HG’s CCM confederates Over the years, thanks to my many collaborators, and particularly those who

undertook amazing exploits in lab and field, including:

Pat Adams Chandra Bellasio Yasmin Baksh-Comeau Mary Bassett John Beardall Hans Bohnert Annie Borland Mark Broadmeadow Susanne von Caemmerer Oli Caspari Lucas Cernusak Asaph Cousins Caroline Crook John Cushman Barney Davies Antony Dodd Mike Fordham Todor Genkov Jim Gillon Jan Girnus Nina Griffiths James Hartwell Kirsty Harwood Richard Haslam Brent Helliker Julian Hibberd Peter Horton Glyn Jones Wanne Kromdijk Gary Lanigan Helen Lee Ulrich Luettge Cristina Maguas Kate Maxwell Ernesto Medina Monica Mejia-Chang Moritz Meyer Maddie Mitchell Nick Owen Simon Pierce Marianne Popp John Raven Fernanda Reinert Casandra Reyes-Garcia Kath Richardson Wendy Robe Andy Roberts Jessica Royles Rowan Sage Christian Schaefer Hans Schepers Ulli Seibt J Andrew C Smith Liz Smith Bob Spreitzer Karl-Heinz Stimmel Tahar Taybi Klaus Winter Louise Wood

And many thanks to the C4CAM 2013 organisers for their kind invitation to launch the festivities (and to Plant Cell and Environment for support)

Drivers of Diversity for the Carbon

Concentrating Mechanism

Confederacy

• Howard Griffiths1, Juan-Carlos Villarreal2 and Moritz Meyer1 • 1Physiological Ecology Group, Department of Plant Sciences, University of Cambridge, U.K.

• 2Department für Biologie, Systematische Botanik und Mykologie, Ludwig-Maximilians-Universität (LMU), München,

• Historical Perspective: under the influence of John Raven and Barry Osmond

• Ancient origins: the CCM confederacy

• Algal and Hornwort CCM systems: origins

• Origins and evolution of C4: insights for metabolic plasticity

• CAM as a model pathway: insights for metabolic plasticity

• Biochemical vs biophysical CCM: challenges for the next generation

1979.....

Orion Face

Direct, with

John Riley

1981..... and so to Trinidad

with Steve Hubbard, John

Riley and J. Andrew Smith

Bromeliad Beginnings in Trinidad,1981

Suggested by Barry Osmond, supported by John

Raven and informed by J Andrew C Smith

With subsequent expeditions in 1983, 1990, 1992,

1995… and 2014?

13C in Organic

material and photosynthetic

pathway

From Stable

Isotopes to

Rubisco

Activate Rubisco and the

planet draws breath • Leaving an inverse signal in

the atmosphere- hence the basis for real-time, on-line

carbon isotope discrimination

Rubisco: flawed, inefficient or at best

confused, and a wasteful predilection for O2

as well as CO2: enter our confederacy of

carbon concentrating mechanisms

• Energetically costly photorespiration

• Requires refixation of fixed carbon and nitrogen

• Suppress by increasing CO2:O2 ratio

RuBP

3P-glycerate

triose/hexose-P

2P-glycolate

glycerate

glycine

CO2

O2

CO2 + NH3

ribulose-5P

storage utilisation

serine

Oxygenase and

photorespiration

Carboxylase and

CBB cycle

Origins and distribution of CCMs both

biophysical and biochemical

Image by J-C Villarreal

1. Ci gradient build up (x1000 in cyanobacteria, x20 in algae)

• HCO3- and CO2 transporters

• Targeted carbonic anhydrases (CA): HCO3- CO2

2. A compartment to package Rubisco, fix Ci and limit CO2 leakage:

cyano. carboxysome and eukaryotic pyrenoid

cyanobacteria Eukaryotic algae

CCM (i) The “biophysical” carbon

concentrating mechanism in algae:

inducible under low ambient [CO2]

CCM- Carbon concentrating

mechanisms: (ii) biochemical • Co-opt an additional carboxylase

• Couple anaplerotic CO2 cycling using PEP Carboxylase

• Spatially separate in two cell types (C4 pathway)

• Temporally separate across day and night, using reciprocal

carbohydrate/organic acid reserves (CAM: Crassulacean acid

metabolism)

• Improve radiation, water and nitrogen use efficiencies

C4: Maize and Miscanthus; CAM: Agave and cacti

Origins of CCMs? (after Badger and colleagues)

Origins of CCMs? (after Badger and Price)

?

?

?

Not forgetting CAM in Isoetes,

Welwitchia etc

The Cyanobacterial CCM

Long et al (2007, 2011) Price

2011 (Photosyn Res): well

defined CCM components,

carboxysome coat and Rubisco

interactions

CCM The “biophysical” CCM in algae:

inducible under low ambient [CO2]

Moroney JV Ynalvez RA (2007) Eukaryotic Cell, 6, 1251–1259

An Algal CCM: many structural, physiological

and molecular correlates are well defined

• Various combinations of bicarbonate pumps and carbonic anhydrases (CA)

• x50- x100 ambient CO2 concentrations delivered to Rubisco packaged within pyrenoid

• Pyrenoid intersected by knotty Intrathylakoid membranes and CA

• Possible role of starch sheath and other proteins as diffusion barriers

We have an understanding of

some of the CCM components in

Chlamydomonas (the ‘knowns’)

Meyer MT and Griffiths H (2013) Origins and Diversity of Eukaryotic CO2-

Concentrating Mechanisms: Lessons for the Future J exp Bot, 64, 769–786

spinach hybrid wildtype

Moritz Meyer and Maddie Mitchell

The Chloroplast pyrenoid is at the heart of the

algal CCM

• The pyrenoid is surrounded by a pronounced starch sheath

• Rubisco is aggregates within the pyrenoid, as shown by imunogold labelling

• Measure photosynthesis as O2 exchange as a function of inorganic carbon (HCO3) supply

• High affinity HCO3 uptake system: WT: +pyrenoid + CCM

• Low affinity HCO3 uptake system: Spinach hybrid: no pyrenoid, no CCM

N-terminus

C-terminus

bA-bB loop

a helix A

a helix B

Previously we have shown that pyrenoid formation

is related to Rubisco SSU alpha helices

• now identifying specific domains associated with Rubisco aggregation

Moritz Meyer and Todor Genkov

also using day night cycles as a means

to investigate CCM induction (as a

simpler alternative to CO2 induction)

Under D/L cycles, Rubisco aggregation and

CCM inducibility occurs on a daily basis

• Transcripts of transporters are linked to pyrenoid and CCM induction

• Other CCM elements (CAs, leak barrier) are constitutively expressed

• Reconstruction of intra-pyrenoid thylakoid tubule network

In addition to CAH3, CCM induction is regulated by

post-translational modifications, and requires

chaperones or linker proteins (we think)

The Pyrenoid, and biophysical CCM, are only found

in Hornworts in terrestrial plant lineages

Vaughn et al. 1992; Renzaglia et al. 2009

Hornwort CCM, Pyrenoid: a Phylogenetic Progression in

Bryophytes ?

Coleochaete

(+CCM/pyr)

Conocephalum

(liverwort)(C3)

Polytrichum

(moss)(C3)

Anthoceros

(hornwort)(+CC

M/pyr)

The CCM in hornworts

improves carboxylation

relative to Pellia but not Marchantia

• Lower CO2

compensation point for

hornwort is consistent

with operation of CCM

• Diffusion limits CO2

uptake in equivalent

(non-ventilated) thalloid

structure (Pellia)

• advantages of internal

air-spaces for

Marchantia and for

progression of C3

terrestrial lineages?

• No selective advantage

for pyrenoid and CCM

in advanced terrestrial

lineages?

Griffiths et al 2004, The Evolution of Plant Physiology eds Poole I and Helmsley A

Recent developments provide a consensus

tree for hornworts using the nad5/rbcL genes

• Villarreal et al (2010: Phytotaxa 9:

150–166)

• The traditional view was a linear

progression from uniplastidic cells

(with a single pyrenoid) to

multiplastidicity (Burr, F.A. (1970)

American Journal of Botany 57: 97–

110)

• This would be consistent with the

pyenoid and CCM as a primitive,

basal condition

Pyrenoid lost and regained?

• The pyrenoid seems to be the

derived condition in Notothyladaceae and

Dendrocerotaceae

• There are examples of pyrenoid loss and gain

• (This rather parallels the situation

found between Chlamydomonas and Chloromonas)

• but the pyrenoid systems are

uniplastidic

Recent

integration of hornwort

Phylogeny,

multiplastidicity,

breeding system

and genome duplication

events

Juan-Carlos Villarreal, unpublished

Divergence of the Hornwort CCM closer to that of C4 and CAM than we thought!

• Leiosporoceros is basal but

pyr-

• the pyrenoid seems to be

basal in the

Anthocerotaceae

• The pyrenoid and

uniplastidicity seem to be

the derived condition in

Notothyladaceae and

Dendrocerotaceae

• The pyrenoid seems to be

a relatively recent

addition, at least over the

past 100 million years

Land plant lineages and origins of a

biophysical CCM

• The CCM in algal lineages probably did not lead directly to the Hornwort system

• Rather than a relict, hornwort pyrenoid and CCM seem to be a new invention

• Why have no other land plant lineages developed a biophysical CCM?

1. Improved diffusive supply of CO2, through stomata and internal air spaces, made CCM unnecessary

2. Energetic cost of CCM unsustainable in low light as canopy cover was generated or shaded habitats initially colonised

3. CCM is helpful to overcome diffusive limitations of external liquid boundary layer and a non-ventilated thallus

4. Are there ultrastructural implications which will require interactions between pyrenoid and thylakoid membrane stacking?

5. We need to define the Hornwort CCM system, particularly in those epiphytic life-forms which can tolerate variable conditions

Rubisco

CO2

PEP

OAA

1-2% CO2

Mesophyll Cell

Bundle Sheath Cell

Malate

Pyruvate

PEP

Carboxylase HCO3

-

C4 photosynthesis involves alterations to

biochemistry, cell biology and development

Kranz anatomy: spatial separation of carboxylase

enzymes between mesophyll and bundle sheath cells

Novel cell-specific targetting mechanisms now

being characterised by Julian and the C4-

Rice/C3C4 consortia • Cis-and Trans- acting elements –now shown to target BS or Mes

• cis-acting elements have been recruited repeatedly within C4

lineages and target gene expression within Bundle Sheath cells

• Bundle Sheath targetting in a variety of tissues:

0

10

20

30

40

50

60

A

240bp 5’

CgNAD-ME1 p35S uidA NosT 5’UTR

ATG TGA +240 -86

M

no

. c

ell

s e

xp

ress

ing

uid

A

0

10

20

30

40

50

60

B

240bp 5’

CgNAD-ME2 p35S uidA NosT 5’UTR

ATG TGA +240 -78

M n

o. c

ell

s e

xp

res

sin

g u

idA

BS BS

unspliced AtNAD-ME1

pNAD-ME1

uidA 3’

region 5’UTR

ATG TGA +5015 -125 -1700 +700

0

10

20

30

40

50

60

D

240bp 5’ AtNAD-ME1

p35S uidA NosT 5’UTR

ATG TGA +240 -125

M

no

. ce

lls e

xp

res

sin

g u

idA

0

10

20

30

40

50

60

E

240bp 5’ CgNAD-ME1

p35S uidA NosT 5’UTR

ATG TGA +240 -86

no

. ce

lls

exp

res

sin

g u

idA

M BS

F

Brown, N.J. et al Hibberd, J.M. (2011) Independent and parallel recruitment of pre-

existing mechanisms underlying C4 photosynthesis. Science, 331: 1436-1439

Also time to re-evaluate origins and

function of Bundle Sheath anatomy

• Origins of Bundle sheath in C3 grasses

perhaps related to maintaining hydraulic

conductance and drought tolerance

Evolution of C4 pathway: consistent with BS

development occurring in advance of

reduced IVD

• With observations now supported by other extensive

phylogenetic studies (Pascal-Antoine, Erika and Colin in PNAS; Ben and Julian)

Griffiths H, Weller G, Toy LFM, Dennis RJ (2013) Plant Cell & Environment, DOI:

10.1111/j.1365-3040.2012.02585.x 36 (2), 249-261

Eddy flux over C4 crop canopies- Maize in the ‘summer’ of

2008: work by Wanne Kromdijk, Gary Lanigan and

colleagues

C4 Physiology and decarboxylase subgroups: why

does Maize (NADP-ME) also have PEPCK activity?

A metabolic model suggests that transamination allows

plasticity in supply and demand for ATP and reductant

between M and BS cells (Chandra Bellasio)

Profiles of light penetration across a leaf were used to

derive the potential ATP supply for M and BS cells

induced by changing light quality (as R, G or B).

• Empirical measurements of leaf-level net CO2 uptake, electron transport rate (JATP under low O2 tension) and carbon isotope discrimination.

• Overall C4 operating efficiency (as BS leakiness,Φ) was not affected by light quality (consistent with studies by Asaph and colleagues).

• As the light availability to BS is increased (effected exptlly by (B-R-RGB-G wavelengths), so proportion of ATP supplied by BS can also increase

• 1. Under blue light (absorbed mostly by Mes cells), ATP and reductant generation by BS are minimal, mal export predominates, PEPCK is lowest and most starch is synthesised in the mesophyll

• 3. Under Green Light, (and combined RGB), the BS is virtually self- sufficient in ATP, PEPCK and starch synthase activities are highest

Modelling BS ATP and NADPH demand matches

partitioning implied empirically under R B G wavelengths

We conclude that metabolic plasticity could be an

important determinant of C4 diversity, as well as

anatomy and environmental interactions

• Model was used to partition proportional metabolic activity between Mes

and BS cells( MAL/ASP, rates of PGA reduction (PR), starch synthesis (SS)

and PEP regeneration

• Relative ATP and reductant demand could be calculated for BS and Mes

cells

• Metabolic plasticity (originally visualized as the confusing combination of

NADP-ME and PEPCK activities in Maize) was an important driver of C4

diversity

• Confirms the importance of both decarboxylase systems in Maize in

allowing metabolic plasticity in balancing ATP and NADPH requirements

between BS and M

• Relevance for during steady-state photosynthesis limited by light intensity

or variable light quality and intensity within a crop canopy.

Modelling energetic and metabolic implications of changing light quality and

intensity in maize leaves

Chandra Bellasio and Howard Griffiths (about to be submitted)

And so to

CAM:

Bromeliads

and Clusia

stranglers

• For many years,

HG and

colleagues have

been defining

CAM metabolic

plasticity as:

• environmental

control of CAM

Phases

• PEPC and Rubisco

activation

• circadian control

etc

Davies B. N. and Griffiths H. (2012). Plant Cell

and Environment 35, 1211–1220

But do stem and leaf succulents have

different ways to regulate CAM expression?

• What matters:

• Degree of succulence?

• Differentiated chlorenchyma and water storage tissue?

• Life cycle?

• Metabolic plasticity?

• Intriguingly (and these

ARE the only measurements Nick has

made so far!) Kalanchoë

and Agave differ in their

degree of succulence

• The leaf succulent (Kd)

has lower air spaces and

stomatal density than the

stem succulent (At)

• Implying that stomatal and mesophyll

conductances will be

lower in dense leaf

succulent life forms

• Otherwise parameterise a systems dynamics model

for CAM from published

data

Then along came Nick Owen…….

Kalanchoë daigremontiana Agave tequilana

• SYSTEMS DYNAMICS: State-dependent feedback mechanisms

operating between state (A-variables) and flow (J- and V- variables)

• Allows temporal separation of carboxylase activities and four

Phases of carbon uptake that characterise CAM expression to

be modelled

• Simulate regulation at key flow junctions (e.g. stomatal

conductance, mesophyll conductance, malic acid transport

across the tonoplast) and their feedback control (e.g. stomatal

aperture, malic acid induced inhibition of PEPC, carboxylase

enzyme kinetics)

To put it simply…. perhaps better to

check out Nick’s poster

The model was initially parameterised using Kalanchoë

kinetic constants, stomatal and mesophyll conductances

and metabolite storage: would it work for Agave?

• Agave has higher nocturnal gas exchange and acid accumulation during ‘Phase I’ of CAM. So ramp up:

• Vacuolar storage capacity (Xvmas x2)

• Stomatal and mesophyll conductances(gsmax, gm x4)

• PEPC Vmax (x2)

• PEPC Km (x0.5)

• And nocturnal activity CO2 fixation and malic acid accumulation increases!

• But the magnitude and plasticity of expression of

CAM daytime Phases (II, III,

IV) is much higher in

Kalanchoe, surely?

• OK, so reduce Rubisco

Vcmax (x 0.5)

• Reduce relative rate of

decarboxylation (Vdmax) (x 0.4)

• Delay timing of PEPC-

Rubisco handover

• And the day-time expression of CAM Phases is reduced!

Park Nobel reported making a CAM Agave C3 by

watering- how well did Nick’s model do? • Simulated CAM expression for the leaf-succulent Kalanchoë

daigremontiana showed a strong correlation with measured gas exchange and malic acid accumulation (R2 = 0.912 and 0.937 respectively)

• And for the stem-succulent, A. tequilana, R2 = 0.928

• So again we see that metabolic plasticity, aided by the succulent ultrastructural framework, allows productivity to be maximised by contrasting lifeforms

Owen N.A. & Griffiths

H (2013) A System

Dynamics model of

Crassulacean Acid

Metabolism

New Phytol. Doi

10.1111/nph.12461

Lessons from the CCM confederacy

(i) were drivers of CCM diversity due to

hydraulic or CO2 limitations? • Colonisation of land actually equates to colonisation of air (JAR)

• Stomata and vascular system were co-incident 420 mya for those earliest land plant life-forms

• There has always been a trade-off between water transport, (hydraulic conductance) evaporative demand to allow CO2 uptake

• Increasingly we recognise that mesophyll conductance limitations represent an additional component

• Neat examples include evolution of 3D venation in succulent tissues*, BS/IVD interactions in semi-arid regions, and limitations to gas exchange imposed by surface water films… which were then colonised by a CCM

(*Ogburn and Edwards Curr. Biol 2013)

Lessons from the CCM confederacy

(ii) why have only Hornworts got a

biophysical CCM on land?

• H1. Uniplastidicity, can be secondarily derived, with pyrenoid plus CCM confers an ecological advantage for an undifferentiated (no air spaces) gametophyte thallus, overcoming mesophyll diffusive limitations in habitats with intermittent water supply.

• H2. Multiplastidicity, in hornworts lacking a CCM, allowed the development of granal stacks and the spatial distribution of photosystems, (more similar to advanced land plants), has ecological advantages for acclimation to high and low light habitats

• H3. The CCM in hornworts is analogous to that in algae and may have shared key inorganic carbon transport components but there are different forms of pyrenoid within a single genus (Nothoceros).

• Maybe epiphytic Nothoceros genus hold a clue to how a pyrenoid

and CCM adapts under variable environmental conditions..

But we need to find out!

Lessons from the CCM confederacy

(iii)will C4 or an algal/cyanobacterial

CCM be introduced into a crop? • Rubisco makes an amazing contribution to carbon

sequestration but we will need more crops for the future

• Rubisco operating efficiency: biological turbochargers (CCMS) have evolved convergently many times in terrestrial and aquatic plants

• Understanding the C4 pathway offers genuine prospects for C4 rice and wheat, but spatial co- organisation and targetting is a real challenge

• Could and an algal/cyanobacterial CCM, in every chloroplast of every cell, offer an alternative turbocharger for a higher plant?

• Would a carboxysome or pyrenoid prove tractable additions to higher plant chloroplasts? In addition to DIC transporters, will we need to engineer Rubisco super- molecular complexes? Would there be issues associated with ionic balance/pH regulation for chloroplasts?

Lessons from the CCM confederacy

(iv) does metabolic plasticity help to

overcome the anatomical straightjacket

imposed by Kranz and succulence?

• There have been over 60 independent origins for

both of C4 and CAM

• Intriguing that optimising light use in C4 seems to be

a driver for metabolic plasticity

• Intriguing that it should prove relatively facile

metabolically modify the transition from leaf

succulent to stem succulent CAM expression

• In the future, we need to look at both metabolic and

anatomical convergence in the evolutionary

progression from C3 systems

Lessons from the CCM confederacy

(v) don’t let your thinking be straight-

jacketed by terminology (a final rant)

• Sorry, but anatomical preconditioning or pre-adaptation are terms which (to me) seem to suggest some direction or logical evolutionary progression towards your favourite pathway

• Instead of some linear progression towards (gain or loss) of a given CCM, remember how many times C4, CAM and biophysical CCMs have independently evolved (and those intermediates that did not make it!)

• Examine the C3 progenitors for anatomical and metabolic plasticity; identify potential environmental effectors; therein will lie the cell-specific regulation, the common drivers for gene expression or development, and ultimately the plasticity needed to cope with contrasting environmental conditions (or life for a new CCM in a new higher plant home!)

To conclude:

• Rubisco makes an amazing contribution to carbon sequestration but we will need more, stress tolerant crops for the future

• Rubisco operating efficiency: biological turbochargers (CCMS) have evolved convergently many times in terrestrial and aquatic plants (and in many more instances than could be covered)

• Understanding the C4 pathway offers genuine prospects for C4 rice and wheat, but spatial co- organisation and targetting offers exciting challenges

• A cyanobacterial, algal or hornwort CCM, in every chloroplast of every cell, could offer an alternative turbocharger for a higher plant, perhaps.

• John Albert, Barry and all the other tremendous C4 and CAM pioneers, there is still plenty for us to do; as for you youngsters, what are you waiting for? There are exciting times ahead!

Thank you for your attention, and now lets get back to

Trinidad with young Jamie Males! Anyone else up for it?

And later with

Littorella in

the Lake

District

Ahh..... the field trip to Loch Brandy in pursuit of Isoetes, with John, Kath, Beardbogles and Malcolm Reid

When the pyrenoid is suppressed, so

too are elements of the CCM

• Transcripts of transporters are linked to pyrenoid and CCM

• Other CCM elements (CAs, leak barrier) are constitutively expressed

O'Leary, BioScience (1988), 38: 5 p328-336

4.4‰ 28‰

High [CO2]/[O2] ratio

12C = 98.89% of atmospheric C 13C = 1.11% of atmospheric C

An unknown portion of CO2 leaks back

?

?

?

-5.7‰

2ATP required per bicarbonate fixed

C4 Physiology: about 20- 30% of concentrated

CO2 retrodiffuses due to BS leakiness (ϕ), which

increases at low light intensities

Wanne Kromdijk...summer 2007

Leakiness increases with decrease in light intensity

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

0 200 400 600 800 1000 1200

PAR, μmol photons m-2 s-1

Ca

rbo

n i

so

top

e d

isc

rim

ina

tio

n (Δ

13C

),

pe

rmil

le ‰

0.0

5.0

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rbo

n i

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top

e d

isc

rim

ina

tio

n (Δ

13C

),

pe

rmil

le ‰

Algae and their symbiotic partners in

lichens- the

biophysical CCM operating in the

aquatic and terrestrial

environment

• Over the past billion years or so, various evolutionary diversions and divergences have led to an array of carbon concentrating mechanisms (CCM), such as the largely aquatic biophysical CCM, and the biochemical C4 and CAM pathways. These processes have helped to overcome the ancient catalytic predilections of Rubisco and improve the CBB cycle operating efficiency over the past 500 million years, perhaps in response to limitations in water and CO2 supply in terrestrial and aquatic habitats. Currently, there is an increasing hope that we can introduce elements from these mechanisms into C3 crop plants as a means to cope with crop security and food-supply stability in a climatically challenged and more populous world. By way of an introduction to C4CAM meeting, the presentation will consider some of the factors likely to have led to the origins of biophysical and biochemical carbon concentrating mechanisms. We will address the convergence in form and function seen within the most commonly found representatives, and the way that anatomical, physiological and molecular divergence provides evidence, in many cases, for multiple origins phylogenetically within a given taxonomic group. We will consider the origins of the cyanobacterial and algal CCM, and whether the Hornworts (a bryophyte sister group to early land plants) offer scope for investigation either as a relict of some aquatic algal CCM, or as a relatively recent diversification only in the past 100 million years. For the biochemical C4 and CAM pathways, we will address the phylogenetic progression and integration of biochemistry (C2, C4) and anatomy (Bundle sheath, succulence) with likely drivers of diversity for those photorespirationally-challenged and hydraulically-constrained precursors. By way of conclusion, we will try to face the original challenge set by one of our august organisers, to generate sage-like suggestions for the relative exclusivity of biophysical and biochemical CCMs between (semi)aquatic and mostly terrestrial habitats. Parenthetically, such musings may also affect those hoping to enhance C3 crop productivity with some form of biophysical or biochemical CCM, and the complexities of marrying such processes within a multicellular, spatial and/or temporal higher plant framework.

The feasibility of transferring C4 photosynthesis

into C3 crops to increase yield? • The challenge for the international C4 Rice Consortium is

immense, but….

• The repeated evolution of C4 implies it should be possible to

understand the mechanisms

• mRNA seq, advances in proteomics, improved model systems,

and systems analysis are providing significant new insights into the C4 pathway

• Components of Kranz anatomy look like they can be manipulated

in C4 sorghum and C3 rice

• Elements controlling cell specificity are present in multiple C3 genes and operate in multiple lineages

• They will discover a huge amount by trying

10 mis-spent years??

Look C Gully, Glen Clova

Lochnagar Plateau

The Ben-Tower Ridge

He infers carbonate

precipitations occurred

adjacent to cyano cell

walls, firstly when CO2

fell to 1% 1400 – 1300

mya, and then again

to 0.4% (4,000 ppm) at

750- 700 mya

Robert Riding (2006) Geobiology 4, 299–316: the cyano CCM evolved before “snowball earth” episodes, perhaps 6-700 mya, or even 1,200 mya

Why do we need to talk about Rubisco? CCM and improved

Rubisco operating efficiency: is range of Rubisco specificity

factors a cause or effect of CCM?

• Has Rubisco “improved”

with age?

• Did low specificity in cyanos

and algae create

requirement for a CCM?

• OR has the occurrence of

the CCM in cyanos and

algae effected the “relaxed” specificity?

Affinity for CO2 (1/Km)

Cyanobacteria Green algae C4 monocots

C4 dicots C3-C4 interm C3

0 2 4 6 8 10 12 140

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Coccomyxa, why a

Chlorophyte outlier??

Potato couch Rubisco?

After André, 2011 including data of Jorden and Ogren, Kubien and Sage

Whither or wither Rubisco kinetic operating efficiency?

When specificity is plotted from an oxygen-o-centric

perspective, against Km for CO2, perhaps this reflects the

time Rubisco has been operating (?relaxing?) in association

with a CCM.

What happened to CCM during invasion of the land?

Coleochaete

(+CCM)

Liverworts:

Pellia (-CCM)

Marchantia (-

CCM)

Anthoceros

(hornwort)

(+CCM)

Use of stable isotopes to determine CCM

activity, efficiency and boundary layer

limitation

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Liverworts

Conocephalum

Lunularia

Marchantia

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Carbon

concentrating

mechanisms

Phaeoceros

carolinus

Coleochaete

orbicularis

Chlamydomon

as reinhardtii Thallus Relative Water Content

Dry Wet Dry Wet

Under trial conditions, C4 Maize (corn) and a

C4 weed Echinochloa outperform rice

• Carbon sequestration

• Food security

• biodiversity

IRRI, Phillippines, image courtesy Julian HIbberd

C4

Maize

C4

weed,

Echino-

chloa

C3 rice