Phosphate Sensing and Signalling in Plants

Question 1

Structure

Answer 1

1.
2.
3.

Question 2

How do plants sense and response to fluctuating Pi levels?

Answer 2

PHR-SPX module

Question 3

PHR1 - the basics

Answer 3

• phosphate starvation response 1
• PSR gene master regulator
• MYB BD Coiled-coil TF
• evolutionary conserved plants -> algae (C. reinhardtii)

Question 4

phr1-1

Answer 4

• forward genetics screen
• transgenic GUS reporter
• only one mutant
• fails to induce IPS1
• defective starch, sugar, anthocyanin accumulation; RSA; PSR gene attenuation

Question 5

IPS1

Answer 5

• PSR marker gene
• induced by PHR1
• interferes w/ miRNA 399 under high phosphate

Question 6

How does PHR1 function?

Answer 6

dimerises w/ GNATATNC, P1BS

Question 7

P1BS

Answer 7

• PHR1 DNA Binding Sequence
• cis promotor of PSR genes

Question 8

PHR1 localisation

Answer 8

• nuclear @ high and low phosphate
• constitutively expressed

Question 9

AtPHR1oe

Answer 9

• increased shoot Pi
• increased PHT/F1

Question 10

GUS

Answer 10

beta-glucorodinase reporter gene system

Question 11

How does PHR sense Pi?

Answer 11

SPX

Question 12

SPX

Answer 12

• highly conserved
• phosphate-sensing module
• At: 4
• Os: 6
• PSR-inducible (GUS transcriptomics)

Question 13

How does PHR regulate SPX?

Answer 13

• negative PSR regulator under high phosphate
• mutant analysis
• binds P1BS

Question 14

spx

Answer 14

• ISP1 expressed under high Pi

Question 15

spx1spx2

Answer 15

• IPS1 highly expressed
• phosphate toxicity

Question 16

phr2spx1spx2

Answer 16

• wt recapitualtion

Question 17

protein-protein interaction studies

Answer 17

• Co-IP on N. benthamiana
• BiFC on tobacco
• SPX1/2 bind PHR2 @ C196 via MYB-CC domain
• inhibits PSR P1BS cis-promotor binding in a Pi-dependent manner

Question 18

PHR2 + radio labelled P1BS DNA probe

Answer 18

• destabilised with high SPX1
• only under high phosphate

Question 19

Mechanism of PHR-SPX module function:

Answer 19

• low Pi: cytosolic SPX4 = degraded (GUS)
• OsSPX4-PHR2 inhibits PHR2 cyto-nuclear transport
• high Pi: InsP7 ligand binds SPX4

Question 20

InsP7

Answer 20

• inositol polyphosphate
• necessary (not Pi)

Question 21

PHO1

Answer 21

• PHOSPHATE 1
• mediates Pi loading from roots -> shoots + organs
• no PHT homology
• broadly expressed (RT-PCR, GUS)
• SPX-EXS protein
• At: 11 homologs
• Os: 3 homologs

Question 22

SPX-EXS

Answer 22

• large gene Family
• Pi sensors

Question 23

PHO1 under PSR?

Answer 23

• P1BS
• PHR1-regulated
• low Pi: PHO1 induction in root xylem (GUS)

Question 24

phr1

Answer 24

• low PHO1

Question 25

PHO1oe

Answer 25

- decreased shoot growth due to phosphate toxicity (micro moles/ gram fresh weight after 30 days) - increased Pi - leaves: 100x greater Pi (xylem exudate)

Question 26

PHO2

Answer 26

- negative regulator of Pi uptake under high Pi - root-expressed - shoot impact - Ub-conjugating E2 - PHO1, PHT/F1 proteolytic degradation

Question 27

Atpho2

Answer 27

- Pi hyper accumulation (mg/g FW) - toxicity under high phosphate

Question 28

reciprocal micrografting

Answer 28

- showing root functionality - pho2s, wtr: decreased Pi - wts, pho2r: increased Pi

Question 29

miRNA 399

Answer 29

- PSR gene - shoot expression unregulated by PHR1 under low phosphate - mobile -> root - PHO2 silencing