ....................................................................................... 38
2.17. Lipid polyester analysis of Arabidopsis seeds ..................................... 39
2.18. Proanthocyanidin analysis ................................................................... 40
2.19. RNA extractions from seeds ................................................................ 40
2.20. cDNA synthesis ................................................................................... 41
2.21. Real-Time quantitative PCR ................................................................ 41
2.22. RNA-Sequencing ................................................................................ 43
2.23. Statistical analysis ............................................................................... 43
3. Isolation of seed lines matured at cool temperature with reduced
dormancy phenotype ..................................................................................... 44
3.1. Introduction ............................................................................................. 44
3.2. Results ................................................................................................... 46
3.2.1. Selection of awake lines................................................................... 46
3.2.2. Identification of seed lines with altered seed coat pigmentation ....... 52
3.2.3. Preliminary response of the putative awake mutants to ABA, PAC,
and NaCl ....................................................................................................... 54
3.2.4. Awe27 displays similar phenotypes to amp1-1 mutant .................... 57
3.3. Discussion and conclusions.................................................................... 59
4. Further characterisation of the putative awake mutant lines .............. 64
4.1. Introduction ............................................................................................. 64
4.2. Results ................................................................................................... 64
4.2.1. Analysis of flowering time of awe mutants ....................................... 66
4.2.2. Evaluation of the inheritance of the awake mutant phenotypes ....... 68
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Table of contents
4.2.3. ABA and PAC dose-response curve of awe seed germination ........ 69
4.2.4. Germination response of awe lines to glucose and under osmotic
stress 71
4.2.5. Germination response to Red and Far-Red light .............................. 73
4.2.6. Temperature regulation of seed dormancy in awe mutants ............. 74
4.2.7. Temperature induction of secondary dormancy in awe mutants ...... 76
4.2.8. Characterisation of seed coat in awe lines ....................................... 79
4.3. Discussion and conclusion ..................................................................... 82
5. Characterisation of awe1 and gpat5 reveals a role for suberin in seed
dormancy regulation by temperature ........................................................... 87
5.1. Introduction ............................................................................................. 87
5.2. Results ................................................................................................... 88
5.2.1. Characterisation of seed dormancy and germination of gpat5 and
awe1 seeds ................................................................................................... 88
5.2.2. TZ permeability and lignin staining of gpat5 and awe1 seed coat .... 90
5.2.3. Suberin staining of seed coat and roots of gpat5 and awe1 seedlings
92
5.2.4. Germination and seedlings sensitivity of gpat5 and awe1 to ABA,
PAC and salts ............................................................................................... 93
5.2.5. Flavonoid quantification in awe1 and gpat5 seeds. .......................... 95
5.2.6. ABA content and DOG1, CYP707a2 and GA3OX1 expression level in
gpat5 and awe1 dry seeds ............................................................................ 96
5.2.7. Awe1 is not a novel allele of the GPAT5 locus ................................. 98
5.2.8. Temperature regulation of suberin biosynthetic genes in seeds .... 101
5.2.9. Suberin quantification in gpat5 and awe1 seeds ............................ 104
5.2.10. Transcriptomic analysis of gpat5 and awe1 ................................ 106
5.2.11. Segregation analysis and construction of F3 segregant population
and genetic analysis .................................................................................... 113
5.3. Discussion and conclusions.................................................................. 116
6. General discussion ................................................................................ 120
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Table of contents
References .................................................................................................... 130

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List of figures
List of figures

Figure 1.2.1 ABA and GA regulation of seed dormancy and germination in
response to the environment ....................................................................... 4
Figure 1.2.2 ABA biosynthetic pathway from zeaxanthin ................................... 5
Figure 1.2.3 Proposed mechanism of ABA signalling ......................................... 8
Figure 1.2.4 Schematic representation of GA biosynthesis in Arabidopsis ........ 9
Figure 1.4.1 Cold temperatures during seed set induce high level of seed
dormancy.. ................................................................................................ 19
Figure 3.2.1 Schematic representation of the forward genetic screen to identify
embryonic and seed coat mutations, which render seeds less sensitive to
cold-induced dormancy in Arabidopsis ...................................................... 48
Figure 3.2.2 Pools of M1 segregating plants after 15 days .............................. 49
Figure 3.2.3 Germination of selected mutant seed lines matured at 16°C with
significant reduction of dormancy. ............................................................. 51
Figure 3.2.4 Dry seeds of putative transparent testa mutants identified from the
screen. ...................................................................................................... 52
Figure 3.2.5 Pictures and germination of tt mutants matured at 16°C in
response to different period of cold stratification at 4°C ............................ 53
Figure 3.2.6 Germination of selected mutant lines in response to ABA, PAC and
NaCl.. ........................................................................................................ 56
Figure 3.2.7 Venn diagram for germination on ABA, PAC and NaCl results. ... 57
Figure 3.2.8 Preliminary characterisation of awe27. ......................................... 59
Figure 4.2.1 10 days awe mutant plants. .......................................................... 65
Figure 4.2.2 20 days awe mutant plants. .......................................................... 66
Figure 4.2.3 30 days awe mutant plants. .......................................................... 66
Figure 4.2.4 Flowering time of awe mutants. .................................................... 67
Figure 4.2.5 F1 germination of eight selected mutant crossed with WT. .......... 69
Figure 4.2.6 Seeds sensitivity to ABA and PAC. .............................................. 70
Figure 4.2.7 Seedlings sensitivity to glucose. ................................................... 72
Figure 4.2.8 Germination of selected lines in response to PEG8000.. ............. 73
Figure 4.2.9 PHYB dependent germination of awake mutant lines. ................. 74
Figure 4.2.10 Germination of selected seed lines in response to temperature. 75
Figure 4.2.11 Germination of selected seed lines in response to different
stratification temperature ........................................................................... 77
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List of figures
Figure 4.2.12 Germination of selected seed lines in response to different
stratification temperature.. ......................................................................... 78
Figure 4.2.13 Freshly harvested awe mutant seeds. ........................................ 79
Figure 4.2.14 TZ staining of awake mutant lines. ............................................. 80
Figure 4.2.15 Autofluorescence of awake mutant seed lines. .......................... 81
Figure 4.2.16 Mucilage staining of selected lines. ............................................ 81
Figure 5.1.1 Autofluorescence of gpat5-1 and awe1. ....................................... 87
Figure 5.2.1 Germination of gpat5 and awe1 seeds matured at 16°C in
response to different temperature and length of stratification. ................... 88
Figure 5.2.2 Germination phenotype of gpat5 and awe1 seeds and maternal
inheritance of germination. ........................................................................ 90
Figure 5.2.3 gpat5 and awe1 seed coat permeability to TZ. ............................. 91
Figure 5.2.4 Phloroglucinol-HCl staining of mature seeds. ............................... 92
Figure 5.2.5 Suberin staining of gpat5 and awe1 in seeds and root. .............. 92
Figure 5.2.6 Response of gpat5 and awe1 seeds to germination inhibitors……..
.................................................................................................................. 93
Figure 5.2.7 Germination of gpat5 and awe1 seeds with different salt
concentration. ............................................................................................ 94
Figure 5.2.8 gpat5 and awe1 seedlings under salt stress. ............................... 95
Figure 5.2.9 Flavonoid quantification in gpat5 and awe1 seeds matured at 22°C
and 16°C. .................................................................................................. 96
Figure 5.2.10 ABA content of gpat5 and awe1 dry seeds matured at 16°C. .... 97
Figure 5.2.11 Expression level of genes involved in seed dormancy in awe1 and
gpat5 seeds. .............................................................................................. 98
Figure 5.2.12 Alignment of TAIR10 GPAT5 cDNA sequence from WT and
awe1........................................................................................................ 100
Figure 5.2.13 Upregulated suberin biosynthetic genes in dry seeds by cool
temperature.. ........................................................................................... 102
Figure 5.2.14 GPAT5 expression level in mature green seeds.. .................... 102
Figure 5.2.15 GPAT4 and GPAT6 expression level in mature green seeds.. . 103
Figure 5.2.16 Expression level of suberin related genes in mature seeds. .... 104
Figure 5.2.17 Lipid polyester monomers profile of awe1 and gpat5 mature dry
seeds....................................................................................................... 106
Figure 5.2.18 Bioanalayser traces of RNA samples for RNAseq experiment.. 107
Figure 5.2.19 CV2 vs log FPKM .................................................................... 108
10
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List of figures
Figure 5.2.20 Differentially expressed genes between seed lines with reduced
dormancy level and dormant Col-0 16°C seeds. ..................................... 110
Figure 5.2.21 Germination of five Colxawe1 (BC1F1) replicates and eighty
BC1F2 seed lines matured at 16°C ......................................................... 115
Figure 5.2.22 Phenotypic distribution in F2-derived BC1F3 population of
Col/awe1 in contrast to in Col-0 and awe1 population……………………115

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