lecture #18 - oregon state university · lecture #18 “carbon concentrating mechanism” rubisco...

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Lecture #18 “Carbon Concentrating

Mechanism”

RubisCO is CO2 limited

RubisCO is a carboxylase and an oxygenase with low affinity for CO2

Conditions that drive the evolution of CO2 Concentrating Mechanisms

Earliest fossil evidence of plants with a CO2 Concentrating Mechanism 12 Mya. Kranz anatomy of C4 plants Very little evidence prior to 12 Mya.

Conditions that drive the evolution of CO2 Concentrating Mechanisms

Both the earth system and biology follow Dollo’s

Principle

• “An organism is unable to return, even partially, to a previous stage already realized in the ranks of its ancestors”

• More generally referred to as contingency

• Both the earth and biology follow this principle

Bridgham, J. T., E. A. Ortlund, AND J. W. Thornton. 2009. An epistatic ratchet constrains the direction of glucocorticoid receptor evolution. Nature 461: 515-519.

C5

C1

2xC3

C3

C1

C4

C3 and “C4 assisted” photosythesis

pyruvate

PPDK

PPDK Pyruvate, Phosphate DiKinase

THE C4 PATHWAY IN PLANTS USES

UBIQUITOUS ENZYMES

CARBOXYLATION DECARBOXYLATION

Pep Carboxylase Malic Enzyme

NAD or NADP

Pep Carboxykinase In a green alga

Pep Carboxykinase

With C4 Intermediate Active Bicarbonate Transport

CO2 Diffusion Active CO2 Transport

HCO3- HCO3

- HCO3-

HCO3-

HCO3-

CO2

CO2 CO2

CO2

CO2 CO2

CO2

CO2

C4 acid

CO2

Biomass Biomass

Biomass Biomass

Chloro- plast CO2

HCO3-

Alternative carbon transport systems

C4 Plants

C3 Plants

Many algae

Does not exist

Carboxylation

Decarboxylation And Fixation by RubisCO

Hatch MD and Slack CR 1966. Photosynthesis by sugar-cane leaves. A new carboxylation reaction and the pathway of sugar formation. Biochemical Journal 101, 103–111

Kranz-less C4 photosynthesis

Bienertia cycloptera CO2

CO2

C4

C3

PPDKp

PEPc

MEm

RubisCO

C3

There are suggestions that single cell C4 may have been the earliest form of CO2 Concentrating Mechanism

C4 Photosynthesis

• Many grasses • Some shrubs • No trees • Many forbs and weeds

• 1% of plant species • 30% of plant photosynthesis

corn

Sugar cane

sorghum

Saltbush (Atriplex)

Pigweed (Amaranthus)

1 2 3 4

Carbonic Anhydrase 5

5

Vasc

ular

Tis

sue

CO2

GAP

Starch

Sucrose

GAP

PEPcase: initial Carbon fixation step; substrate is HCO3 and thus no affinity for O2

C4 Photosynthesis

With C4 Intermediate Active Bicarbonate Transport

HCO3- HCO3

- HCO3- HCO3

-

CO2 CO2

CO2 CO2

C4 acid

CO2

Biomass Biomass

Chloro- plast CO2

HCO3-

Acid Stable Acid Labile

14C 14C

Questions: What is the nature of carbon pool in marine diatoms? Can we determine that the CCM has an organic intermediate pool?

Nota Bene: Inhibit decarboxylation using MPA

Carbon influx Si oil centrifugation

sample of cells

fixative silicon oil

add 14C label and mix

10 sec and centrifuge

freeze in LN2

Scintillation Counter

cut off bottom Acidify half

Varied fixative and added MPA

Difference = organic carbon

About 70% of the carbon taken up in 10 s is stored as organic carbon

With C4 Intermediate Active Bicarbonate Transport

HCO3- HCO3

- HCO3- HCO3

-

CO2 CO2

CO2 CO2

C4 acid

CO2

Biomass Biomass

Chloro- plast CO2

HCO3-

What is the nature of carbon pool in marine diatoms? Organic Carbon

Question: • What is the importance of C4 carbon fixation in diatom photosynthesis? Straightforward methodology. Inhibit carbon fixation with DCDP (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-

propenoate)

-OOC -OOC OPO32- CH2PO3

2-

C C C

Cl Cl CH2 PEP DCDP

control

+DCDP

HCl +DCDP

T. weissflogii (diatom)

Chlamydomonas sp. (C3 Chlorophyte)

control

Effect of PEPCase

inhibition by DCDP*

on diatom photosynthesis

control

+DCDP

+DCDP high CO2

+DCDP low O2 control

+DCDP

T. pseudonana PEPC1 (mito) ++ PEPC2 (CER/PPS) ++ ME 0 PEPCK 0 T. weissflogii ME 0 PEPCK 0

Key transcripts upon shift from high to low CO2

Quantitative Proteomics MudPIT

Tryptic Lys-C Digest HPLC fractions

T. pseudonana PEPC2 (CER/PPS) ++ ME 0

PEPCK - CA (3) ++ Pyruvate Carboxylase ++ plastid targeted

Proteins under low CO2

C3 & C4 Photosynthesis

Calvin Cycle

Rubisco CO2 CO2

stomata

Calvin Cycle

Rubisco CO2 CO2

stomata

C3

C4 C4 acid

• C4 has significant impacts on the photosynthesis-irradiance relationship and water use efficiency

• Minimal loss from photorespiration • Better water use efficiency (for a given CO2 uptake, C4 plants can keep

stomata more closed)

• Enhanced high-light photosynthetic rate

Advantages of C4 Photosynthesis

note

C3 C4

Advantages of C4 Photosynthesis

4

5

Vasc

ular

Tis

sue

CO2

GAP

Starch

Sucrose

GAP

Disadvantages of C4 Photosynthesis Pyruvate,orthophosphate dikinase (PPDK) in C4 plants is cold sensitive - mechanism of inactivation not fully resolved. [Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C3 and C4 plants, Chinthapalli, Murmu and Raghavendra. Journal of Experimental Botany, 2003 54:707±714]

C3 C4

Disadvantages of C4 Photosynthesis

C4 Photosynthesis

CO2

CO2

HCO3 C4 CO2

NADPH

C3

NADPH

C3

ATP

Advantages • Minimal loss from

photorespiration • Better water use

efficiency

Disadvantages • Temperature

sensitivity • Still dependent on gas

exchange during the day

CAM Plants CAM: Crassulacean acid metabolism

The ‘other C4’ or ‘C4-PM’

Basic principles: • Another ‘patch’ on typical C3

photosynthesis • As with C4, initial carbon fixation in CAM is

through PEPCase • The primary objective of CAM, however, is

to allow the stomata to be CLOSED for most (or all) of the day – thus addressing a weakness of standard C4

• This is achieved by storing carbon at night

CAM Photosynthesis

C3, C4, & CAM

Calvin Cycle

Rubisco CO2 CO2

stomata

Calvin Cycle

Rubisco CO2 CO2

stomata

C3

C4 C4 acid

Calvin Cycle

Rubisco CO2 CO2

stomata CAM C4 acid

Night Day

Night

CAM Photosynthesis

The quantity of acid produced at night can be very high (up to 1.4 M H+) – the cytoplasm could not function properly at the pH this would create

CAM Photosynthesis

Day One major difference between C4 and CAM is that CAM does not require separation of pathway between different cells. It all happens within a single cell.

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Time for a break…