Photosynthesis: Assimilate partitioning Getting the...

Photosynthesis: Assimilate partitioning

Getting the building blocks for growth and cell wall biosynthesis from the leaf tissue to actively growing organs.

Bob Turgeon

Water

proton-coupled symporters

amino acids amino acids

PM-ATPase

chloroplast

SucroseSucrose

Vacuole

Plasmodesmata

mass flow

ATP

pH = 7.3∆E = -180 mV

CO2

Phloem

Mesophyll pH = 5.5

H+

SucroseH+

Apoplastic phloem loading

pH jump forms trans-membrane pH gradient

potassium & valinomycinform defined membrane potential

Κ

14C-substrate

+pH = 8.0

pH = 6.014C-substrate

pHi = pHo

Negative membrane potential

6.04.02.00.00.00

400

800

1200

Time (min)

Sucr

ose

Tra

nspo

rtpm

ol/ m

g p

CCCP

Sucrose

Cytoplasm Extracellular

Plasma Membrane

+H

C

CHSucCHSuc

CSucrose

H+

(∆ sensitive?)Ψ

(∆ sensitive?)Ψ

DEPC

SH

PCMBS

Binds to a substrate protectable site

Properties of the Proton-Sucrose Symporter

m

5050

Apparent KmSucrose 1.0 mMProtons 0.7 µM

InhibitorsDEPC I = 750 µMPCMBS I = 30 µM

pH Optimum < 6.0

Stoichiometry 1:1

Location Plasma Membrane

Specificity Low Affinity For:glucose, fructoseraffinose, maltose mannose, lactoseand melibose

5050

Proton-sucrosesymporter

Amino Acid Import

Amino Acid Import

Amino Acid ImportAmino Acid ExportPrimary AssimilationAmino Acid Cycling (phloem to xylem)

Amino Acid ExportPrimary AssimilationAmino Acid Cycling (xylem to phloem)

Amino Acid Import

Mature leaves

Developing leaves, fruit, and seed

Root

Developing rootsand meristem Phloem

Xylem

Amino acids are the currencyof nitrogen allocation in

multicellular plants.

Strategies for identifying symporter proteins and genes ….

• BiochemicalDifferential labelingPhotoaffinity labelingSolubilization & reconstitution

• MolecularPCRMutantsFunctional complementation

Biochemically limited andtransport incompetent

Yeast Mutant

MolecularCloning via Functional Complementation

Transform mutants with a plant cDNAlibraryconstructed in a yeast expression vector

Screen for restored growthon a limiting medium

H

ExpressionVector

+

Limiting Substrate

Positive Transformant

With successful transcription, translation, and insertion, we select for plant transporter-dependent growth.

ATP

6.04.02.00.00.0

1.0

2.0

Time (min)

Ala

nine

Tra

nspo

rt (n

mol

/ mg

cells

)

B

16.012.08.04.00.00.0

0.2

0.4

0.6

0.8

Time (min)

His

tidin

e Tr

ansp

ort

(nm

ol/ m

g ce

lls)

A

100

100

200

200

300

300

400

400

3 3 2 2 1 1 0 0 -1 -1-2 -2-3 -3* * *

Amino Acid Position, N to C Terminal

Hydrophobic

Hydrophilic

Structure no. 1

N

1 2 3 4 5 6 7 8 9 10

C

CYTOPLASMStructure no. 2

N

1 2 3 4 5 6 7 8 9 10 11

C

CYTOPLASM

Proton-Sucrose Symporter

CYTOPLASMN C

1 2 3 4 5 6 7 8 9 10 11 12

Cytoplasmic side

Random Mutagenesis

silent mutations, nonsense mutations, substitutions, premature stops

Transform into yeast

Screen for altered transport phenotypes

Construct

0.1

0.2

0.3

0.4

0.5

0.6

0Wt Mal9 (G334S)

Mal

tose

tran

spor

t (n

mol

/ m

g FW

. min

)

0.2 mM

0.5 mM

1 mM

2 mM

H65

CytoplasmicN C

1 2 3 4 5 6 7 8 9 10

L461

11 12

N155

G334

Q249W250

Plasmodesmata

Sucrose

Sink

Tissu

e

Phloem

Sucrose

Mesophyll

amino acids

chloroplast

Sucrose

amino acids

amino acids

H+

hexose

sucrose

H+

Vacuole

Vacuole

Vacuole

Amino acids

Amino acids

Sucrose

amino acids

ATP

symplastic

NO3

mass flow

water

water

CO2

1. Sucrose symporters serve both source and sink tissues

2. BUT, there is at least one other way to generate the hydrostatic pressure difference

Phloem Loading in a “Symplastic System”

Also depends on accumulating high concentrations of solutes, but in this case, the “sugar” is synthesized in place, not transported into the CC/SE complex.

X

Synthesize high concentrations of raffinose &/or stachyose in the companion cell (intermediary cell). Raffinose is too large to diffuse back into bundle sheath cell via plasmodesmata. This generates a high osmotic concentration that results in water influx and high hyrodstatic pressure that drives phloem transport to sinks.

H2O

Sucrose diffuses into the companion cell

Direct evidence of pressure flow: the aphid.

Sapdroplet

Aphid feeding Stylet in sieve-tubemember (LM)

Stylet

Severed stylet exuding sap

Sap droplet

Sieve-tubemember

25 µm

What happens when sucrose gets to the import-dependent organs?

It is unloaded from the companion cell/ sieve element complex via apoplastic and symplasticpathways.

The movement of sucrose from sources to sinks is called assimilate partitioning. Controlling this process could have a profound impact on crop yield.

Plasmodesmata

Sucrose

Sink

Tissu

e

Mesophyll

amino acids

chloroplast

sucrose

Phloem

SucroseSucrose

amino acids

amino acids

H+

H+

hexose

Vacuole

Vacuole

Vacuole

Amino acids

Sucrose

ATP

symplastic

NO3

mass flow

water

Apoplastic model of phloem loading

?

?

TP

H+

water

Amino acids

CO2

H+

amino acids

SUCROSESYMPORTER

PHOTOSYNTHETICACTIVITY SINK ACTIVITY

H2O

CO2

time

transpirational feeding

Tran

spor

t Act

ivity

(pm

ol /

min

/ mg

prot

ein)

0

500

1000

1500

2000

2500

0 100 200

mM Sucrose Fed 24 Hours

SucGlucAla

• Are changes in sucrose transport activity due to osmotic effects?

No. KCl, sorbitol, mannitol have no effect.

• Is the observed regulation sucrose-specific?

Yes. Hexoses and hexose analogs are not effective.

• What happens to sucrose transport activity?

Km remains unchanged. Vmax decreases.

• How?

BvSUT1 mRNA and protein decrease with 2 hr half-livesTranscription is controlled by a sucrose sensing protein-phospho relayand overall transport capacity is directly proportional to transcription

Jen Chiou, Matt Vaughn, Wendy Ransom-Hodgkins, Greg Harrington

Sucrose SensorSucrose

Protein Turnover

Sucrose Symporter

Transcription

mRNA Turnover

A

Kinase

Phosphatase

B

PhloemLoading

All within the companion cell

Sucrose

H+

High rate of symporter transcription

High rate of sucrose export

Abundant symporter protein

Sucrose

Low Sink Demand

H+

Sucrose accumulation in the phloem

Low rate of sucrose export

Down-regulation of symporter transcription

Less symporter protein

PH

LOE

M

PH

LOE

M

Sucrose-signalingresponse pathway

High Sink Demand

How can we identify players in the sucrose signaling pathway?

1. Mutagenize SUT1promoter::reporter plants and screen for mutants that lack the sucrose signaling pathway

2. Use expression profiling of companion cells to identify specific kinases and phosphatases, then look at phenotypes of knockouts

3. Look for a sucrose response that is more tractable for genetic analysis

Four day old seedlings induced with 90 mM sugar treatments and photographed three days later.

Water

Anthocyanin Content

012345678

wat

er

sucr

ose

gluc

ose

fruct

ose

gluc

+fru

c

man

nose

mal

tose

sorb

itol

A53

0-A

657/

gFW

H20

Suc

rose

PAP1

ACTING

luco

se

Fruc

tose

Glu

c +

Fruc

Man

nose

Mal

tose

Sor

bito

l

PAP1 (Production of Anthocyanin Pigment 1) Transcription factor ‐ known “master”regulator of anthocyanin biosynthesis and part of a multi‐peptide complex

Pro 8 Pro 62 Pro 63

H2O SUC H2O SUC H2O SUC

Col-0 35S-GUS

1 mm

H2O SUC H2O SUC

H2O SUC H2O SUC H2O SUC

Pro 8 Pro 62 Pro 63

Wg 34 Wg 35 Wg 43

H2O SUC H2O SUC H2O SUC

Col-0 35S-GUS

H2O SUC H2O SUC1 mm

H2O SUC H2O SUC

ΔIntron1 2A ΔIntron1 3B

ΔIntron2 3A ΔIntron2 5B

H2O SUC H2O SUC

ΔIntron1&2 4A ΔIntron1&2 6A

H2O SUC H2O SUC

SURE-1

5’-upstream sequence

200 bps = exon = intron

ATG

I II III

ISURE-2

PAP1 gene

PAP1wg_mut1-GUSGUSI II III

SURE-1 (TTTTCTATT) mutated to TTTGAGATT

PAP1wg_mut2-GUSGUSI II III

SURE-1 (TTTTCTATT) mutated to SURE-2 (AATACTAAT)

PAP1wg_mut3-GUSGUSI II III

205 bp deletion (includes SURE-1)

PAP1wg_mut4-GUSGUSI II III

205 bp deletion (excludes SURE-1)

H20

SU

C

∆ SURE-1

PAP1

GUS

ACTIN

GLU

H20

SU

C

∆ SURE-1

GLU

H20

SU

C

Minus SURE-1

GLU

H20

SU

C

+ SURE-1, - 5’

GLU

4 day old plants, 4 hr induction

Biotech Approach to Increase Yield: Use sugar beet as a model biofuel crop by by manipulating sugar allocation in the plant’s vascular system.

Why sugar beet as a model for carbon partitioning

• Root yields of over 40 tons per hectare at 15.5-18% sucrose content (6-7 tons of sugar per hectare)

• Record yields approach 25% sucrose, so there is somewhere to put “extra” sucrose

Approach: Constitutively express hyper-active symporter

Hypothesis: This will draw down sugars in photosynthetic cells which will,1) Increase photosynthesis 2) Delay senescence

Leaf mesophyllPlasmodesmata

Sucrose

Phloem

Sucrose

chloroplast

SucroseH+

H+

vacuole

Sucrose

CO2

ATP

mass flow

?

?

AtSUC1H65K TTCmGAS1p

The modified SUT is expressed in the transgenic lines

Independent transgenic lines

Con

trol 1

Con

trol 2

13 121

8 43 46 139

202

226

230

239

248

257

Average tuber FW of control and transgenic lines

0100200300400500600700800900

aver

age

tube

r FW

(g)

Con

trol

SU

T-8

SU

T-13

SU

T-43

SU

T-46

SU

T-12

1

SU

T-13

9

SU

T-20

2

SU

T-22

6

SU

T-23

0

SU

T-23

9

SU

T-24

8

SU

T-25

7

**

*

Independent transgenic lines

p-value ≤ 0.01

Average above ground biomass of control and transgenic lines

05

101520253035

** * *

aver

age

abov

e gr

ound

bi

omas

s (g

)

Con

trol

SU

T-8

SU

T-13

SU

T-43

SU

T-46

SU

T-12

1

SU

T-13

9

SU

T-20

2

SU

T-22

6

SU

T-23

0

SU

T-23

9

SU

T-24

8

SU

T-25

7

Independent transgenic lines

p-value ≤ 0.05p-value ≤ 0.01

aver

age

tube

r FW

/leaf

DW

Average tuber FW/leaf DW of control and transgenic lines

Con

trol

SU

T-8

SU

T-13

SU

T-43

SU

T-46

SU

T-12

1

SU

T-13

9

SU

T-20

2

SU

T-22

6

SU

T-23

0

SU

T-23

9

SU

T-24

8

SU

T-25

7

Independent transgenic lines

05

101520253035404550

Rice as a model crop for identifying biomass genes

Rationale:The Green Revolution was about seed production, not biomassRice is a great model for new energy crops because ……….

Simple grassGene syntenyAg-infrastructure well establishedPowerful genetic, molecular, and genomic tools

OryzaSNP set20 diverse rice lines• resequenced for SNP discovery

PNAS 106:12273-12278 2009

Approach:• Define morphological and physiological differences

• Forward genetic screens

• QTL mapping

• Association mapping

• Transgenic manipulation of candidate genes

knockout, over‐expression, truncated proteins

OryzaSNP set• Sampling

– Morphological• leaf length & width, plant height, tiller number, and total above ground biomass (3‐fold) and seed yield (inverse of biomass)

– Physiological• Leaf area index, photosynthetic rates, cellulose and lignin content of leaves and stems

OryzaSNP set

• Significant genetic variation for both morphological and physiological data

• Heritability• Photosynthesis

– M2O2 and Cypress• Highest photosynthetic rate

– Pokkali• Lowest photosynthetic rate, largest biomass

– Why PS not correlated to total biomass

Mutant populations: chemical and fast neutron

Amino Acid Transporters & Nitrogen regulation of gene expressionLishan ChenHui-Chu ChangAdriana Ortiz-LopezAaron SchmitzEkrem DundarMengjuan GuoXianan LiuVince StoergerChristian HermansSilvana Porco

Sucrose Transporters and gene regulationTzyy-Jen ChiouJade LuMatt VaughnWendy Ransom-HodgkinsGreg HarringtonAnshuman Kumar

RICEJan LeachJohn McKayHei Leung - IRRIBettina BroecklingCourtney Jahn

USDA-ARS, DOE, & NSF