the land pyrenoid: a silurian way to deal with heat and light?

8/8/2019 The land pyrenoid: A Silurian way to deal with heat and light?

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The land pyrenoid:

A Silurian way to deal with heat

and light?

Richardson, D.A. (2001) Cambridge University: Plant science Dept.incomplete referencing

ABSTRACT

Early Silurian land plants (450MYA), inferably similar to the extant hornwort, could have made use of a pre-

existing pyrenoid CCM, (inherited from an algal ancestor), not because CO2 was in short supply in the aerial

environment; (7000ppm CO2; 20 times greater than today's CO2 conc.), but in response to higher temperatureand more intense light conditions compared to the water column. The land CCM, in the Silurian atmosphere

could have responded to high PAR as modern day hornwort CCMs do; by increasing efficiency for CO2 at the

cost of a reduced light use efficiency; the CCM acting as - a ''light use efficiency reducer''(based on results) -

and in high temperatures the CCM up regulates, also increasing efficiency for CO2, acting as - an ''oxygenase

reaction reducer''- (based on results). The hornwort CCM can be likened to a primitive stomata, regulating

CO2 uptake, in response to light, temperature and thallus water content variations. The eventual loss of the

pyrenoid CCM, and the move towards a more advanced morphology, (as seen in the C3 liverworts), meant CO2

was no longer being pumped around the active site of Rubisco, thus early C 3 liverwort Rubisco kinetics would

have improved efficiency, moreover, early C3 liverworts evolved new chloroplast architecture to deal with high

PAR in a more efficient way. Also, the evolution of pores, to reduce water loss, made the early C3 liverworts

less dependant on water to photosynthesise, than the desiccation prone solid thallus of the hornwort.

INTRODUCTION

Bryophyte Phylogeny

Liverworts and hornworts are a successful group of non-

flowering, rootless lower plants, grouped as bryophytes. They

possess a polykiohydric nature, namely their water content

tends to adjust to the moisture conditions of their environment

(Deltoro et al 1998). This condition is markedly different from

that of the tracheophytes, which are homohydric, where water

supply (from roots) and water status are maintained by

stomatal apparatus which prevent the desiccation of the

photosynthetic tissue. Unusually though, an ancient living

group of bryophytes named hornworts possess stomata in the

sporophyte but not the gametophyte (REF).

Polykiohydry in land plants appears to be a much more

ancient state than homoihydry, with Mega-fossil evidence of

the early land plants shown to be liverwort-like in ultra structure

(Niklas 1997; Edwards et al 1998). However within the range

of surviving orders of bryophytes, progressive specialisation oftheir morphology show evidence of a move towards greater

control over water relations; from simple solid thallus (Pellia

spp.) to the more complex differentiated structures with pores

(Marchantia spp.) that show more control over water status.

In terms of the origin and evolution of early land plants,

modern day bryophytes quite possibly resemble the

characteristics of the earliest plants on land. The first good

evidence for the existence of bryophyte-like land plants

(Eoembryophytes) is seen in spore tetrads (comprising four

membrane-bound spores), found over a broad geographic

area in the mid-Ordovician period, 476 Myrs (Gray 1993).

The combination of decay resistant walls (implying the

presence of sporopollenin) and tetrahedral configuration

(implying haploid meiotic products) are further diagnostics of

land plants. Further evidence lay in spore wall ultra structure

and the structure of fossil cuticles from the Late Silurian and

Devonian mega fossils, leading Kendrick and Crane (1997)to suggest the above palaeobotanical evidence would


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support previous arguments that land flora during these times

was liverwort-like. According to Kendrick and Crane (1997),

land plants (embrophytes) are most closely related to the

Charophyceae, a small group of predominantly freshwater

green algae. Within this group, either Coleochaetales (15

living species) or Charales (400 living species) or a groupcontaining both, is a sister group to land plants. Land plant

monophyly is supported by comparative morphology and

gene sequences (18S cox iii).

Relationships among the major basal living groups are

uncertain. But the best supported hypothesis resolves

liverworts as basal and either mosses or hornworts as the

living sister group to vascular plants (tracheophytes).

From water to air

The transition from an aqueous medium, in which the

ancestral Charophyceae group lived, to a gaseous medium,

exposed the early land plants to new physical conditions.

For instance, in place of the structural support of unlimited

water the first land plants in an aerial environment faced

desiccation exposure and the compressive effects of

gravity. The early thalli would have also been exposed to

relatively higher photon flux density, which would previously

have been exponentially attenuated by a water column, and

a 104 gain in diffusion rate of CO2 as water places such

diffusive limits (Osmund et al 1982). Consequently, key

physiological and structural adaptations, over time, needed

to occur in early land plants. Development of cutins to

reduce water loss probably evolved from pre-existing

elements of the primary metabolism in the ancestral

charophycean algae (REF). Additionally, photoprotective

mechanisms possibly developed to cope with higher photon

fluxes in aerial environments utilizing aspects of the already

existing photo respiratory pathways (Kendrick and Crane

1997). However it is unknown whether the photoprotective

role of photorespiration evolved in photosynthetic organisms

in shallow water at high light intensity or in the early land

plants.

Rubisco

The effect of the land invasion by early land plants probably

had a profound effect on the carboxylating enzyme ribulose

1,5 bisphosphate carboxylase / oxygenase, Rubisco, a

photosynthetic enzyme, that had evolved in an aquatic

media for some 3,800 million years into an aerial CO2

concentration during the late Ordovician early Silurian of

approx. 5400 7000ppm CO2 (Berner 1998), without the

diffusive barriers placed on CO2 by water.

Rubisco catalyses the first step of the dark reaction side of

the photosynthetic reaction termed the Calvin cycle (REF).

The first step of this reaction involves Rubisco covalently

attaching CO2 to a 5 carbon sugar, RuBP, and the

simultaneous hydrolysis of the six carbon intermediate to

form 2 molecules of PGA, of which one bears the carbon

introduced from CO2. However, Rubisco has a poor ability

to distinguish between CO2 from the O2 molecule, perhapsbecause there is no formal binding site for CO2.

Consequently, when RuBP is bound to an active site of

Rubisco, it can be attacked by O2, producing the two

products; 2 phosphoglycolate (P-glycolate) and PGA, a

process termed an oxygenase reaction. To salvage lost C

as a result of oxygenation of RuBP, the photorespiratory

cycle (evolved earlier) recycles P-glycolate to release C at

an energetic cost (REF). The poor kinetic properties of

Rubisco, along with diffusive limitation to the passage of

CO2 through water and cell boundary layers were all limiting

factors to photosynthesis in the aquatic medium placing a

strong selection pressure towards the development of

mechanisms for increasing CO2 concentrating around the

Rubisco molecule, mechanisms termed, Carbon

Concentrating Mechanisms (CCMs), possibly evolving 3400

MYA. The effect of raising the internal CO2 environment

using CCMs (sometimes by 50 100 fold would and does

counteract the above constraints). Whether the early land

plants retained the CCM is uncertain, however, an inference

may be made to suggest a pyrenoid-based CCM was still

present in some, if not all, early land plants (before c3

orientation), as the pyrenoid (only found in microalage) is

found also in a group of byrophytes from the Class

Anthocerotae.

The biophysical CCM

The most extensively studied CCMs have been in

cyanobacteria which use a carboxsome-based CCM, small

polyhedral-shaped protein bodies containing both Rubisco

and the enzyme Carbonic anhydrase (CA) which catalyses

the dehydration of HCO3- to CO2 (Badger & Andrews 1987;

Bowes 1993).

CA

H+ + HCO3- CO2 + H2O

In micro algae and some hornworts, an equivalent structure

to the carboxysome is the pyrenoid, a starch coated

protinaceous structure present in the chloroplast, believed to

play a similar role (Badgeret al1993). to microalage, with a

pyrenoid-based CCM (Pronina & Semenko 1992; Badger

1998).

To summarize the makeup of an generalized CCM, 4

components are needed; (1) a mechanism whereby rapid

interconversion between CO2 and HCO3- can take place,


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extracellularly and intracellularly, the latter occurring at

typical stromal pH values within the Rubisco-containing

compartment or more effectively, at low pH in the thylakoid

lumen; (2) a Ci-transport mechanism at plasma membrane,

chloroplast envelope or both; (3) ATP energy to power Ci

transport; (4) a diffusion barrier to prevent CO2 fromdiffusing away from Rubisco (Smith & Griffiths 2000). It has

been established that the first two features of the above list

have been shown to involve CA in a range of eukaryotic

algae and cyanobacteria which utilise CCMs and it has

recently been suggested that CA might function as a

diffusion barrier (Raven 1997).The pyrenoid structure

thought to be associated with the CCM found in many micro

algae (Badger et al 1994; Amoroso et al 1998; Woods

1999) and lichenised algae (Palmqvist 1993), has also

been observed in some species of byrophytes from the

class Anthocerotae (REF). Moreover, Vaughn (1992)

established, using immuno-gold labelling, that the enzyme

Rubisco was found within the pyrenoid structure. Smith &

Griffiths (1996b), further established that Anthoceros

crispus and Phaeoceros laevis (Smith & Griffths 2000)

exhibited low compensation points, low K0.5 and a CO2

uptake and release pool after photosynthesis had been

inhibited, using the C-reduction cycle inhibitor,

glycoladehyde, (a method term light-dark transients),

revealing a CO2 pumping mechanism termed a Dissolved

Inorganic Carbon pump (DIC pump). All of which have been

used as physiological CCM diagnostics in cyanobacteria

and micro algae. To date, Phaeoceros laevis and

Anthoceros crispus have been shown to possess an

operative CCM showing similar characteristics to micro

algal CCMs. But why retain this ancient microalgal relic in

the aerial environment? Considering approx 450-500 million

years ago CO2 Concentrations were (5400 7000ppm) or

indeed independently evolve the same (or similar)

mechanism, at a latter period, during a drop in atmospheric

CO2?

With or without a CCM

It has been observed that not all species from the

Anthocerotae class possess a pyrenoid state (Vaughn

1990). For example all genus of the multiplastidic

Megaceros spp. and two uniplastidic species, Anthoceros

fusiformis and Phaeoceros coriaceus, do not possess a

pyrenoid-based CCM. This is further borne out by carbon

isotope studies where the delta values for Megaceros

moandreus and Megaceros endivifolis (herbarium samples)

show C3 responses. Whereas on-line measurements on

Anthoceros crispus and Anthoceros agrestis (sporophyte)

showed a C4-like response, although there is no evidence

C4 or CAM biochemistry ( pers com). This situation raises

some intriguing questions as to the evolutionary significance

of the "no pyrenoid condition" in uniplastidic cells of some

hornworts and liverworts that have assumed a C3

orientation.

Hornwort CCM variability

Hanson, Andrews and Badger (Functional Plant Biology

Volume 29 Number 2 & 3 2002)examined hornwort CCM

function by using a combined fluorometer/mass

spectrometer based technique to compare pyrenoid-

containing (Phaeoceros Prosk. and Notothylas Sull.) and

pyrenoid-lacking (Megaceros Campbell) hornworts, with the

liverwort Marchantia polymorphaL. that has standard C3

photosynthesis and a thalloid growth form similar to

hornworts. They found that Notothylas has more CCM

activity than Phaeoceros, and that Megaceros has the least

CCM activity. Notothylas and Phaeoceros had

compensation points from 1113 parts per million (ppm)

CO2, lower K0.5(CO2) than Marchantia, with negligible

photorespiration, and they accumulate a pool of dissolved

inorganic carbon (DIC) between 19108 nmol mg1

chlorophyll. Megaceros had an intermediate compensation

point of 31 ppm CO2 (compared with 64 ppm CO2 in

Marchantia), a lowerK0.5 (CO2) than Marchantia, and some

photorespiration, but no DIC pool. They also determined the

catalytic rate of carboxylation per active site of Rubisco for

all four species (Marchantia, 2.6 s1; Megaceros, 3.3 s1;

Phaeoceros, 4.2 s1; Notothylas 4.3 s-1), and found that

Rubisco content was 3% of soluble protein for pyrenoid-

containing species, 4% for Megaceros and 8% for

Marchantia.

Diffusive limitations

Physiological reasons for the retention of a CCM on land, or

indeed its recent evolution in response to falling atmospheric

CO2, is still unclear, as the diffusive limitations of water are

no longer limiting and unlike algae they are not moving up

and down a water column, facing sudden shifts in HCO3-

and light. However, diffusive limitations might have still been

acting on the early liverwort-like plants due to heavily

cuticlised solid thalli (prior to air chamber evolution), to

reduce water loss. Furthermore without pores or stomata

(as seen in modern day C3 liverworts) the conductance of

CO2 within the solid thallus might have been low enough to

require the retention of a CCM.

Robe, Richardson & Griffiths, (unpublished) conducted a

detailed comparative study to evaluate the relative costs


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and benefits of a biophysical CCM in Phaeoceros laevis

(one chloroplast per cell with an unventilated thalli) as

opposed to photosynthesis being based on the diffusive

supply of CO2 in a number of liverworts ranging from Pellia

spp. representing the unventilated thalloid structure similar

to Phaeoceros, together with liverworts showing increasinglevels of complexity and ventilation (viz Conocephalum,

Marchantia and Lunularia). The results showed that the

limitations to gas exchange in an unventilated thallus such

as Pellia, were so great as to render minimal rates of CO2

assimilation, with a high internal conductance to CO2 (gi);

meanwhile, the advantages of the CCM in Phaeoceros

were to restore the rates of CO2 assimilation and electron

transport to those equivalent to the highly ventilated

bryophyte thallus ofLunularia. In conclusion, the operation

of the CCM certainly does ensure that most of the

Anthocerotae can compare with other more advanced

bryophytes,

Land pyrenoid CCM and Low Nitrogen

Apart from a possible advantage a CCM might provide a

solid pore-less thallus over CO2 conductance, (Beardall et

al. 1982; Raven et al 1985) suggest that the CCM may

confer another advantage, namely improved nitrogen use

efficiency of growth. This is hypothesised to result from the

idea that less nitrogen has to be diverted to the synthesis of

Rubsico, and possibly photrespiratory enzymes. In an early

Silurian aerial environment, nitrogen availability and

acquisition may have been limiting compared to a water

column as the case may still be today in certain

environments (Crittenden, Katucka & Oliver 1994). As the

Rubisco molecule has a low turnover rate then retention of a

CCM that conveys Nitrogen efficiency would be

advantageous (Raven and Lucus 1985). However, this has

not been conclusively demonstrated.

Land pyrenoid CCM - reducing oxygenation

Another advantage of a CCM in a land plant such as the

Anthocerotae would be to reduce the oxygenase reaction in

the enzyme Rubisco by elevating the CO2 Concentration

around its active site, reducing competition from oxygen.

The unique chloroplast architecture of the more advanced

Anthocerotae posses thylakoids that cross the pyrenoid,

termed channel thylakoids, Burr (1970), which have been

speculated (through inference from work with algae) by

Makay & Gibbs (1991) to be dominant in photosystem I and

not Photosystem II (water splitting side of the light reaction,

releasing oxygen), therefore reducing further oxygenationevents.

Land pyrenoid CCM and high light

Growth in high light conditions might be another

environmental factor acting to retain or effect the operation

of a CCM on land. This factor has been noted in a study by

Smith et al (1998) whom investigated a hypothesis that

cyanobiont lichens (with CCM) growing under contrasting

microhabitats show inter-specific and intra-specific variation

in photosynthetic responses, which could be correlated with

the variations in the degree of expression of the biophysical

CCM. It was found that populations of Peltigera

membranaecea, from exposed crags showed more

pronounced CCM activity (by the accumulation of a larger

Ci pool) than populations from shaded deciduous oak

woodland. A possible explanation for these observations

were that an active CCM will effectively reduce the light-

utilization efficiency of photosynthesis (Palmqvist et al

1994c), therefore, increased CCM activity as a strategy of

optimising the supply of CO2 to Rubisco, might be most

profitable in environments where CO2 is a more limiting

resource than light, i.e exposed habitats where lichens are

subject to high PAR. An investigation into the way liverworts

(with increasing morphological specialisation, including the

hornwort Phaeoecros laevis) deal with excess of light has

so far not been in studied. Moreover how theAnthocerotae

CCM responses to increased light intensity needs to be

evaluated.

Land pyrenoid CCM and desiccation

The retention of pyrenoid CCM on land may be useful in

the response of these polykiohydric organisms to variations

in environmental stresses, such as high light, but also more

importantly at fluctuating thallus water contents. Smith et al

1998, conducted a investigation on the effect of the uptake

and release pools of CO2 in the lichen Peltigera

membranaecea at varying thallus water contents using a

method termed light dark transients. It can be inferred from

the data that a 10 fold drop in CO2 up take and release pool

sizes occur when thallus water contents decrease from

optimal (5.1- 6.2 mg g-1 d.wt) to (2.3 mg g-1 d.wt) a 63%

reduction in water content.

Additionally, calculations by Green & Snelger (1982), show

that when Monoclea spp. (a liverwort with a solid thallus,

with no air pores) is compared to Marchantia spp.(with

water proof cuticle penetrated with air pores), maximum

photosynthesis is only slightly greater in Marchantia, but air

spaces giving greater advantage over Monoclea for water

relations. However, the solid thallus of Monclea showed

superior photosynthetic ability in very moist environments,

possibly like the hornwort (with CCM).


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The pyrenoid CCM and high/low CO2

Finally, fluctuations in external CO2 concentrations have

been a popular line of investigation in marine and freshwater

algae with CCMs (Matsuda, Bozzo and Colman 1998;

Woods 1999; Colman 2000) It has been recently

demonstrated that in cells of Chlamydomonas reinhardii,

external CO2 concentrations can affect the active uptake of

CO2 and HCO3- and has been show to be suppressed in

Chlamydomonas reinhardiigrown in high external CO2 after

about 8 days in 5% CO2 (pers com Colman 2000). After this

period the new generation of cells lost the CCM capacity.

However when transferred to a low CO2 external

environment, active transport of CO2 and HCO3- within 2hrs

of acclimation, and CA ext activity increased 10 fold after 6

hours after acclimation to 0.035% Co2. It has been

proposed that the CCM is induced when the CO2

concentration in the medium is reduced to a critical level.

Matsuda, Bozzo and Colman (1998) have suggested the

possibility of a CO2 sensor at the green algal cell surface,

which under high CO2 growth conditions would cause

repression of the CCM, whereas under CO2 depletion the

sensing mechanism would initiate a signaling cascade

culminating in the derepression of the CCM. Woods (1999)

also was able to down regulate the CCMs in Chlorellaspp.

and Trebuxiaspp .with a 2 day 5% CO2 treatment and then

upregulated it with low CO2 treatment only after 2 hours.

Carbonic anhydrase and the land pyrenoid

The possibility of being able to switch off or down regulate

the operation of theAnthoceroate CCM is not unrealistic in

the light of recent work by Smith and Griffiths (2000) who

established that CA plays a role in the operation of the CCM

in Phaeoceros laevis , as in all microalage investigated.

Smith and Griffiths (2000) conducted an investigation into

the role of CA in photosynthesis and the activity of the CCM

in Anthocerotae by using the membrane-permeable CA

inhibitor ethoxzolamide (EZ), drawing a comparison to a

range of liverworts and mosses. The results showed that

inhibition of assimilation occurred in all bryophyte treated

with EZ, however the degree of inhibition was greatest in

Phaeoceros laevis. Furthermore, there was a pronounced

decline in Ci-uptake efficiency and a decrease in the initial

slope of CO2-affinity curve at low external levels where Ci-

uptake efficiency in other liverworts were unaffected. There

were no significant differences between the convexities of

the light response curves in Phaeoceros which would

indicate a diversion of ATP to energise the CCM. In studies

on light dark transients on Phaeoceros treated with EZ a

speculation made was that although active transport of CO2

was still occurring (due to the appearance of a CO2 release

pool), the Ci transported to the stroma is not being utilized

by Rubisco when CA is suppressed by EZ. This point may

raise the question as what the exact mechanism of Citransport is if EZ does not suppress the active uptake of

CO2 in the Anthocerotae. The major drawback with using

EZ to manipulate the operation of theAnthocerotae CCM in

future studies is that CA is involved in other non CCM

photosynthetic processes, as shown by the depressions of

gross assimilation rates in mosses of 65 % and 50% in

other non CCM based liverworts treated with EZ.

The usefulness of being able to "switch of " or down

regulate the Phaeoceros CCM with a high CO2

environment will be critical in elucidating the operation of the

Anthocerotae CCM. However in terms of the ecology of

Phaeoceros, a relatively rapid response (of approx. 14

days) to variations in external CO2 (initially 5% CO2) may

not be seen, due to the fact that fluctuations in CO2 in the

aerial environment is relatively minimal, or at least not as

irritate as in a water column,.

Aims

There are two aims of this investigation, the first is to

measure photon utilisation (elucidated from fluorescence

measurements (see appendix) in a range of liverworts with

increasing morphological specialization viz Pellia spp.,

Conocephalum spp, Marchantia polymorpha L and

Lunularia spp. ( all without a CCM ) compared to the same in

Phaeoceros laevis (with CCM). The following

measurements will be made;

Electron transport rate (ETR)

Non photochemical quenching (NPQ)

The second aim of this investigation is to attempt to

manipulate the operation of the hornwort CCM by

introducing the following environmental conditions;

1. Desiccation

2. High external CO2

3. High/ low light

4. High temperature

The control plant chosen is Marchantia polymorpha L,

mainly because Pmax is usually high, and it is easy to grow.

Pellia ssp. would have been an ideal choice, having a solid

thallus like Phaeoceros laevis, but it struggles in

unfavourable conditions and results can appear erratic.

HoweverPellia will be used for the desiccation experiment.


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MATERIALS AND METHODS

Acclimation procedures

Measuring Florescence characteristics

Specimens of Pellia ssp, Conocephalem ssp, Lunalaria

spp, Marchantia polymorphaL (all C3 biochemistry) and

Phaeoceros laevis. (with CCM) were collected from Devon

in late March. For 14 days each species was grown on peat

and misted at least 3 times a day in a Fissons growth

cabinet with a photoperiod of 12 hours 40mol photons m-2

s-1 @15 0C and 12 hours no light @10oC. Measurement of

FV/FM, Electron Transport Rate (ETR) and Non

photochemical Quenching (NPQ) were made using PAM

florometer (see Maxwell).

Thallus drying at 30% RH over 3 hours

Specimens of Pellia spp Marchantia polymorpha L and

Phaeoceros laevis were adapted to the same conditions as

for measuring Florescence characteristics.After this period,

thallus (at 100% thallus water content ) were dab dried and

weighed. 2mg of thallus from each the plants were placed

on tissue in a fissons growth cabinet at low RH, and 40mol

photons m-2 s-1, 15oC. Thallus water content was then

weighed every hour, Pmax was also determined along with

chlorophyll content.

Acclimation procedure for plants grown at two

different light regimes.

Specimens of Marchantia polymorpha L. and Phaeoecros

laevis were collected from Devon, in late March. Each

species was grown in seed trays on peat and misted three

times a day under two different light regimes (in two

different Fissons growth cabinets) 5mol photons m-2 s-1

and 120mol photons m-2 s-1. Photoperoid was 12 hours

and the temperature regime was set at 15oC in light and

10oC without light. Each species were adapted to these

conditions for 14 days before chlorophyll florescence

measurements were made using PAM florometer (see

theory and method), with light response, carbon dioxide

compensation points, light dark transients,and K0.5 CO2.

Acclimation procedure for plants grown at 5%CO2, low CO2, and ambient CO2 regimes.

Specimens ofMarchantia polymorpha L and Phaeoceros

laevis were collected from Devon and acclimated in

Newcastle botanical gardens during August and September

(natural photoperoid at 25 oC). The Phaeoceros and

Marchantia mats were divided into 3 plastic boxes each

(20cm by 20cm) containing peat, where the open top of the

box were covered and sealed with cling film, with a ambient

CO2 (400ppm) supply feed through one side creating a

positive pressure released from a hole in the opposite side

of the box. The boxes were set up in a Fissons growth

cabinet under 40mol photons m-2 s-1 with a photoperoid of

12 hours and a temperature regime of 15oC in light and

10oC without light. These specimens were acclimated under

these conditions and sprayed three times a day with distilled

water for 14 days. After acclimation in the growth cabinet

conditions pieces of thalli were removed for measurements

of chlorophyll f lorescence (see theory and method)., light

response, carbon dioxide compensation points, light dark

transients, K0.5 CO2 and chlorophyll analysis,. After all

measurements were completed, the CO2 supply was

changed from ambient (400ppm) to 5% CO2. All other

conditions in the growth cabinet remained unchanged. After

14 days in 5% CO2 (pers com Colman 2000 after 8 days of

acclimation at 5% CO2 cells of Chlamydomonas showed

loss of CCM characteristics) the same measurements were

performed for plants at 0.0175% CO2.

Acclimation procedure for plants grown at 30oC

Specimens of Marchantia polymorpha L and Phaeoecros

laevis were collected from Devon, in late March. Each

species was grown in seed trays on peat and misted three

times a day under two different temperature regimes 10oC

and 30oC in two different growth cabinets. PAR was 40

mol photons m-2 s-1 and photoperoid for 12 hours light

and 12 hours darkness, plants was misted with distilled

water over 3 times a day. Plants were grown under these

conditions for 14 days, after which, pieces of thallus were

removed, excess water was dab dried and CCM diagnostics

were performed; light response, carbon dioxide

compensation points, light dark transients, K0.5 CO2, and

chlorophyll analysis.

Light response curvesAn ADC IRGA (ADC 225 Mk 3 ADC Hoddesdon, UK) in

absolute mode was connected to a modified Hansatech

Oxygen electrode (LD2, Hansatech, Norwich UK) with

water jacket maintained at 18 degrees C. The cuvette was

illuminated by a KL 1500 electronic light source (Hansatech,

Norwich UK) which delivered a range of light intensities

from 0 to 2000mol photons m-2 s-1. The lowest average

light intensity available was 15 +- 4 mol photons m-2.s-1 and

the highest average value 2000 +- 400 mol photons m-2. s-

1. CO2 (350ppm) was supplied in compressed air from gas

cylinder via IRGA to the cuvette. A clean piece of thallus


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was sprayed with distilled water, blotted and placed within

the cuvette, ensuring self shading did not occur (Smith PhD

thesis 1995). Thallus was dark adapted within the cuvette in

a closed system initially, and dark respiration recorded

through a chart recorder. The system was opened and

purged, in preparation from the following light intensities; 15,55, 97, 205, 325, 456, 660, 832, 1335, 1911, 2000mol

photons m-2. S-1, purging after each exposure. The samples

were also weighed before and after each experiment to

ensure that the water content had not dropped below that

which is optimal for photosynthesis (Smith PhD thesis

1995).

The above procedure was repeated 3 times for each

species.

CO2 response curves and K0.5 CO2

An ADC IRGA (ADC 225 Mk 3 ADC Hoddesdon, UK) in

absolute mode was connected to a modified Hansatech

Oxygen electrode (LD2, Hansatech, Norwich UK) with

water jacket maintained at 18 degrees C. The cuvette was

illuminated by a KL 1500 electronic light source (Hansatech,

Norwich UK) which delivered saturating light; 320mol

photons.m-2.s-1 for Marchantia polymorpha L (Pmax) and

660mol photons. m-2.s-1 forPhaeoceros laevis . (Pmax). A

clean piece of thallus was sprayed with distilled water,

blotted and placed within the cuvette, ensuring self shading

did not occur (Smith PhD thesis 1995). CO2 was supplied to

the thalli from a compressed air cylinder (350ppm CO 2) via

a gas diluter where the air passes through carbosorb

(ADC Hoddesdon, UK) to dilute 350ppm CO2 (100%) to

0%, 13%, 28%, 46%, 64% of system was initially purged

with the required % CO2 as an 350ppm CO2, supplied via

the IRGA to the cuvette. The open system. The system was

subsequently closed and the thallus left to deplete the CO2

concentration within the closed system, the values were

recorded on a chart recorder. The procedure was repeated

at all of the above CO2 concentrations, purging after each

exposure. Chlorophyll analysis was preformed on each

sample almost immediately. The above process was

repeated 3 times for each species and the average values

calculated together with SE. The averaged values at each

CO2 concentration were plotted against nmol.chl.mg-1.s-1, a

line of best fit that dissected the x axis of the plot, was then

read off to be the CO2 compensation point. K0.5 CO2 was

also calculated from the CO2 response curves by reading

off CO2 concentration at the Pmax at both ambient, and

5% CO2 treatments.

Light dark transient releases of DIC pools.

An ADC IRGA (ADC 225 Mk 3 ADC Hoddesdon, UK) in

differential mode was connected in an open system with a

modified Hansatech Oxygen electrode (LD2, Hansatech,

Norwich UK) (Badger & Price, 1992). Hansatech LS-2

(Hansatech, Norwich, UK) lamp supplying red light was

used as the light source and temperature was maintained

by a water jacket at 15 degrees C - water circulated and

supplied (Grant refrigerated flow heater / cooler system).

Compressed air (350ppm CO2) was supplied from cylinder

(1.7m height, 0.23m width) which maintained a constant

partial pressure of CO2 and flow rate of 300L/min

compressed air. The relative humidity of the air flowing

through the system remained within the range 50 70%

throughout the experiment. A piece of thallus from either

Phaeoceros laevis (sample) or Marchantia polymorpha L

(control). was immersed in distilled water, in darkness for a

period of half an hour to ensure the complete metabolism of

any remaining DIC pool, prior to being blotted and weighed

(Smith, PhD Thesis 1995). The piece of thallus was then

placed in the cuvette and left to attain a steady rate of dark

respiration, recorded using a chart recorder. Saturating light

was then used to illuminate the thallus (Marchantia 320mol

photons.m-2.s-1, Phaeoceros 660mol photons. m-2.s-1) for a

peroid of up to twenty minutes using the red light source

detailed above, after which the maximum rate of

photosynthesis was attained. On turning the light off, the

release of an internal pool of CO2 was detectable in the

Phaeoceros laevis thallus and recorded as a short-lived (< 1

min) burst of CO2 within the system by the chart recorder.

The reaction time of the system to the changes in light was

minimised by ensuring that the tubing between the cuvette

and the IRGA were as short as possible (Smith, PhD

Thesis 1995). Chlorophyllanalysis was performed on each sample almost

immediately. The experiments were repeated using thalli

which had been immersed in 50mM glycolaldehyde (which

inhibits photosynthetic CO2 fixation by inhibiting the

regeneration from ribulose-5-phosphate) (Miller and

Canvin, 1989), for up to half and hour depending on the

hydroscopic properties of the thalli (Badger, et al. 1993).

Chlorophyll analysis

Chlorophyll analysis was carried out according to Porra et al

(1989). Approximately 20mg (FW) sample was immersed in

chilled 80% acetone (10ml) and ground with pestol and

mortar. The extraction was spun at 4 degrees C for 10mins

at 5000rpm, supernatant was poured into 1 mm glass

cuvettes and absorbency readings were taken at 664nm


8/12

and 646nm against an acetone blank. The following

equation was used:

In ug chl ml-1

Chl a = 12.25 A 664 2.55 A 647

Chl b = 20.31 A 647 - 4.91 A 664

Total = 17.76 A 647 + 7.34 A 664

RESULTS

Effect of decreasing Thallus Water Content over Time, on Net Assimilation Max

When the Photosynthetic Maximum (Pmax) of Phaeoceros laevis with a CCM, (solid thallus), is compared to Marchantia

polymorpha L with pores, and Pellia spp .with a solid thallus without a CCM, it appears that Phaeoceros is extremely sensitive to

water loss. After only 1 hour of drying at 30% RHPmax drops by 60% compared to only a 20% drop in Pmax for the other two

liverworts. After 2 hours Phaeoceros is no longer photosynthesising and the size of its DIC pools (not shown) is zero. Therefore

one can conclude that the capacity of the Phaeoceros CCM is integrally connected to Thallus Water Content. Whereas,

Marchantia with a pore and internal air spaces, can withstand over 3 hours of drying, and surprisingly Pellia with a solid thallus (like

Phaeoceros) only drops 50% TWC after 3 hours and can go on photosynthesising. This obviously must influence habitat, making

Phaeoceros dependent on an environment where water is always available.

Comparative fluorescence characteristics in a range of liverwort gametophytes

and the hornwort Phaeoceros laevisgametophyte under 40mol photons m-2 s-1 for

14 days (n=5).


9/12

It is clear that the Phaeoceros laevis (CCM) has the highest Electron Transport Rate (Jmax), being twice that of the nearest

liverwort. However, Pmax (from light response curves) in Phaeoceros is lower than the more specialized liverworts; Marchantia

polymorpha L and Lunalaria spp, so an assumption could be made that the high ETR ofPhaeoceros is being diverted to power the

Ci pump. Therefore a high Jmax could be used as a diagnostic for a CCM.

Non photochemical Quenching (NPQ) shows Phaeoceros to have the greatest angle of NPQ slope (1600) compared to the other

liverworts, probably as a consequence of the high ETR interacting as an electron sink supplying power to the DIC pump. A steep

angle of NPQ might also mean the CCM is interacting with the zanthopyhll cycle, where (H + ) protons; that acidify the thylakoid

lumen at high light, (used to convert violaxanthin (V) to zeaxanthin so binding Z to LHCH monomers), are being used to supply the

reaction; H+ + HCO3-- CA CO2 + H2O, consequently, the thylakiod lumens protonation rate of Violaxanthin (V) to

Zeaxanthin (Z) and other Xanthophylls might be low, causing comparatively unusual low NPQ. Furthermore, photosynthesis and

ETR in the hornwort continues to 2000mol photons m-2 s-1 before Photoinhibtion (PI), compared to the other liverworts, which

were photinhibited at 1000mol photons m-2 s-1). Resultantly, a high degree of PSII damage, (Fo I - Fo) equaling (4), occurred in

the hornwort, compared to (1-2) in other liverworts (observed from fluorescence data).

Effect of low and high PAR (for 14 days respectively) on photosynthetic

characteristics of Phaeocerosand Marchantia gametophytes (n=4).

Species Treatment Max Net Assimilation CO2 K0.5 CO2 Magnitude

mol photons (nmol CO2 (l l-1) (l l-1) of DIC pool m-2 s-1

mg -1 Chl s-1) (nmol CO2mg-1 Chl)

Phaeoceros 5 6.3 2.1 22 3.2 122 4.2 18.3 3.8

120 3.4 1.4 14 3.1 113 3.8 27.8 3.2

Marchantia 5 6.2 1.6 88 5.1 183 10.1 no pool

120 2.8 1.3 88 4.3 185 9.2 no pool

ANOVA Significant difference

There is a sig. diff. in photosynthetic characteristics in Phaeoceros laevis plants grown under the 120 mol photon m-2 s-1

treatment and the5 mol photon m-2 s-1 treatment, after 14 days. The three diagnostic tools used to measure CCM operation are

low CO2, low K 0.5 CO2 and large DIC pool, all show sig. diff. in high light grown Phaeooceros. Resultantly there was anincreased efficiency for CO2 in high PAR grown Phaeoceros. Unusually the magnitude of the DIC pool (27.8nmol CO2 mg-1 Chl)

was induced at 1000mol photons m-2 s-1 as opposed to (18.3 nmol CO2 mg-1 Chl) at 100mol photons m-1 s-1, showing CCM

plasticity to different light regimes. Conversely, Marchantia polymorphaL shows no sig. diff. apart from low Net Assimilation (as

with Phaeoceros), which could be explained by loss of chlorophyll due to the 120 mol photon m-2 s-1 treatment, moreover

FV/FM in Phaeoceros and Marchantia was 0.68, showing they were under stress.


10/12

Phaeoceros laevis Marchantia polymorpha L

The low net assimilation in high light grown plants in both Marchantia and Phaeoceros could be put down to chlorophyll loss due to

high PFD conditions. However the Phaeoceros CCM is up-regulated in high light grown plants, showing higher CO2 utilisation

efficiency in high PAR, than in low light grown plants, however the increased efficiency for CO 2 at high PAR is apparently obtained

at the cost of a reduced light use efficiency seen by the decline of the convexity of the light response curve and low net

assimilation. Therefore, it can be established that the Phaeoceros CCM lowers light-utilization efficiency at high PAR, allowing for

short term high PAR adaptability, but low PAR is preferred, giving higher Pmax, Moreover, in Phaeoceros grown under the high

light regime, photosynthesis continued to well past 1500 mol photons m-2 s-1 and did not photo-inhibit. Consequently, NPQ was

low (not shown), and the hornworts sustained a comparative higher degree of PSII damage, Fo I Fo = (5+) and low FV/FM

observed from florescence data. Finally, it can be established that a CCM lowers light-utilization efficiency at high PAR, allowing

for short term high PAR adaptability, but low PAR is preferred, giving higher Pmax.

Effect of differing CO2 regimes (for 14 days respectively) on photosynthetic

characteristics of Phaeocerosand Marchantiagametophytes (n=4).

Species Treatment Max Net Assimilation CO2 K0.5 CO2 Magnitude

(nmol CO2 (l l-1) (l l-1) of DIC poolmg Chl m-2 s-1) (nmol CO2

mg-1 Chl)

Phaeoceros Ambient CO2 5.5 2.3 22 4.2 123 2.1 18.2 3.8

5% CO2 5.7 3.6 29 7.1 123 1.1 17.3 2.1

0.0175 % CO2 5.2 2.9 21 5.4 120 3.1 19.3 4.1

Marchantia Ambient CO2 5.8 2.6 85 4.1 184 4.8 no pool

5% CO2 5.4 3.3 94 6.3 194 6.5 no pool

0.0175 % CO2 5.3 2.3 81 4.3 180 8.5 no pool


Surprisingly, Phaeoceroslaevis showed no sig. diff. in CO2, K 0.5 CO2 , or DIC pool size, (the three diagnostics), meaning that

the 5% CO2 regime (14 days) did not down-regulate the CCM, as compared to an ambient CO2 level, and the low CO2 regimedid not up regulate the CCM. In Marchantia polymorphaL, the control, a sig. diff. was showed in CO2 and K 0.5 CO2, at 5% CO2,

6

4

2

0

6

4

2

0

mol photons m-2 s-1 mol photons m-2 s-1

Grown at 5mol

photons m

-2

s

-1

Grown at 120molphotons m-2 s-1

Grown at 5molphotons m-2 s-1

Grown at 120mol

photons m-2 s-1


11/12

however FV/FM was low, (florescence data) and the plants were struggling at 5% CO2. It can be stated that differing external

CO2 concentration has not affected the capacity of the CCM in Phaeoceros after 14 days; maybe more time is required at these

CO2 concentrations for a tissue response.

Effect of high temperature (for 14 days) on photosynthetic characteristics of

Phaeocerosand Marchantiagametophytes (n=4).

Species Temperature Max Net Assimilation CO2 K0.5 CO2 Magnitude

degrees (nmol CO2 (l l-1) (l l-1) `of DIC poolCentigrade mg Chl m-2 s-1) (nmol CO2

mg-1 Chl)

Phaeoceros 15 5.1 1.1 23 2.8 123 4.2 19.2 4.1

30 7.2 2.9 16 2.3 113 3.4 28.3 3.3

Marchantia 15 5.8 1.1 82 4.8 183 4.1 no pool

30 6.0 1.3 78 3.1 180 5.1 no pool


Phaeoceros laevis showed sig. diff. in CCM diagnostics as lower CO2, lower K 0.5 CO2 , and a large DIC pool, in the 30o C

treated plants compared to the 15 oC treatment plants, whereas MarchantiapolymorphaL showed no sig. diff in photosynthetic

characteristics. Therefore it can be seen that high temperature can effect the Phaeoceros CCM after a 14 day treatment

by up-regulating the CCM to increase the CO2 concentration around Rubisco, lowering the oxygenase reaction as oxygen

out-competes CO2 in the active site of Rubisco at high temperature (observed as low dark respiration rates not shown)

DISCUSSION

The following investigation revealed that when the

gametophyte of extant hornworts is exposed to external

CO2 concentrations of either 5%, ambient or 0.00175%,

(for 14 days), the CCM showed no effect in its operation,

unlike algal CCMs where 5% CO2 after only 2-8 days

switches the CCM off (REF). This indicates that a

hornwort tissue response, to differing external CO2

concentrations, has not occurred, (maybe more time is

needed in these CO2 environments).However, in hornworts grown at 300C for 14 days

(compared to those at grown at lower temperatures) there

was a sig. diff. in the operation of the CCM, appearing to

increase the capacity of dissolved inorganic carbon (DIC)

uptake and lower the CO2 compensation point and, lower

K 0.5 CO2, (with hydrated thallus).

Therefore an advantage of a CCM in a land plant such as

the Anthocerotae would be to reduce the oxygenase

reaction in the enzyme Rubisco by elevating the CO2

Concentration around its active site, reducing competition

from oxygen. The unique chloroplast architecture of the

more advanced Anthocerotae possess thylakoids that

cross the pyrenoid, termed channel thylakoids, Burr

(1970), which have been speculated (through inference

from work with algae), by Makay & Gibbs (1991), to be

dominant in PSI, not PSII (water splitting side of the light

reaction, releasing oxygen), therefore reducing further

oxygenation events, possibly making up for a Rubisco

with low specificity.

Furthermore, high light intensity (compared to low light

intensity grown plants), can have a sig. effect on thecapacity of the hornwort CCM; with a large dissolved

inorganic carbon (DIC) uptake and low CO2

compensation point and low K 0.5 CO2. An active CCM will

effectively reduce the light-utilization efficiency of

photosynthesis, therefore increased CCM activity will

optimise the supply of CO2 to Rubisco helping it deal with

high PAR.

The hornwort appeared to be able to photosynthesise at

Photon Flux Densities (PFD) above 1500umols m2. s2, (in

those grown at 120umol m2. s2 for 14 days); However a

constituently expressed CCM at high PAR kept NPQ low,

causing PSII damage, maybe acting as an electron sink


12/12

to power the DIC pump, and/or as a proton sink in the

thylakiod lumen, suplying protons to the reaction; (H+ )+

HCO3- CA CO2 + H2O, so lowering protonation of

the xanthophylls reducing NPQ. However, at an extremely

low light treatment, Pmax was greater than that of high

light treated plants (lower Pmax could have been causedby chlorophyll loss), indicting growth at low PFD is

preferred in the hornwort. However light adaptability is

possible with use of the CCMs plasticity as a light use

efficiency reducer a high PAR and a oxygenase reaction

reducer at high temperature.

Early land plants in the Silurian atmosphere, 450MYA,

may well have made use of the already existing pyrenoid

CCM (as possessed by the ancestral Coleochales), not

because external CO2 was in short supply (Silurian aerial

environment; [CO2] 5400 - 7000ppm) (REF), but as a way

to deal with high PAR by increasing efficiency for CO2 at

the cost of a reduced light use efficiency, the CCM acting

as - a ''light use efficiency reducer'' - and in high

temperatures the CCM up regulates, also increasing

efficiency for CO2 acting as - a ''oxygenase reaction

reducer''. The hornwort CCM can be likened to a primitive

stomata, regulating CO2 uptake, in response to light,

temperature and thallus water content variations.

The eventual loss of the pyrenoid CCM, and the move

towards a more advanced morphology as seen in the C3

liverworts, meant a change in shape of the chloroplast

from band shaped to discoid (REF). The discoid shape

would allow more light adaptability; an example of this is

the unusual light dependant changes in the chloroplast

morphology in species of hornworts without pyrenoids

(Burr 1968). Brown a lemon (19--) suggests the evolution

of the multiplastic cell (away from the uniplastic cell found

in Phaeoceros) may have meant the end of pyrenoid

containing plastids. In the mutiplastidic cell the division of

a number of plastids would have been more difficult to

co-ordinate. An even distribution of Rubisco in the stromawould not require a pyrenoid division to insure that

Rubisco is present in every chloroplast. With Rubisco no

longer being pumped CO2 around the active site, the

early C3 liverwort Rubisco kinetics would have improved

efficiency, coupled with morphological specialisation to

aid CO2 influx, and pores to reduce water loss, thus

making the early C3 liverworts less dependant on water

to aid CO2 in flux.

To retain a pyrenoid CCM on land meant adaptability to

the Silurian atmosphere; higher temperatures and

stronger light conditions (REF) than experienced in the

water column. To retain a pyrenoid CCM on land today

means hornworts with CCMs have the plasticity to adapt

to high and low temperatures and very low light, to higher

light environments, ranging from the Australian and India

rain forests, to, ditches in Scotland and Canada.

However, full hydration of the thallus is essential for

efficient CCM activity, so a damp to wet environment is

the habitat where they are found. If environmental

conditions are no longer favourable, hornwort tuber

formation can occur and the thallus dies (REF); the plant

to be resurrected when environmental conditions are

suitable. This is another useful adaptation to cope with

the high PAR and drying summer conditions experienced

by Phaeoceros growing in the Mediterranean area (REF).

the land pyrenoid: a silurian way to deal with heat and light?

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