The role of HAK/KUP/KT family of transporters and potassium channels in abiotic stress responses

184 Pages·2017·11.67 MB·English

The role of HAK/KUP/KT family of
transporters and potassium channels
in abiotic stress responses in
Arabidopsis and rice

RADWA MORAD MOHSEN EBRAHIM
MOHAMED

PhD

University of York
Biology

October 2016
Abstract
Abiotic stress is a worldwide problem that affects the productivity of crops. Salinity
stress for example, affects about 950 million hectares of arable lands while about 64%
of the global land is affected by water deficit (drought). Drought and salinity are the
most deleterious stresses, resulting in nutritional imbalance. Therefore, the main
mechanism of improving a plant tolerance to stresses is by improving nutritional
acquisition such as K+ uptake.
The Two Pore K+ vacuolar Channel (TPK1) was reported to function in K+ release
from guard cell vacuoles, therefore stomatal closure. However, attpk1 showed weak
phenotype, suggesting a good level of redundancy by other transporters predicted to
localize in the guard cell tonoplast. The K+ high affinity uptake permeases
HAK/KUP/KT transporters AtKUP12 and AtHAK8 are among these candidates. We
test this hypothesis in the model plant Arabidopsis thaliana. Loss of function of
AtKUP12 mutants showed severe inhibition of germination, seedling establishment
and growth of plants during K+ deficiency resulted from high NH +/K+ ratio.
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Therefore, we suggest a potential role for this transporter in K+ flux during osmotic,
salinity and K+ starvation stresses. We also suggest a possible epistasis relationship
between AtKUP12 and AtTPK1. Possibly, they both function in K+ efflux from
vacuoles to cytoplasm during stress. Our results also suggest no functional relationship
between AtTPK1 and AtHAK8. AtKUP12 was able to rescue the growth of K+
deficient yeast strain during K+ starvation.
In rice, we study the role of the Stelar K+ Outwardly Rectifying channel (SKOR) in
K+ transport from roots to shoots under different stresses. We aim at improving K+
transport to shoots therefore the overall plant response to abiotic stress, using different
overexpressor lines. Overexpressing the TPKb (an isoform of the TPK channel
localized in the storage vacuoles) resulted in higher K+ levels in the transgenic plants
during K+ starvation. We test the possibility of the involvement of other transporters
in this phenomenon. Expression levels of the HAK1 and KAT1 as well as the GORK
transporter and channel genes were measured in TPKb overexpressor lines. OsHAK1
showed slight increase in expression while other transporters did not show significant
differences.
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Table of Contents
Abstract............................................................................................................................ II
Table of Contents ............................................................................................................ III
List of tables ................................................................................................................. VIII
List of figures .................................................................................................................. IX
Acknowledgments .......................................................................................................... XII
Author’s declaration .................................................................................................... XIII
Chapter 1. General introduction ..................................................................................1
1.1 Abiotic stress as a worldwide problem affecting the productivity of plants ............1
1.2 K+ as a major ion in plant physiology ...................................................................2
1.3 Mechanisms of K+ uptake and distribution in plants ..............................................3
1.3.1 Shaker-type channels ........................................................................................4
1.3.2 The two-pore K+ channels (TPK) family ...........................................................6
1.3.2.1 The AtTPK1 K+ channel and its role in the abiotic stress responses ...........7
1.3.3 The HAK/KUP/KT family of transporters .........................................................9
1.3.3.1 The phosphorylation of the HAK/KUP/KT family members...................... 11
1.3.3.2 Physiological roles of family members .................................................... 12
1.3.4 The high affinity K+ transporters (HKT) ......................................................... 14
1.3.5 Other channels/transporters ............................................................................. 14
1.4 The role of K+ in plant responses to different stresses.......................................... 15
1.5 How K+ deficiency affect plants ......................................................................... 15
1.5.1 Morphological effects ..................................................................................... 15
1.5.2 Physiological effects ....................................................................................... 16
1.6 K+ deficiency sensing and signaling .................................................................... 17
1.6.1 How a plant senses K+ deficiency ................................................................... 17
1.6.2 Reactive oxygen species (ROS) ...................................................................... 18
1.6.3 Calcium signaling ........................................................................................... 19
1.6.4 Hormones ....................................................................................................... 19
1.7 Salt stress ........................................................................................................... 20
1.7.1 Sodium entry into plants ................................................................................. 21
1.7.2 Long distance Na+ transport ............................................................................ 22
1.7.3 The compartmentation of Na+ into vacuoles and removal to the apoplast ......... 22
1.7.4 Salinity sensing and signaling ......................................................................... 23
1.7.4.1 How a plant senses salinity ..................................................................... 23
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1.7.4.2 The SOS pathway of salinity signaling ..................................................... 24
1.8 Drought Stress .................................................................................................... 24
1.8.1 Types of plant responses to drought ................................................................ 25
1.9 How drought and salinity affect plants ................................................................ 26
1.9.1 Growth of plants ............................................................................................. 26
1.9.2 Germination of seeds ...................................................................................... 27
1.9.3 Germination under stress, a harmony of hormones .......................................... 28
1.9.4 Stomatal conductance and photosynthesis ....................................................... 29
1.9.4.1 Stomatal closure under abiotic stress ...................................................... 30
1.9.4.2 Vacuolar ion channels/transporters involved in the stomatal closure ....... 31
1.9.5 Reactive oxygen species and oxidative stress (ROS) ....................................... 32
1.10 Physiological mechanisms of drought and salinity tolerance ............................... 33
1.10.1 Osmotic adjustment ........................................................................................ 33
1.10.2 Aquaporins and water uptake .......................................................................... 33
1.11 Arabidopsis and rice as model plants .................................................................. 34
1.12 Aims of the thesis ............................................................................................... 35
Chapter 2. The role of AtKUP12, AtTPK1 and AtHAK8 in the abiotic stress responses
37
2.1 Introduction ........................................................................................................ 37
2.1.1 AtKUP12 predicted localization and expression .............................................. 37
2.1.2 Interactomes and regulation by hormones ....................................................... 38
2.1.3 AtHAK8 transporter previous work and expression......................................... 39
2.2 Aims of study ..................................................................................................... 41
2.3 Materials and methods ........................................................................................ 43
2.3.1 Genotyping of T-DNA insertion lines ............................................................. 43
2.3.1.1 DNA extraction ....................................................................................... 44
2.3.1.2 Polymerase chain reaction analysis (PCR) .............................................. 45
2.3.2 Semi-quantitative reverse transcription analysis (RT-PCR) of mutants ............ 47
2.3.2.1 RNA isolation.......................................................................................... 47
2.3.2.2 cDNA synthesis ....................................................................................... 47
2.3.2.3 RT-PCR of mutants ................................................................................. 47
2.3.3 Germination and stress experiments ................................................................ 48
2.3.4 Generating double and triple mutants .............................................................. 49
2.3.5 Growth rescue of the K+ deficient yeast by the AtKUP12 transporter .............. 50
2.3.5.1 Amplifying full coding sequence .............................................................. 50
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2.3.5.2 The plasmid vector pYES2 ....................................................................... 51
2.3.5.3 Double digestion (DD) of AtKUP12 and pYES2 using BamHI and NotI
restriction enzymes ..................................................................................................... 52
2.3.5.4 Purification of the digested plasmid from gel........................................... 52
2.3.5.5 Ligation of AtKUP12 into pYES2 ............................................................ 53
2.3.5.6 Transformation of Escherichia coli competent cells ................................. 53
2.3.5.7 Colony PCR ............................................................................................ 53
2.3.5.8 Isolation of plasmids ............................................................................... 53
2.3.5.9 Diagnostic digestion of the construct ....................................................... 54
2.3.5.10 Sequencing of isolated plasmids .............................................................. 54
2.3.5.11 Yeast transformation ............................................................................... 54
2.3.5.12 K+ starvation assays of yeast on agarose plates ....................................... 55
2.3.6 Stomatal conductance under ABA treatment ................................................... 56
2.3.7 Water loss measurements ................................................................................ 56
2.4 Results ............................................................................................................... 57
2.4.1 Genotyping of tpk1, hak8 and kup12 single mutants using PCR analysis ......... 57
2.4.2 Generation of double and triple knockouts ...................................................... 58
2.4.3 atkup12 germination is severely inhibited by K+ deficiency and AtTPK1 is
epistatic to AtKUP12 .................................................................................................... 60
2.4.3.1 Germination rates after 48 hours ............................................................ 62
2.4.3.2 Germination rates after 72 hours ............................................................ 63
2.4.3.3 Germination rates after 96 hours ............................................................ 66
2.4.3.4 atkup12-2 mutants response resembles atkup12-1 ................................... 67
2.4.3.5 Effect of 200 µM K+ treatment on germination of mutant seeds ............... 68
2.4.4 Loss of function of AtKUP12 inhibits seedling establishment during K+
deficiency when combined with 10 mM NH + ............................................................... 69
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2.4.5 Analysis of the role of AtKUP12 transporter in the K+ deficient yeast strain .... 72
2.4.5.1 Yeast transformation ............................................................................... 74
2.4.5.2 AtKUP12 complements yeast growth under low K+ conditions ................ 74
2.4.5.3 Growth of K+ deficient yeast on galactose contained medium .................. 75
2.4.6 Effect of different stresses on seedlings of mutants ......................................... 77
2.4.6.1 Effect of abiotic stress treatments on tpk1, hak8 and hak8tpk1 mutants.... 77
2.4.6.2 Effect of abiotic stress treatments on tpk1, kup12 and kup12tpk1 mutants
and a role for AtKUP12 in salinity stress response ..................................................... 78
2.4.6.3 Effect of abiotic stress treatments on hak8kup12 and hak8kup12tpk1
mutants 79
2.4.7 Effect of K+ starvation on seedlings ................................................................ 80
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2.4.8 Transpirational water loss of mutants .............................................................. 81
2.4.9 Stomatal conductance under ABA treatment ................................................... 85
2.5 Discussion .......................................................................................................... 87
2.5.1 Germination of seeds during K+ starvation is significantly inhibited by the
presence of NH + and Na+ ............................................................................................. 88
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2.5.2 K+ relieves NH + toxicity and Na+ cannot substitute K+ in high NH +/K+ conditions
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90
2.5.3 The AtKUP12 is important in the early stages of germination during K+ starvation
resulted from high NH +/K+ ratio .................................................................................. 91
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2.5.4 Behavior of tpk1, hak8, hak8kup12 and hak8kup12tpk1 under treatments ........ 93
2.5.5 Combining knockout mutants suggests epistasis in function between AtTPK1 and
AtKUP12...................................................................................................................... 94
2.5.6 AtKUP12 is important for seedling establishment as for germination under low
K+ treatment ................................................................................................................. 96
2.5.7 AtKUP12 rescued the growth of the mutant yeast on K+ deficient medium ...... 97
2.5.8 Role of transporters in seedlings under stress treatments ................................. 99
2.5.9 Water loss and conductance measurements ................................................... 101
Chapter 3. The role of potassium channels in rice responses to abiotic stress ........ 103
3.1 Introduction ...................................................................................................... 103
3.1.1 The SKOR channel and its role in abiotic stress tolerance ............................. 103
3.1.2 The TPKb vacuolar channel and its role in abiotic stress tolerance in rice ...... 104
3.2 Aims of the chapter .......................................................................................... 105
3.3 Materials and methods ...................................................................................... 105
3.3.1 Plant material ............................................................................................... 105
3.3.2 Hygromycin media selection ......................................................................... 106
3.3.3 Growth medium and conditions .................................................................... 106
3.3.4 Extraction of DNA and RNA ........................................................................ 106
3.3.5 PCR analysis of overexpressor SKOR lines ................................................... 107
3.3.6 RT-PCR and qPCR analysis.......................................................................... 107
3.3.7 Relative growth rate of OXSKOR lines under stress treatments ...................... 108
3.3.8 Measurement of ion contents in rice tissues................................................... 109
3.3.9 Statistical analysis ........................................................................................ 109
3.4 Results ............................................................................................................. 109
3.4.1 Hygromycin selection of OXTPKb homozygous lines ................................... 109
3.4.2 Identification of transgenic OXSKOR lines using hygromycin primers ........... 110
3.4.3 RGR of OXSKOR lines under different abiotic stress treatments.................... 111
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3.4.4 Ion contents of OXSKOR lines under different abiotic stress treatments ......... 112
3.4.4.1 Ion contents of shoots ............................................................................ 113
3.4.4.2 Ion contenets of roots ............................................................................ 114
3.4.5 RT-PCR analysis of OXSKOR lines: ............................................................. 115
3.4.6 qPCR analysis of the OXSKOR rice lines ...................................................... 116
3.4.7 Expression levels of the major K+ channels/transporters under the overexpression
of TPKb in rice ........................................................................................................... 117
3.5 Discussion ........................................................................................................ 120
3.5.1 The SKOR channel: is overexpression always recommended? ...................... 120
3.5.2 TPKb relieves K+ deficiency by increasing K+ release from PSV ................... 120
Chapter 4. Final conclusions ..................................................................................... 124
4.1 Abiotic stress: the challenges, the achievements and the ambitions ................... 124
4.2 Aims of the thesis and research outcomes ......................................................... 125
4.3 AtKUP12 functions in seed germination and seedling establishment during K+
starvation and a possible epistasis between AtKUP12 and AtTPK1 ............................... 126
4.4 Proposed model of AtKUP12 role in K+ transport and epistasis relationship ...... 129
4.5 AtKUP12 complements the growth of the K+ deficient yeast during K+ starvation
131
4.6 atkup12 mutants showed salt sensitive and possibly a stomatal phenotype ........ 132
4.7 AtHAK8 did not play a role in seed germination or seedling establishment ....... 132
4.8 Combining tpk1, hak8 and kup12 mutants ......................................................... 133
4.9 The SKOR channel and its role in abiotic stress responses in rice ...................... 134
4.10 The role of TPK family of channels under K+ deficiency in rice ........................ 135
4.11 Future work ...................................................................................................... 135
Appendix ........................................................................................................................ 137
References ...................................................................................................................... 140

VII
List of tables
Table 2-1: Reagents used in PCR analyses. ............................................................ 46
Table 2-2: Primers used in the genotyping single, double and triple mutants. ......... 46
Table 2-3: Standard PCR analysis conditions ......................................................... 46
Table 2-4: Primers used in the semi-quantitative RT-PCR to detect any transcripts in
the single, double and triple mutants. ..................................................................... 48
Table 2-5: Primers used in amplifying the full coding sequence of AtKUP12 and the
addition of restriction sites BamHI and NotI. ......................................................... 51
Table 2-6: Primers used in sequencing plasmids. .................................................... 54
Table 3-1: Primers used in PCR and RT-PCR analysis of OXSKOR and OXTPKb lines.
............................................................................................................................ 108

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List of figures
Figure 1-1: Topological structure of the TPK1 sub-unit, a member of the TPK K+
channels family. ....................................................................................................... 7
Figure 1-2: Clusters of the HAK/KUP/KT family of transporters in Arabidopsis. ... 10
Figure 1-3: Suggested topology and function of the HAK/KUP/KT family
transporters. ........................................................................................................... 12
Figure 1-4: Mechanism of low K+ sensing and signaling in Arabidopsis plants....... 20
Figure 1-5: Vacuolar channels/transporters functioning in stomatal closure. ........... 32
Figure 2-1: Expression levels of AtKUP12 in different seed tissues and time points and
in comparison with AtHAK8 and AtTPK1 during imbibition of seeds. .................... 40
Figure 2-2: Predicted locations and roles of candidate transporters during the stomatal
closure. .................................................................................................................. 43
Figure 2-3: Diagram of the mutant lines used in experiments and positions of T-DNA
insertions. .............................................................................................................. 44
Figure 2-4: The pYES2 vector plasmid (Invitrogen). .............................................. 52
Figure 2-5: Confirming T-DNA insertions in single mutants using standard PCR and
RTPCR. ................................................................................................................. 58
Figure 2-6: PCR analysis of double and triple mutants. .......................................... 59
Figure 2-7: Stages of seed germination and inhibition of seedlings during K+ starvation
.............................................................................................................................. 61
Figure 2-8: Effect of K+ starvation on the germination of seeds under high NH +/K+
4
levels after 48 hours. .............................................................................................. 64
Figure 2-9: Effect of K+ starvation on the germination of seeds under high NH +/K+
4
levels after 72 hours. .............................................................................................. 65
Figure 2-10: Effect of K+ starvation on seed germination under high NH +/K+ levels
4
after 96 hours. ........................................................................................................ 67
Figure 2-11: Seeds germination of atkup12-2 after 48 hours of sowing................... 68
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Figure 2-12: Germination under 200 µM K⁺ average and normalized to control (10
mM K+). ................................................................................................................ 69
Figure 2-13: Effect of high NH +/K+ ratio on seedling establishment. Cotyledons
4
emergence was counted 7 days of sowing............................................................... 71
Figure 2-14: Seedling establishment under high K+ (10 mM) and low K+ (50 µM)
combined with 10 mM NH4+. ................................................................................. 71
Figure 2-15: Amplifying AtKUP12 full coding sequence using PCR. ..................... 72
Figure 2-16: Double digestion of the pYES2 plasmid using BamHI and NotI restriction
enzymes. ................................................................................................................ 73
Figure 2-17: Colonies PCR. Transformed colonies were selected on LB medium
supplied with 100 mg/L carbenicillin. .................................................................... 73
Figure 2-18: Diagnostic double digestions of the isolated AtKUP12-pYES2 construct.
.............................................................................................................................. 74
Figure 2-19: AtKUP12 complements the growth of K+ deficient yeast in glucose
plates. .................................................................................................................... 75
Figure 2-20: AtKUP12 complements the growth of K+ deficient yeast in galactose
plates. .................................................................................................................... 76
Figure 2-21: Effect of different stress treatments on fresh weights of Arabidopsis
wildtype tpk1, hak8 and hak8tpk1 seedlings. .......................................................... 78
Figure 2-22: Effect of different stress treatments on fresh weights of Arabidopsis
wildtype and tpk1, kup12 and kup12tpk1 seedlings. ................................................ 79
Figure 2-23: Effect of different stress treatments on fresh weights of Arabidopsis
wildtype and hak8kup12 and hak8kup12tpk1 seedlings. ......................................... 80
Figure 2-24: Effect of K+ starvation on fresh weights of Arabidopsis wildtype and
mutant seedlings. ................................................................................................... 81
Figure 2-25: Transpirational water loss in rosettes. ................................................. 83
Figure 2-26: Transpirational water loss in rosettes. ................................................. 85
Figure 2-27: Stomatal conductance of whole leaves in mutants .............................. 86
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