The Effect of Modifying the Higher-Order Structure of Plant High-Affinity Phosphate Transporters

142 Pages·2017·2.87 MB·English

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LSU Doctoral Dissertations Graduate School
2014
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Elena Batista Fontenot
Louisiana State University and Agricultural and Mechanical College
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Fontenot, Elena Batista, "The Effect of Modifying the Higher-Order Structure of Plant High-Affinity
Phosphate Transporters" (2014). LSU Doctoral Dissertations. 1256.
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THE EFFECT OF MODIFYING THE HIGHER-ORDER STRUCTURE
OF PLANT HIGH-AFFINITY PHOSPHATE TRANSPORTERS

A Dissertation

Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy

in

The Department of Biological Sciences

by
Elena B. Fontenot
B.S., Loyola University New Orleans, 2008
December 2014
Table of Contents
List of Tables ...................................................................................................................... v

List of Figures .................................................................................................................... vi

List of Abbreviations ....................................................................................................... viii

Abstract .............................................................................................................................. ix

Chapter 1. Introduction ....................................................................................................... 1

Incentive to understand plant nutrient uptake ................................................................. 1

N:P:K .......................................................................................................................... 2

Downstream impacts of excavating and utilizing phosphorus ................................... 3

Removal of fertilizer run-off and its heavy metal residues ......................................... 4

Phytoremediation ........................................................................................................ 5

Plant arsenic uptake .................................................................................................... 6

Plant membrane transport proteins ................................................................................. 8

Pumps, Channels, and Transporters .......................................................................... 10

Transporter kinetics .................................................................................................. 12

Major Facilitator Superfamily transporters ............................................................... 14

Nitrate and ammonium transporters .......................................................................... 17

Sucrose transporters .................................................................................................. 19

Phosphate transporters .............................................................................................. 20

Multi-functional Pht1 family members ......................................................................... 22

Regulation of Pht1 transporters and PHO1 in response to Pi ....................................... 25

miRNAs modulate the expression of Pht1 transporters and PHO1 .............................. 26

miRNA399 and miRNA827-mediated regulation of PHO2 and NLA ..................... 27

Post-translational regulation of PHO1 and Pht1 transporters ................................... 29

Physiological and additional molecular effects in response to Pi limitation ................ 30

Oligomerization of proteins .......................................................................................... 32

Obligate and non-obligate protein interactions ......................................................... 33

Approaches to study protein-protein interactions ..................................................... 35

Goals of studying the oligomerization of plant phosphate transporters ....................... 38

Chapter 2. Materials and Methods .................................................................................... 40

General Material and Methods ...................................................................................... 40

PCR ........................................................................................................................... 40

Plasmid minipreps ..................................................................................................... 41

Plasmid midipreps ..................................................................................................... 42

DNA quantification and sequencing ......................................................................... 43

Cloning with the Gateway® system ......................................................................... 44

ii
Transformation of electro-competent E. coli ............................................................ 45

Transformation of heat-shock E. coli ........................................................................ 46

Transformation of electro-competent Agrobacterium tumefaciens .......................... 46

DNA Electrophoresis ................................................................................................ 47

Overlap extension PCR for mutagenesis .................................................................. 47

Materials and methods for Chapter 3 ............................................................................ 49

Protoplast transformations and split luciferase complementation assay ................... 49

Yeast transformation for mbSUS .............................................................................. 51

Selection of mbSUS yeast transformants .................................................................. 51

mbSUS assay ............................................................................................................ 53

Materials and methods for Chapter 4 ............................................................................ 53

Preparation of plasmids for yeast complementation assays ...................................... 53

Yeast transformation for yeast complementation assays .......................................... 54

Selection of PAM2 yeast transformants ................................................................... 55

Yeast growth assays .................................................................................................. 57

Yeast RNA extraction ............................................................................................... 57

32P uptake assay ........................................................................................................ 58

Preparation for localization assays ............................................................................ 59

Agroinfiltration of tobacco leaves ............................................................................ 59

Materials and methods for Chapter 5 ............................................................................ 61

Stable transformation of plants ................................................................................. 61

Selection of homozygous stable lines ....................................................................... 61

Seed sterilization and stratification ........................................................................... 62

Seedling growth on phosphate and arsenate ............................................................. 63

Anthocyanin extraction and quantification ............................................................... 64

Arsenate growth assays and uptake assays ............................................................... 65

Chapter 3. The in vivo and in vitro oligomeric interactions of Pht1;1 and Pht1;4. .......... 66

Introduction ................................................................................................................... 66

Results ........................................................................................................................... 68

In vivo and in vitro analyses of Pht1;1 and Pht1;4 protein interactions ................... 68

In vivo analyses of Pht1;1 and Pht1;4 protein interactions in yeast systems ............ 71

Discussion ..................................................................................................................... 72

Chapter 4. A site-directed mutant of Pht1;1 that blocks dimerization but enhances
phosphate transporter activity. .......................................................................................... 75

Introduction ................................................................................................................... 75

Results ........................................................................................................................... 77

The role of a conserved Pi transporter motif in oligomerization .............................. 77

Tyr312 plays a role in Pht1;1 interaction and function ............................................. 81

Y312D mutation increases phosphate uptake ........................................................... 83

Discussion ..................................................................................................................... 87

iii
Chapter 5. How does altering Tyr312 in Arabidopsis Pht1;1 affect in planta phosphate
and arsenate uptake? ......................................................................................................... 90

Introduction ................................................................................................................... 90

Results ........................................................................................................................... 92

Growth of transformants on low phosphate .............................................................. 92

Pht1;1Y312D may induce a tolerance to arsenate ........................................................ 96

Discussion ................................................................................................................... 101

Chapter 6. Conclusions ................................................................................................... 105

References ....................................................................................................................... 110

Appendix ......................................................................................................................... 129

Vita .................................................................................................................................. 131

iv
List of Tables
Table 1. Student’s t-test of growth coefficient values ...................................................... 84

Table 2. Kinetic properties of Pht1;1 and Pht1;1Y312D ...................................................... 86

Table 3. Kinetic properties of 32P uptake assays with arsenate ...................................... 100

Appendix Tables:
Appendix Table 1. Gateway compatible PCR primer sequences for pDONR ............... 129

Appendix Table 2. pYES2 vector compatible PCR primer sequences ........................... 129

Appendix Table 3. Primer sequences for mutagenesis constructs .................................. 129

Appendix Table 4. RT-PCR gene-specific primers ........................................................ 130

v
List of Figures
Figure 1. The structures of phosphate, arsenate, and arsenite ............................................ 7

Figure 2. Three classes of plasma membrane transport proteins ........................................ 9

Figure 3. Biphasic kinetics of a nitrate transporter from Arabidopsis .............................. 13

Figure 4. Schematic of a typical 12 TM MFS transporter ................................................ 14

Figure 5. Crystal structures of MFS transporters .............................................................. 16

Figure 6. Model of Pi uptake by Pht1 transporters in Arabidopsis roots .......................... 21

Figure 7. Expression patterns of the Arabidopsis Pht1 transporter family ....................... 24

Figure 8. Modulation of Pht1 transporters and PHO1 by regulatory components at
different levels of regulation ..................................................................................... 27

Figure 9. Nitrate transporter NRT1.1 can form a non-obligate homomeric complex ...... 34

Figure 10 Pht1;1 and Pht1;4 interact to form homomeric and heteromeric oligomers .... 70

Figure 11 Split assays in yeast confirm homomeric and heteromeric interactions of Pht1;1
and Pht1;4 ................................................................................................................. 70

Figure 12 Identification of a conserved motif among PHTs ............................................. 79

Figure 13 PiPT crystal structure with motif of interest ..................................................... 79

Figure 14 Interaction of the Pht1;1 homo-oligomer is abolished by mutation of Tyr312 80

Figure 15 Co-expression of Pht1;1 with Tyr312 mutants shows reconstituted luciferase
activity with Y312A and Y312F ............................................................................... 80

Figure 16 Intracellular plasma membrane localization of Pht1;1 Tyr312 mutations ....... 82

Figure 17 Growth rate of Y312D exceeds the growth rate of Pht1;1, Y312A, and Y312F
under low Pi conditions ............................................................................................ 84

vi
Figure 18 RT-PCR of RNA isolated from yeast shows similar transgene transcript
abundance. ................................................................................................................ 85

Figure 19 Uptake assay profile of Pht1;1 and Pht1;1Y312D ............................................... 86

Figure 20 Shoot and root growth study of homozygous transformants in a wild-type
Columbia background on different phosphate concentrations .................................. 94

Figure 21 Shoot and root growth study of homozygous transformants in a Δpht1;1
Columbia background on different phosphate concentrations .................................. 95

Figure 22 Anthocyanin accumulation in Pht1;1:WT and Pht1;1Y312D:WT ...................... 96

Figure 23 Over-expressed wild-type homozygous lines grown on arsenate .................... 97

Figure 24 Pht1;1Y312D: Δpht1;1 homozygous lines show tolerance to arsenate ................ 98

Figure 25 Growth of Pht1;1 and Pht1;1Y312D in arsenate ................................................ 100

Figure 26 Arsenate tolerance of yeast strains ................................................................. 101

vii
List of Abbreviations

Å angstrom MYB a plant transcription
ABC ATP-binding cassette factor family
transporters N nitrogen
ADP adenosine diphosphate Na+ sodium ion
Al aluminum NLA nitrogen limitation
AMT ammonium transporter adaptation
APase acid phosphatases NRT nitrate transporter
As arsenic P phosphorus
AsIII arsenite PAP purple acid phosphatases
AsV arsenate Pb lead
ATP adenosine triphosphate PHF1 phosphate transporter
BiFC bimolecular fluorescence traffic facilitator1
complementation PHL1 PHR1-LIKE1
Ca2+ calcium ion PHO1 phosphate 1
Cd cadmium PHO2 phosphate 2
Co-IP co-immunoprecipitation PHR1 phosphate starvation
Cu copper response regulator 1
dH O deionized water Pht phosphate transporter
2
EXS ERD1-XPR1-SYG1 Pi inorganic phosphate
EtBr ethidium bromide pK logarithmic measure of
a
Fe iron the acid dissociation
FRET fluorescence resonance constant
energy transfer PM plasma membrane
FW fresh weight PPI protein-protein
GFP green fluorescent protein interaction
H+ proton ppm parts per million

ICP-MS Inductively coupled PSR phosphate starvation
plasma mass responsive genes
spectrometry SLC split luciferase
IPS1 induced by phosphate complementation system
starvation 1 SPX SYG1/Pho81/XPR1
K potassium proteins
K+ potassium ion SUT sucrose transporters
K binding affinity TM transmembrane
m
mbSUS mating-based split UTR untranslated regions
ubiquitin system WRKY plant transcription factor
MFS major facilitator family
superfamily Y2H yeast two-hybrid system
µ micro YFP yellow fluorescent protein
miRNA microRNA Zn zinc

viii
Abstract
Phosphorus is an essential nutrient taken up by plants in the form of phosphate
and is a component of key molecules, such as ATP, DNA, and RNA, and is one of the
most frequently limiting macronutrients for plant growth. More than 80 % of phosphorus
found in soil exists as non-inorganic forms and is unavailable for plant uptake,
demonstrating the importance for genes induced to cope with phosphate starvation,
including high-affinity phosphate transporters (Phts). Phts are membrane-localized
transporters involved in the transport of phosphate and arsenate and have a high capacity
for transporting phosphate in areas where phosphate concentrations are low, behaving as
the main mechanism of phosphate uptake by plants. The Pht1 family in Arabidopsis
thaliana contains nine members, of which AtPht1;1 (Pht1;1) and AtPht1;4 (Pht1;4) have
been shown to be the major transporters involved in phosphate acquisition from soil.
Membrane transporters often function as oligomeric complexes. In plants, there is
evidence that oligomerization of transporter isoforms is an important aspect of transport
function and influences dynamic regulation. Prior studies have illustrated the importance
of the role of higher-ordered plant membrane transporters in uptake regulation however,
the potential oligomerization and functions of oligomerization among Phts has not been
formally investigated. This research demonstrates the formation of Pht higher-order
structures and suggests a function for this association in phosphate and arsenate uptake.
In vivo and in vitro protein interaction assays that comprised of split luciferase
complementation of transformed protoplasts, yeast-based split ubiquitin and yeast-two-
hybrid assays, and immunoprecipitation assays of root microsomal proteins confirm that
Pht1;1 and Pht1;4 form homomeric and heteromeric structures. Furthermore, the
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