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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

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 temperature
and 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 CCM’s 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 C 3 liverworts), meant CO2
was no longer being pumped around the active site of Rubisco, thus early C3 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 C 3 liverworts
less dependant on water to photosynthesise, than the desiccation prone solid thallus of the hornwort.


Bryophyte Phylogeny
Liverworts and hornworts are a successful group of non- control over water relations; from simple solid thallus (Pellia
flowering, rootless lower plants, grouped as bryophytes. They spp.) to the more complex differentiated structures with pores
possess a polykiohydric nature, namely their water content (Marchantia spp.) that show more control over water status.
tends to adjust to the moisture conditions of their environment In terms of the origin and evolution of early land plants,
(Deltoro et al 1998). This condition is markedly different from modern day bryophytes quite possibly resemble the
that of the tracheophytes, which are homohydric, where water characteristics of the earliest plants on land. The first good
supply (from roots) and water status are maintained by evidence for the existence of bryophyte-like land plants
stomatal apparatus which prevent the desiccation of the (Eoembryophytes) is seen in spore tetrads (comprising four
photosynthetic tissue. Unusually though, an ancient living membrane-bound spores), found over a broad geographic
group of bryophytes named hornworts possess stomata in the area in the mid-Ordovician period, 476 Myrs (Gray 1993).
sporophyte but not the gametophyte (REF). The combination of decay resistant walls (implying the
Polykiohydry in land plants appears to be a much more presence of sporopollenin) and tetrahedral configuration
ancient state than homoihydry, with Mega-fossil evidence of (implying haploid meiotic products) are further diagnostics of
the early land plants shown to be liverwort-like in ultra structure land plants. Further evidence lay in spore wall ultra structure
(Niklas 1997; Edwards et al 1998). However within the range and the structure of fossil cuticles from the Late Silurian and
of surviving orders of bryophytes, progressive specialisation of Devonian mega fossils, leading Kendrick and Crane (1997)
their morphology show evidence of a move towards greater to suggest the above palaeobotanical evidence would
support previous arguments that land flora during these times The first step of this reaction involves Rubisco covalently
was liverwort-like. According to Kendrick and Crane (1997), attaching CO2 to a 5 carbon sugar, RuBP, and the
land plants (embrophytes) are most closely related to the simultaneous hydrolysis of the six carbon intermediate to
Charophyceae, a small group of predominantly freshwater form 2 molecules of PGA, of which one bears the carbon
green algae. Within this group, either Coleochaetales (15 introduced from CO2. However, Rubisco has a poor ability
living species) or Charales (400 living species) or a group to distinguish between CO2 from the O2 molecule, perhaps
containing both, is a sister group to land plants. Land plant because there is no formal binding site for CO2.
monophyly is supported by comparative morphology and Consequently, when RuBP is bound to an active site of
gene sequences (18S cox iii). Rubisco, it can be attacked by O2, producing the two
Relationships among the major basal living groups are products; 2 – phosphoglycolate (P-glycolate) and PGA, a
uncertain. But the best supported hypothesis resolves process termed an oxygenase reaction. To salvage lost C
liverworts as basal and either mosses or hornworts as the as a result of oxygenation of RuBP, the photorespiratory
living sister group to vascular plants (tracheophytes). cycle (evolved earlier) recycles P-glycolate to release C at
an energetic cost (REF). The poor kinetic properties of

From water to air Rubisco, along with diffusive limitation to the passage of
CO2 through water and cell boundary layers were all limiting
The transition from an aqueous medium, in which the
factors to photosynthesis in the aquatic medium placing a
ancestral Charophyceae group lived, to a gaseous medium,
strong selection pressure towards the development of
exposed the early land plants to new physical conditions.
mechanisms for increasing CO2 concentrating around the
For instance, in place of the structural support of unlimited
Rubisco molecule, mechanisms termed, Carbon
water the first land plants in an aerial environment faced
Concentrating Mechanisms (CCMs), possibly evolving 3400
desiccation exposure and the compressive effects of
MYA. The effect of raising the internal CO2 environment
gravity. The early thalli would have also been exposed to
using CCMs (sometimes by 50 – 100 fold would and does
relatively higher photon flux density, which would previously
counteract the above constraints). Whether the early land
have been exponentially attenuated by a water column, and
plants retained the CCM is uncertain, however, an inference
a 104 gain in diffusion rate of CO2 as water places such
may be made to suggest a pyrenoid-based CCM was still
diffusive limits (Osmund et al 1982). Consequently, key
present in some, if not all, early land plants (before c3
physiological and structural adaptations, over time, needed
orientation), as the pyrenoid (only found in microalage) is
to occur in early land plants. Development of cutins to
found also in a group of byrophytes from the Class
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 The biophysical CCM
fluxes in aerial environments utilizing aspects of the already The most extensively studied CCMs have been in
existing photo respiratory pathways (Kendrick and Crane cyanobacteria which use a carboxsome-based CCM, small
1997). However it is unknown whether the photoprotective polyhedral-shaped protein bodies containing both Rubisco
role of photorespiration evolved in photosynthetic organisms and the enzyme Carbonic anhydrase (CA) which catalyses
in shallow water at high light intensity or in the early land the dehydration of HCO3- to CO2 (Badger & Andrews 1987;
plants. Bowes 1993).

Rubisco H+ + HCO3-  CO2 + H2O

The effect of the land invasion by early land plants probably In micro algae and some hornworts, an equivalent structure
had a profound effect on the carboxylating enzyme ribulose to the carboxysome is the pyrenoid, a starch coated
1,5 bisphosphate carboxylase / oxygenase, Rubisco, a protinaceous structure present in the chloroplast, believed to
photosynthetic enzyme, that had evolved in an aquatic play a similar role (Badger et al 1993). to microalage, with a
media for some 3,800 million years into an aerial CO2 pyrenoid-based CCM (Pronina & Semenko 1992; Badger
concentration during the late Ordovician early Silurian of 1998).
approx. 5400 – 7000ppm CO2 (Berner 1998), without the
To summarize the makeup of an generalized CCM, 4
diffusive barriers placed on CO2 by water.
components are needed; (1) a mechanism whereby rapid
Rubisco catalyses the first step of the dark reaction side of
interconversion between CO2 and HCO3- can take place,
the photosynthetic reaction termed the Calvin cycle (REF).
extracellularly and intracellularly, the latter occurring at C4 or CAM biochemistry (pers com). This situation raises
typical stromal pH values within the Rubisco-containing some intriguing questions as to the evolutionary significance
compartment or more effectively, at low pH in the thylakoid of the "no pyrenoid condition" in uniplastidic cells of some
lumen; (2) a Ci-transport mechanism at plasma membrane, hornworts and liverworts that have assumed a C3
chloroplast envelope or both; (3) ATP energy to power Ci orientation.
transport; (4) a diffusion barrier to prevent CO2 from
diffusing away from Rubisco (Smith & Griffiths 2000). It has
been established that the first two features of the above list Hornwort CCM variability
have been shown to involve CA in a range of eukaryotic
Hanson, Andrews and Badger (Functional Plant Biology
algae and cyanobacteria which utilise CCMs and it has
Volume 29 Number 2 & 3 2002) examined hornwort CCM
recently been suggested that CA might function as a
function by using a combined fluorometer/mass
diffusion barrier (Raven 1997).The pyrenoid structure
spectrometer based technique to compare pyrenoid-
thought to be associated with the CCM found in many micro
containing (Phaeoceros Prosk. and Notothylas Sull.) and
algae (Badger et al 1994; Amoroso et al 1998; Woods
pyrenoid-lacking (Megaceros Campbell) hornworts, with the
1999) and lichenised algae (Palmqvist 1993), has also
liverwort Marchantia polymorphaL. that has standard C3
been observed in some species of byrophytes from the
photosynthesis and a thalloid growth form similar to
class Anthocerotae (REF). Moreover, Vaughn (1992)
hornworts. They found that Notothylas has more CCM
established, using immuno-gold labelling, that the enzyme
activity than Phaeoceros, and that Megaceros has the least
Rubisco was found within the pyrenoid structure. Smith &
CCM activity. Notothylas and Phaeoceros had
Griffiths (1996b), further established that Anthoceros
compensation points from 11–13 parts per million (ppm)
crispus and Phaeoceros laevis (Smith & Griffths 2000)
CO2, lower K0.5(CO2) than Marchantia, with negligible
exhibited low compensation points, low K0.5 and a CO2
photorespiration, and they accumulate a pool of dissolved
uptake and release pool after photosynthesis had been
inorganic carbon (DIC) between 19–108 nmol mg–1
inhibited, using the C-reduction cycle inhibitor,
chlorophyll. Megaceros had an intermediate compensation
glycoladehyde, (a method term light-dark transients),
point of 31 ppm CO2 (compared with 64 ppm CO2 in
revealing a CO2 pumping mechanism termed a Dissolved
Marchantia), a lower K0.5 (CO2) than Marchantia, and some
Inorganic Carbon pump (DIC pump). All of which have been
photorespiration, but no DIC pool. They also determined the
used as physiological CCM diagnostics in cyanobacteria
catalytic rate of carboxylation per active site of Rubisco for
and micro algae. To date, Phaeoceros laevis and
all four species (Marchantia, 2.6 s–1; Megaceros, 3.3 s–1;
Anthoceros crispus have been shown to possess an
Phaeoceros, 4.2 s–1; Notothylas 4.3 s-1), and found that
operative CCM showing similar characteristics to micro
Rubisco content was 3% of soluble protein for pyrenoid-
algal CCMs. But why retain this ancient microalgal relic in
containing species, 4% for Megaceros and 8% for
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 Diffusive limitations
CO2? Physiological reasons for the retention of a CCM on land, or
indeed its recent evolution in response to falling atmospheric
With or without a CCM CO2, is still unclear, as the diffusive limitations of water are

It has been observed that not all species from the no longer limiting and unlike algae they are not moving up

Anthocerotae class possess a pyrenoid state (Vaughn and down a water column, facing sudden shifts in HCO3-

1990). For example all genus of the multiplastidic and light. However, diffusive limitations might have still been

Megaceros spp. and two uniplastidic species, Anthoceros acting on the early liverwort-like plants due to heavily

fusiformis and Phaeoceros coriaceus, do not possess a cuticlised solid thalli (prior to air chamber evolution), to

pyrenoid-based CCM. This is further borne out by carbon reduce water loss. Furthermore without pores or stomata

isotope studies where the delta values for Megaceros (as seen in modern day C3 liverworts) the conductance of

moandreus and Megaceros endivifolis (herbarium samples) CO2 within the solid thallus might have been low enough to

show C3 responses. Whereas on-line measurements on require the retention of a CCM.

Anthoceros crispus and Anthoceros agrestis (sporophyte) Robe, Richardson & Griffiths, (unpublished) conducted a

showed a C4-like response, although there is no evidence detailed comparative study to evaluate the relative costs
and benefits of a biophysical CCM in Phaeoceros laevis
(one chloroplast per cell with an unventilated thalli) as Land pyrenoid CCM and high light
opposed to photosynthesis being based on the diffusive
Growth in high light conditions might be another
supply of CO2 in a number of liverworts ranging from Pellia
environmental factor acting to retain or effect the operation
spp. representing the unventilated thalloid structure similar
of a CCM on land. This factor has been noted in a study by
to Phaeoceros, together with liverworts showing increasing
Smith et al (1998) whom investigated a hypothesis that
levels of complexity and ventilation (viz Conocephalum,
cyanobiont lichens (with CCM) growing under contrasting
Marchantia and Lunularia). The results showed that the
microhabitats show inter-specific and intra-specific variation
limitations to gas exchange in an unventilated thallus such
in photosynthetic responses, which could be correlated with
as Pellia, were so great as to render minimal rates of CO2
the variations in the degree of expression of the biophysical
assimilation, with a high internal conductance to CO2 (gi);
CCM. It was found that populations of Peltigera
meanwhile, the advantages of the CCM in Phaeoceros
membranaecea, from exposed crags showed more
were to restore the rates of CO2 assimilation and electron
pronounced CCM activity (by the accumulation of a larger
transport to those equivalent to the highly ventilated
Ci pool) than populations from shaded deciduous oak
bryophyte thallus of Lunularia. In conclusion, the operation
woodland. A possible explanation for these observations
of the CCM certainly does ensure that most of the
were that an active CCM will effectively reduce the light-
Anthocerotae can compare with other more” advanced”
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
Land pyrenoid CCM and Low Nitrogen profitable in environments where CO2 is a more limiting
Apart from a possible advantage a CCM might provide a resource than light, i.e exposed habitats where lichens are
solid pore-less thallus over CO2 conductance, (Beardall et subject to high PAR. An investigation into the way liverworts
al. 1982; Raven et al 1985) suggest that the CCM may (with increasing morphological specialisation, including the
confer another advantage, namely improved nitrogen use hornwort Phaeoecros laevis) deal with excess of light has
efficiency of growth. This is hypothesised to result from the so far not been in studied. Moreover how the Anthocerotae
idea that less nitrogen has to be diverted to the synthesis of CCM responses to increased light intensity needs to be
Rubsico, and possibly photrespiratory enzymes. In an early evaluated.
Silurian aerial environment, nitrogen availability and
acquisition may have been limiting compared to a water Land pyrenoid CCM and desiccation
column as the case may still be today in certain
The retention of pyrenoid CCM on land may be useful in
environments (Crittenden, Katucka & Oliver 1994). As the
the response of these polykiohydric organisms to variations
Rubisco molecule has a low turnover rate then retention of a
in environmental stresses, such as high light, but also more
CCM that conveys Nitrogen efficiency would be
importantly at fluctuating thallus water contents. Smith et al
advantageous (Raven and Lucus 1985). However, this has
1998, conducted a investigation on the effect of the uptake
not been conclusively demonstrated.
and release pools of CO2 in the lichen Peltigera
membranaecea at varying thallus water contents using a
Land pyrenoid CCM - reducing oxygenation method termed light dark transients. It can be inferred from
the data that a 10 fold drop in CO 2 up take and release pool
Another advantage of a CCM in a land plant such as the
sizes occur when thallus water contents decrease from
Anthocerotae would be to reduce the oxygenase reaction in
optimal (5.1- 6.2 mg g-1 d.wt) to (2.3 mg g-1 d.wt) a 63%
the enzyme Rubisco by elevating the CO2 Concentration
reduction in water content.
around its active site, reducing competition from oxygen.
Additionally, calculations by Green & Snelger (1982), show
The unique chloroplast architecture of the more “advanced”
that when Monoclea spp. (a liverwort with a solid thallus,
Anthocerotae posses thylakoids that cross the pyrenoid,
with no air pores) is compared to Marchantia spp.(with
termed channel thylakoids, Burr (1970), which have been
water proof cuticle penetrated with air pores), maximum
speculated (through inference from work with algae) by
photosynthesis is only slightly greater in Marchantia, but air
Makay & Gibbs (1991) to be dominant in photosystem I and
spaces giving greater advantage over Monoclea for water
not Photosystem II (water splitting side of the light reaction,
relations. However, the solid thallus of Monclea showed
releasing oxygen), therefore reducing further oxygenation
superior photosynthetic ability in very moist environments,
possibly like the hornwort (with CCM).
The pyrenoid CCM and high/low CO2 on light dark transients on Phaeoceros treated with EZ a
speculation made was that although active transport of CO2
Finally, fluctuations in external CO2 concentrations have
was still occurring (due to the appearance of a CO2 release
been a popular line of investigation in marine and freshwater
pool), the Ci transported to the stroma is not being utilized
algae with CCMs (Matsuda, Bozzo and Colman 1998;
by Rubisco when CA is suppressed by EZ. This point may
Woods 1999; Colman 2000) It has been recently
raise the question as what the exact mechanism of Ci
demonstrated that in cells of Chlamydomonas reinhardii,
transport is if EZ does not suppress the active uptake of
external CO2 concentrations can affect the active uptake of
CO2 in the Anthocerotae. The major drawback with using
CO2 and HCO3- and has been show to be suppressed in
EZ to manipulate the operation of the Anthocerotae CCM in
Chlamydomonas reinhardii grown in high external CO2 after
future studies is that CA is involved in other non CCM
about 8 days in 5% CO2 (pers com Colman 2000). After this
photosynthetic processes, as shown by the depressions of
period the new generation of cells lost the CCM capacity.
gross assimilation rates in mosses of 65 % and 50% in
However when transferred to a low CO2 external
other non CCM based liverworts treated with EZ.
environment, active transport of CO2 and HCO3- within 2hrs
The usefulness of being able to "switch of " or down
of acclimation, and CA ext activity increased 10 fold after 6
regulate the Phaeoceros CCM with a high CO2
hours after acclimation to 0.035% Co2. It has been
environment will be critical in elucidating the operation of the
proposed that the CCM is induced when the CO2
Anthocerotae CCM. However in terms of the ecology of
concentration in the medium is reduced to a critical level.
Phaeoceros, a relatively rapid response (of approx. 14
Matsuda, Bozzo and Colman (1998) have suggested the
days) to variations in external CO2 (initially 5% CO2) may
possibility of a CO2 sensor at the green algal cell surface,
not be seen, due to the fact that fluctuations in CO2 in the
which under high CO2 growth conditions would cause
aerial environment is relatively minimal, or at least not as
repression of the CCM, whereas under CO2 depletion the
irritate as in a water column,.
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 Chlorella spp. Aims
and Trebuxia spp .with a 2 day 5% CO2 treatment and then There are two aims of this investigation, the first is to
upregulated it with low CO2 treatment only after 2 hours. measure photon utilisation (elucidated from fluorescence
measurements (see appendix) in a range of liverworts with

Carbonic anhydrase and the land pyrenoid increasing morphological specialization viz Pellia spp.,
Conocephalum spp, Marchantia polymorpha L and
The possibility of being able to “switch off” or down regulate
Lunularia spp. ( all without a CCM ) compared to the same in
the operation of the Anthoceroate CCM is not unrealistic in
Phaeoceros laevis (with CCM). The following
the light of recent work by Smith and Griffiths (2000) who
measurements will be made;
established that CA plays a role in the operation of the CCM
in Phaeoceros laevis , as in all microalage investigated.  Electron transport rate (ETR)

Smith and Griffiths (2000) conducted an investigation into  Non photochemical quenching (NPQ)

the role of CA in photosynthesis and the activity of the CCM The second aim of this investigation is to attempt to

in Anthocerotae by using the membrane-permeable CA manipulate the operation of the hornwort CCM by

inhibitor ethoxzolamide (EZ), drawing a comparison to a introducing the following environmental conditions;

range of liverworts and mosses. The results showed that 1. Desiccation

inhibition of assimilation occurred in all bryophyte treated 2. High external CO2

with EZ, however the degree of inhibition was greatest in 3. High/ low light

Phaeoceros laevis. Furthermore, there was a pronounced 4. High temperature

decline in Ci-uptake efficiency and a decrease in the initial The control plant chosen is Marchantia polymorpha L,

slope of CO2-affinity curve at low external levels where Ci- mainly because Pmax is usually high, and it is easy to grow.

uptake efficiency in other liverworts were unaffected. There Pellia ssp. would have been an ideal choice, having a solid

were no significant differences between the convexities of thallus like Phaeoceros laevis, but it struggles in

the light response curves in Phaeoceros which would unfavourable conditions and results can appear erratic.

indicate a diversion of ATP to energise the CCM. In studies However Pellia will be used for the desiccation experiment.
MATERIALS AND METHODS (20cm by 20cm) containing peat, where the open top of the
box were covered and sealed with cling film, with a ambient
Acclimation procedures
CO2 (400ppm) supply feed through one side creating a

Measuring Florescence characteristics positive pressure released from a hole in the opposite side

Specimens of Pellia ssp, Conocephalem ssp, Lunalaria of the box. The boxes were set up in a Fissons growth

spp, Marchantia polymorphaL (all C3 biochemistry) and cabinet under 40µmol photons m-2 s-1 with a photoperoid of

Phaeoceros laevis. (with CCM) were collected from Devon 12 hours and a temperature regime of 15oC in light and

in late March. For 14 days each species was grown on peat 10oC without light. These specimens were acclimated under

and misted at least 3 times a day in a Fissons growth these conditions and sprayed three times a day with distilled

cabinet with a photoperiod of 12 hours 40µmol photons m-2 water for 14 days. After acclimation in the growth cabinet

s-1 @15 0C and 12 hours no light @10oC. Measurement of conditions pieces of thalli were removed for measurements

FV/FM, Electron Transport Rate (ETR) and Non of chlorophyll florescence (see theory and method)., light

photochemical Quenching (NPQ) were made using PAM response, carbon dioxide compensation points, light dark

florometer (see Maxwell). 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
Thallus drying at 30% RH over 3 hours
conditions in the growth cabinet remained unchanged. After
Specimens of Pellia spp Marchantia polymorpha L and
14 days in 5% CO2 (pers com Colman 2000 after 8 days of
Phaeoceros laevis were adapted to the same conditions as
acclimation at 5% CO2 cells of Chlamydomonas showed
for measuring Florescence characteristics. After this period,
loss of CCM characteristics) the same measurements were
thallus (at 100% thallus water content ) were dab dried and
performed for plants at 0.0175% CO2.
weighed. 2mg of thallus from each the plants were placed
on tissue in a fissons growth cabinet at low RH, and 40µmol
Acclimation procedure for plants grown at 30oC
photons m-2 s-1, 15oC. Thallus water content was then
weighed every hour, Pmax was also determined along with Specimens of Marchantia polymorpha L and Phaeoecros

chlorophyll content. 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
Acclimation procedure for plants grown at two
and 30oC in two different growth cabinets. PAR was 40
different light regimes.
µmol photons m-2 s-1 and photoperoid for 12 hours light
Specimens of Marchantia polymorpha L. and Phaeoecros
and 12 hours darkness, plants was misted with distilled
laevis were collected from Devon, in late March. Each
water over 3 times a day. Plants were grown under these
species was grown in seed trays on peat and misted three
conditions for 14 days, after which, pieces of thallus were
times a day under two different light regimes (in two
removed, excess water was dab dried and CCM diagnostics
different Fissons growth cabinets) 5µmol photons m-2 s-1
were performed; light response, carbon dioxide
and 120µmol photons m-2 s-1. Photoperoid was 12 hours
compensation points, light dark transients, K0.5 CO2, and
and the temperature regime was set at 15oC in light and
chlorophyll analysis.
10oC without light. Each species were adapted to these
conditions for 14 days before chlorophyll florescence
Light response curves
measurements were made using PAM florometer (see
theory and method), with light response, carbon dioxide An ADC IRGA (ADC 225 Mk 3 – ADC Hoddesdon, UK) in
compensation points, light dark transients,and K0.5 CO2. 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
Acclimation procedure for plants grown at 5%
CO2, low CO2, and ambient CO2 regimes. illuminated by a KL 1500 electronic light source (Hansatech,
Norwich UK) which delivered a range of light intensities
Specimens of Marchantia polymorpha L and Phaeoceros
from 0 to 2000µmol photons m-2 s-1. The lowest average
laevis were collected from Devon and acclimated in
light intensity available was 15 +- 4 µmol photons m-2.s-1 and
Newcastle botanical gardens during August and September
the highest average value 2000 +- 400 µmol photons m-2. s-
(natural photoperoid at 25 o
C). The Phaeoceros and
. CO2 (350ppm) was supplied in compressed air from gas
Marchantia mats were divided into 3 plastic boxes each
cylinder via IRGA to the cuvette. A clean piece of thallus
was sprayed with distilled water, blotted and placed within also calculated from the CO2 response curves by reading
the cuvette, ensuring self shading did not occur (Smith PhD off CO2 concentration at ½ the Pmax at both ambient, and
thesis 1995). Thallus was dark adapted within the cuvette in 5% CO2 treatments.
a closed system initially, and dark respiration recorded
through a chart recorder. The system was opened and Light –dark transient releases of DIC pools.
purged, in preparation from the following light intensities; 15, An ADC IRGA (ADC 225 Mk 3 – ADC Hoddesdon, UK) in
55, 97, 205, 325, 456, 660, 832, 1335, 1911, 2000µmol differential mode was connected in an open system with a
photons m-2. S-1, purging after each exposure. The samples modified Hansatech Oxygen electrode (LD2, Hansatech,
were also weighed before and after each experiment to Norwich UK) (Badger & Price, 1992). Hansatech LS-2
ensure that the water content had not dropped below that (Hansatech, Norwich, UK) lamp supplying red light was
which is optimal for photosynthesis (Smith PhD thesis used as the light source and temperature was maintained
1995). by a water jacket at 15 degrees C - water circulated and
The above procedure was repeated 3 times for each supplied (Grant refrigerated flow heater / cooler system).
species. Compressed air (350ppm CO2) was supplied from cylinder
(1.7m height, 0.23m width) which maintained a constant
CO2 response curves and K0.5 CO2 partial pressure of CO2 and flow rate of 300L/min
An ADC IRGA (ADC 225 Mk 3 – ADC Hoddesdon, UK) in compressed air. The relative humidity of the air flowing
absolute mode was connected to a modified Hansatech through the system remained within the range 50 – 70%
Oxygen electrode (LD2, Hansatech, Norwich UK) with throughout the experiment. A piece of thallus from either
water jacket maintained at 18 degrees C. The cuvette was Phaeoceros laevis (sample) or Marchantia polymorpha L
illuminated by a KL 1500 electronic light source (Hansatech, (control). was immersed in distilled water, in darkness for a
period of half an hour to ensure the complete metabolism of
Norwich UK) which delivered saturating light; 320µmol
any remaining DIC pool, prior to being blotted and weighed
photons.m-2.s-1 for Marchantia polymorpha L (Pmax) and
(Smith, PhD Thesis 1995). The piece of thallus was then
660µmol photons. m-2.s-1 for Phaeoceros laevis . (Pmax). A
placed in the cuvette and left to attain a steady rate of dark
clean piece of thallus was sprayed with distilled water,
respiration, recorded using a chart recorder. Saturating light
blotted and placed within the cuvette, ensuring self shading
was then used to illuminate the thallus (Marchantia 320µmol
did not occur (Smith PhD thesis 1995). CO2 was supplied to
photons.m-2.s-1, Phaeoceros 660µmol photons. m-2.s-1) for a
the thalli from a compressed air cylinder (350ppm CO 2) via
peroid of up to twenty minutes using the red light source
a gas diluter – where the air passes through carbosorb –
detailed above, after which the maximum rate of
(ADC Hoddesdon, UK) to dilute 350ppm CO2 (100%) to
photosynthesis was attained. On turning the light off, the
0%, 13%, 28%, 46%, 64% of system was initially purged
release of an internal pool of CO2 was detectable in the
with the required % CO2 as an 350ppm CO2, supplied via
Phaeoceros laevis thallus and recorded as a short-lived (< 1
the IRGA to the cuvette. The open system. The system was
min) burst of CO2 within the system by the chart recorder.
subsequently closed and the thallus left to deplete the CO2
The reaction time of the system to the changes in light was
concentration within the closed system, the values were
minimised by ensuring that the tubing between the cuvette
recorded on a chart recorder. The procedure was repeated
and the IRGA were as short as possible (Smith, PhD
at all of the above CO2 concentrations, purging after each
Thesis 1995). Chlorophyll
exposure. Chlorophyll analysis was preformed on each
analysis was performed on each sample almost
sample almost immediately. The above process was
immediately. The experiments were repeated using thalli
repeated 3 times for each species and the average values
which had been immersed in 50mM glycolaldehyde (which
calculated together with SE. The averaged values at each
inhibits photosynthetic CO2 fixation by inhibiting the
CO2 concentration were plotted against, a
regeneration from ribulose-5-phosphate) (Miller and
line of best fit that dissected the x axis of the plot, was then
Canvin, 1989), for up to half and hour depending on the
read off to be the CO2 compensation point. K0.5 CO2 was
hydroscopic properties of the thalli (Badger, et al. 1993).

Chlorophyll analysis
mortar. The extraction was spun at 4 degrees C for 10mins
Chlorophyll analysis was carried out according to Porra et al
at 5000rpm, supernatant was poured into 1 mm glass
(1989). Approximately 20mg (FW) sample was immersed in
cuvettes and absorbency readings were taken at 664nm
chilled 80% acetone (10ml) and ground with pestol and
and 646nm against an acetone blank. The following Chl a = 12.25 A 664 – 2.55 A 647
equation was used: Chl b = 20.31 A 647 - 4.91 A 664
In ug chl ml-1 Total = 17.76 A 647 + 7.34 A 664


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% RH Pmax 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 laevis gametophyte under 40µmol photons m-2 s-1 for
14 days (n=5).
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 of Phaeoceros 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 lumen’s 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 2000µmol photons m-2 s-1 before Photoinhibtion (PI), compared to the other liverworts, which
were photinhibited at 1000µmol 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 Phaeoceros and Marchantia gametophytes (n=4).

Species Treatment Max Net Assimilation CO2 K 0.5 CO2 Magnitude

µmol photons (nmol CO2 (µl l-1) (µl l-1) of DIC pool m-2 s-1
mg -1 Chl s-1) (nmol CO2
mg-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 µmol photon m-2 s-1
treatment and the 5 µmol 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 an
increased efficiency for CO2 in high PAR grown Phaeoceros. Unusually the magnitude of the DIC pool (27.8 nmol CO2 mg-1 Chl)
was induced at 1000µmol photons m-2 s-1 as opposed to (18.3 nmol CO2 mg-1 Chl) at 100µmol 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 µmol photon m-2 s-1 treatment, moreover
FV/FM in Phaeoceros and Marchantia was 0.68, showing they were under stress.
Grown at 5µmol
6 photons m-2 s-1
6 Grown at 5µmol
photons m-2 s-1
Grown at 120µmol
photons m-2 s-1
2 Grown at 120µmol 2
photons m-2 s-1

µmol photons m-2 s-1 µmol photons m-2 s-1

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 CO2 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 µmol 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 Phaeoceros and Marchantia gametophytes (n=4).

Species Treatment Max Net Assimilation CO2 K 0.5 CO2 Magnitude

(nmol CO2 (µl l-1) (µl l-1) of DIC pool
mg 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

† ANOVA Significant difference

Surprisingly, Phaeoceros laevis 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 CO 2 level, and the low CO2 regime
did 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,
however FV/FM was low, (florescence data) and the plants were struggling at 5% CO 2. 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

Phaeoceros and Marchantia gametophytes (n=4).

Species Temperature Max Net Assimilation CO2 K 0.5 CO2 Magnitude

degrees (nmol CO2 (µl l-1) (µl l-1) `of DIC pool
Centigrade 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

† ANOVA Significant difference

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 Marchantia polymorphaL 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)

The following investigation revealed that when the cross the pyrenoid, termed channel thylakoids, Burr
gametophyte of extant hornworts is exposed to external (1970), which have been speculated (through inference
CO2 concentrations of either 5%, ambient or 0.00175%, from work with algae), by Makay & Gibbs (1991), to be
(for 14 days), the CCM showed no effect in its operation, dominant in PSI, not PSII (water splitting side of the light
unlike algal CCM’s where 5% CO2 after only 2-8 days reaction, releasing oxygen), therefore reducing further
switches the CCM off (REF). This indicates that a oxygenation events, possibly making up for a Rubisco
hornwort tissue response, to differing external CO2 with low specificity.
concentrations, has not occurred, (maybe more time is Furthermore, high light intensity (compared to low light
needed in these CO2 environments). intensity grown plants), can have a sig. effect on the
However, in hornworts grown at 30 C for 14 days
capacity of the hornwort CCM; with a large dissolved
(compared to those at grown at lower temperatures) there inorganic carbon (DIC) uptake and low CO2
was a sig. diff. in the operation of the CCM, appearing to compensation point and low K 0.5 CO2. An active CCM will
increase the capacity of dissolved inorganic carbon (DIC) effectively reduce the light-utilization efficiency of
uptake and lower the CO2 compensation point and, lower photosynthesis, therefore increased CCM activity will
K 0.5 CO2, (with hydrated thallus). optimise the supply of CO2 to Rubisco helping it deal with
Therefore an advantage of a CCM in a land plant such as high PAR.
the Anthocerotae would be to reduce the oxygenase The hornwort appeared to be able to photosynthesise at
reaction in the enzyme Rubisco by elevating the CO2 Photon Flux Densities (PFD) above 1500umols m2. s2, (in
Concentration around its active site, reducing competition those grown at 120umol m2. s2 for 14 days); However a
from oxygen. The unique chloroplast architecture of the constituently expressed CCM at high PAR kept NPQ low,
more “advanced” Anthocerotae possess thylakoids that causing PSII damage, maybe acting as an electron sink
to power the DIC pump, and/or as a proton sink in the (Burr 1968). Brown a lemon (19--) suggests the evolution
thylakiod lumen, suplying protons to the reaction; (H )+
of the multiplastic cell (away from the uniplastic cell found
HCO3- CA CO2 + H2O, so lowering protonation of in Phaeoceros) may have meant the end of pyrenoid
the xanthophylls reducing NPQ. However, at an extremely containing plastids. In the mutiplastidic cell the division of
low light treatment, Pmax was greater than that of high a number of plastids would have been more difficult to
light treated plants (lower Pmax could have been caused co-ordinate. An even distribution of Rubisco in the stroma
by chlorophyll loss), indicting growth at low PFD is would not require a pyrenoid division to insure that
preferred in the hornwort. However light adaptability is Rubisco is present in every chloroplast. With Rubisco no
possible with use of the CCM’s plasticity as a light use longer being pumped CO2 around the active site, the
efficiency reducer a high PAR and a oxygenase reaction early C3 liverwort Rubisco kinetics would have improved
reducer at high temperature. efficiency, coupled with morphological specialisation to
Early land plants in the Silurian atmosphere, 450MYA, aid CO2 influx, and pores to reduce water loss, thus
may well have made use of the already existing pyrenoid making the early C3 liverworts less dependant on water
CCM (as possessed by the ancestral Coleochales), not to aid CO2 in flux.
because external CO2 was in short supply (Silurian aerial To retain a pyrenoid CCM on land meant adaptability to
environment; [CO2] 5400 - 7000ppm) (REF), but as a way the Silurian atmosphere; higher temperatures and
to deal with high PAR by increasing efficiency for CO 2 at stronger light conditions (REF) than experienced in the
the cost of a reduced light use efficiency, the CCM acting water column. To retain a pyrenoid CCM on land today
as - a ''light use efficiency reducer'' - and in high means hornworts with CCM’s have the plasticity to adapt
temperatures the CCM up regulates, also increasing to high and low temperatures and very low light, to higher
efficiency for CO2 acting as - a ''oxygenase reaction light environments, ranging from the Australian and India
reducer''. The hornwort CCM can be likened to a primitive rain forests, to, ditches in Scotland and Canada.
stomata, regulating CO2 uptake, in response to light, However, full hydration of the thallus is essential for
temperature and thallus water content variations. efficient CCM activity, so a damp to wet environment is
The eventual loss of the pyrenoid CCM, and the move the habitat where they are found. If environmental
towards a more advanced morphology as seen in the C3 conditions are no longer favourable, hornwort tuber
liverworts, meant a change in shape of the chloroplast formation can occur and the thallus dies (REF); the plant
from band shaped to discoid (REF). The discoid shape to be resurrected when environmental conditions are
would allow more light adaptability; an example of this is suitable. This is another useful adaptation to cope with
the unusual light dependant changes in the chloroplast the high PAR and drying summer conditions experienced
morphology in species of hornworts without pyrenoids by Phaeoceros growing in the Mediterranean area (REF).