CO2 diffusion & concentration: aspects of stomatal conductance and intercellular CO2 concentration in plants

Welcome
Brahmesh Reddy B R
PAMB 2088

CO2
Concentration
Improving diffusion

Topics
1. Stomatal conductance
2. Mesophyll conductance
3. Mesophyll tissue thickness
4. VPD response of gs

Stomatal
conductance
Stomatal conductance
(gs) is the diffusion of
gas, such as carbon
dioxide, water vapor, and
oxygen, through the
stomata of a plant.
It also functions as the
measure of stomatal
opening in response to
environmental
conditions.

Stomatal
conductance
Stomatal conductance
occurs specifically through
the stomata when they are
open;
The reverse is known as
stomatal resistance.

The stoma is an aperture formed by two
guard cells. Connected to the guard cells are
subsidiary cells.
Flaccid - Close
Turgid - Open

The shape of guard cells varies based on species. Based on the shape of guard cells and the
number and arrangement of subsidiary cells

Stomatal traits vary between species. The eudicots (A) Arabidopsis thaliana and (B) Phaseolus vulgaris display kidney-shaped guard
cells (colored in green). The grasses (C) Oryza sativa and (D) Triticum aestivum show dumbbell-shaped guard cells (solid green) and
specialized subsidiary cells (light green gradient). Clear differences in stomatal size and stomatal density can be observed

Internal, plant-level factors consistently affect the need and application of stomatal
conductance.
1. Signals from the guard cell and stomatal density
2. Changes in leaf water potential
3. The concentration of the plant hormone abscisic acid (ABA) in the xylem sap
4. Need to take in CO2 for photosynthesis
5. Association with arbuscular mycorrhizal fungi (AMF), which form symbiotic associations
with 80% of plant species, can change stomatal conductance. The exact mechanism and
mode of action are yet unclear.
Internal Influences

Internal, plant-level factors consistently affect the need and application of stomatal
conductance.
1. Light is the primary external factor that determines stomatal conductance, as stomata are
activated and open during daylight.
2. Humidity influences stomatal conductance. Low humidity reduces stomatal conductance
to preserve water. Stomata will open in high humidity even if the leaf water content is less.
3. Soil water and nutrient status will also impact stomatal conductance, which will
decrease when soil moisture is less.
External Influences

4. Air temperature rises will increase stomatal conductance, independent of plant water
status and photosynthesis. While this helps plants cool through evaporation and increases
photosynthesis, plants lose more water.
5. Elevated atmospheric CO2 concentration decreases stomatal conductance, which can end
up reducing photosynthesis.
6. Salinity stress also reduces stomatal conductance–this is significant as nearly 7% of the
global land is saline.
External Influences

● Daylight - CO2 / Photosynthesis
O2 / Respiration
● Open - water vapour / Transpiration
● rs - Prevent Transpiration
● Stoma - VOC release
Functions of gs

Nocturnal Conductance
Leaf stomata are closed at night as no photosynthesis is triggered without
daylight.
However, nocturnal conductance (gn) does occur through cuticles. Some
nocturnal conductance also happens through stomata.

Why?
Hypothesis
● Removal of excess CO2 formed during respiration
● Transportation of oxygen mixed in xylem sap to leaves in tall trees
● Transportation of nutrients
● Leakage due to improper closure of stomata
● Predawn controlled opening of stomata allowing for an early start
in photosynthesis, which is helpful in cases or phases of rapid
growth
Nocturnal Conductance

So what is that we want to do?

Leaf Cross-Section
Wheat (C3) - 400x magn

Leaf Cross-Section
Corn (C4) - 400x magn

Leaf Cross-Section
Leaf Cross-Section

Leaf Cross-Section
Leaf Cross-Section
C3 plants have a normal leaf anatomy where the palisade mesophyll forms a
row of closely packed cells just beneath the upper epidermis of the leaf
In C4 plants, the palisade mesophyll cells form a ring around the bundle
sheath cells and veins of the leaves

We’ll first understand the importance
of stomatal conductance

GSNO -> S-nitrosoglutathione
NO-> Nitric Oxide

NO released or transferred by GSNO targets protein kinases that
ultimately affect the stomatal conductance in sugarcane plants
sprayed with GSNO
GSNO-sprayed plants exhibited increases in photosynthesis under
water deficit, which was a consequence of high stomatal
conductance and increased apparent carboxylation efficiency

GSNO treatment increased by seven times the stomatal conductance at the
maximum water deficit as compared with plants sprayed with water

Stomata continually adjust aperture in response to
external environmental cues (e.g. light),
plant water status
hormonal (e.g. ABA)
circadian
to maintain an appropriate balance between CO2 uptake and
water loss.

short-term dynamic changes in the environment result in a
lack of synchrony between gs and A,
as stomatal responses to changing environmental cues are often
substantially slower than those observed in A, resulting in a
temporal disconnect between A and gs
that can limit photosynthetic carbon assimilation and reduce plant
water use efficiency

the control of stomatal opening and closure determine ‘operational’ or
measured gs, that is the fraction of gsmax at which the leaf operates
McElwain, J.C., Yiotis, C. and Lawson, T. (2016), Using modern plant trait relationships between observed and theoretical maximum stomatal
conductance and vein density to examine patterns of plant macroevolution. New Phytol, 209: 94-103. https://doi.org/10.1111/nph.13579
r2 = 0.5446

Anatomical characteristics
determine the rate of gs

stomatal density (SD)
size and maximum pore area
determine the calculated theoretical maximum stomatal conductance (gsmax)

A positive relationship between SD and gs
MUCHOW, R. C., & SINCLAIR, T. R. (1989). Epidermal conductance, stomatal density and stomatal size among genotypes of
Sorghum bicolor (L.) Moench. Plant, Cell and Environment, 12(4), 425–431. doi:10.1111/j.1365-3040.1989.tb01958.x

r2=0.3255
r2=0.6675
A positive relationship between SD and gs
McElwain, J.C., Yiotis, C. and Lawson, T. (2016), Using modern plant trait relationships between observed and theoretical maximum stomatal
conductance and vein density to examine patterns of plant macroevolution. New Phytol, 209: 94-103. https://doi.org/10.1111/nph.13579
gop
gmax

How to improve stomatal density?
Now we know that, the gs can be improved by increasing the stomatal
density. Hence we need to now increase the stomatal density.

Differential interference contrast (DIC) images of the abaxial epidermis of
mature first leaves of wild type, a STOMAGEN-silenced line (STOMAGENRNAi10)
and a STOMAGEN-overexpressing line (STOMAGEN-OX10).

Stomagen is expressed in inner tissues of immature organs and
secreted out of the cells to mediate inter-tissue signalling
RT–PCR showing STOMAGEN mRNA levels in mature (m) and immature (i) organs

The stomatal lineage is triggered by
SPEECHLESS (SPCH)
which is a basic helix–loop–helix (bHLH) type transcriptional factor.

overexpression of STOMAGEN in a spch mutant and application of
purified stomagen to spch failed to induce stomatal formation

the stomata-inducing activity of stomagen is dependent on the
SPCH pathway
SPCH regulates the expressions of both the receptor-like protein
TMM and its putative ligands EPF1 and EPF2

Stomatal development in leaves is negatively regulated by
TMM and ER, ERL1 and ERL2
putative cell-surface receptors TOO MANY MOUTHS (TMM) and
ERECTA family receptor-like kinases (ER, ERL1 and ERL2)

ligands of these receptors, EPIDERMAL PATTERNING FACTOR
EPF1 and EPF2
act as negative signalling factors at distinct steps during stomatal development

wild-type (WT)
STOMAGEN-overexpressing (ST-OX)
STOMAGEN-silencing (ST-RNAi)

Now the size and density of stomata

the influence of changes in stomatal anatomy (density and size; left panels, stomatal clustering; lower panels) on
stomatal conductance (gs, arrows) and the rate of gs response (red lines). The impact of anatomical traits on
carbon gain (A, dashed lines), the limitation of A by gs (green area) and water use efficiency (Wi) are illustrated.
The influence of stomatal density and size (vertical arrow) and stomatal clustering (horizontal arrow) on the rate of
gs response and the maximum or operational value of gs is highlighted.

Leaves with a greater number of smaller stomata would be expected to have more
rapid stomatal responses and a higher overall gs compared with leaves that had
lower density and larger stomata.

stomatal patterning defects (i.e. stomatal clustering) have been reported to result in
slower gs responses and lower gs values

the maximum rate of stomatal opening is driven by the surface-to-volume ratio of
stomata, attributed to changes in SD and size, as species with higher stomatal
densities and smaller stomata exhibited more rapid gs kinetics

slow stomatal opening - 10% limitation on carbon assimilation,
Therefore, losses in carbon gain over the course of the day,
potentially negatively impacting productivity and yield

slow stomatal closure results in a significant decrease in
intrinsic water use efficiency (Wi)
and resource use thus potentially accelerating early soil water exhaustion

Using already present
Genetic variation and
genetic control for selection

Aminian, R., Mohammadi, S., Hoshmand, S. et al.
Chromosomal analysis of photosynthesis rate and
stomatal conductance and their relationships with grain
yield in wheat (Triticum aestivum L.) under water-
stressed and well-watered conditions. Acta Physiol Plant
33, 755–764 (2011). https://doi.org/10.1007/s11738-
010-0600-0
Wang, S.G., Jia, S.S., Sun, D.Z., Wang, H.Y., Dong, F.F.,
Ma, H.X., Jing, R.L. and Ma, G., 2015. Genetic basis of
traits related to stomatal conductance in wheat cultivars
in response to drought stress. Photosynthetica, 53(2),
pp.299-305.
https://doi.org/10.1007/s11099-015-0114-5

Akihiro Ohsumi, Tomomi Kanemura, Koki Homma,
Takeshi Horie & Tatsuhiko Shiraiwa (2007) Genotypic
Variation of Stomatal Conductance in Relation to
Stomatal Density and Length in Rice (Oryza sativa L.),
Plant Production Science, 10:3, 322-328,
https://doi.org/10.1626/pps.10.322
Junfei Gu, Xinyou Yin, Paul C. Struik, Tjeerd Jan Stomph,
Huaqi Wang, Using chromosome introgression lines to
map quantitative trait loci for photosynthesis parameters
in rice (Oryza sativa L.) leaves under drought and well-
watered field conditions, Journal of Experimental Botany,
Volume 63, Issue 1, January 2012, Pages 455–469,
https://doi.org/10.1093/jxb/err292

That's it!!!
We’ll meet again for
Mesophyll conductance
Mesophyll tissue thickness
VPD response of gs

Brahmesh Reddy B R
PAMB 2088
Welcome
back

Photosynthesis in plants has been considered to be limited only by
two factors:
1. the velocity of diffusion of CO2 through stomata and mesophyll
2. the capacity of photosynthetic machinery to convert light energy
to biochemical energy and fix CO2 into sugars.

Transmission electron micrograph illustrating the liquid pathway for CO2 diffusion within a leaf of Nicotiana
https://doi.org/10.1093/jxb/erp117

The photosynthetic limitation imposed by mesophyll conductance is
large, and under certain conditions can be the most significant
photosynthetic limitation.
Anatomical traits, such as cell wall thickness and chloroplast
distribution are amongst the stronger determinants of mesophyll
conductance

Rapid variations in response to environmental changes might be
regulated by other factors such as aquaporin conductance

Mesophyll resistance is generally divided as
1. gas phase resistance
2. Liquid phase resistance
http://dx.doi.org/10.19103/AS.2022.0119.10

Mesophyll resistance is generally divided as
1. Gas phase resistance
CO2 movement through intercellular airspaces
1. Liquid phase resistance
cell wall; plasma membrane; cytosol; chloroplast envelope;
chloroplast stroma
http://dx.doi.org/10.19103/AS.2022.0119.10

Liquid phase resistance accounts for up to 90%
of mesophyll resistance to CO2 diffusion through the leaf

Factors determining the liquid path length that CO2 must
travel from the intercellular air spaces to the chloroplasts:
1. shape, size and density (packing) of mesophyll cells
2. position and orientation of the chloroplasts

1. The arrangement of the mesophyll layer
mesophyll can be considered a porous medium, where CO2
diffuses through intercellular airspace and into the cells
containing the chloroplasts. Some of the largest physical
determinants of gm are the surface area

1. The arrangement of the mesophyll layer
(Sm) - the surface area of the cells exposed to the
intercellular airspace
(Sc) - the surface area of the chloroplasts exposed to the
intercellular airspace

Sc /Sm
expresses the relative fraction of available area that is
occupied by chloroplasts through which CO2 can diffuse
towards the site of Rubisco

chloroplast
Sc
Mesophyll cell
Sm

The presence of many small cells within the mesophyll
increases both Sm and Sc

The lobed cell shape of rice leaves is an example of a
mesophyll cell anatomy that can increase cell surface to
volume ratio and enhance Sm as well as light absorption

https://doi.org/10.1093/pcp/pcp033
Light micrographs illustrating mesophyll tissue of O. sativa
The rice mesophyll tissue is composed of lobed chlorenchyma cells that are
approximately twice as long as they are wide

the chloroplast volume ranges between 50% the of cell
volume for some cells within the interior of the mesophyll
to 83% of the cell volume for the remaining chlorenchyma
cells.

the dense, chloroplast-rich cytosol and tight mesophyll cell
packing explains in part how rice can have a high
photosynthetic capacity
The greater chloroplast density per unit volume in the rice
leaf allows for more photosynthetic protein per volume,
offsetting the reduced thickness of the mesophyll layer

https://doi.org/10.1093/pcp/pcp033
Scanning electron microscope image illustrating mesophyll tissue of O. sativa
The rice mesophyll tissue is composed of lobed chlorenchyma cells that are
approximately twice as long as they are wide

Rice and its relatives stand out as a group of C3 plants that thrive
in warm to hot environments where photorespiration is favored
There are two potential mechanisms for photorespiratory
compensation are indicated by the structure of the leaf mesophyll

First, the extensive lobing and small cell size, coupled with a near
complete coverage of the cell periphery by the chloroplasts and
stromules, indicate that the rice mesophyll is specialized to
maximize the diffusive conductance of CO2 into the stroma.

Secondly, the interior location of the mitochondria and peroxisomes,
and the extension of the stromules to complete a barrier between
mitochondria and the intracellular space, could be specializations to
maximize the refixation of photorespired CO2.

Peroxisomes and mitochondria are predominantly situated in files adjacent to the
main body of both the chloroplast and chloroplast stromules.
Stromules

Peroxisomes and mitochondria sandwiched between chloroplasts

By creating a stroma-filled barrier (stromule) along the periphery of
the mesophyll cell, the photorespired CO2 will have to pass by
RuBisCO to exit the cell.
The addition of this CO2 supply to that coming from the intercellular
spaces should increase the stromal CO2 concentration, and improve
the carboxylation capacity of RuBisCO.

CO2 transfer conductance in the mesophyll
it is primarily a function of membrane permeability and the surface
area of chloroplasts exposed to the intercellular spaces

Rice has high values in both mesophyll surface area and
chloroplast/stromule coverage. Exposed mesophyll surface area in
rice ranges between 19 and 44 m2 m–2, while in wheat the values
range between 8 and 24 m2 m–2

The high degree of lobing coupled with small cell sizes explains the
high surface area exposure of the rice mesophyll tissue.
This arrangement would also ensure that rice has 30% greater
surface area coverage than wheat, where the exposed surface area of
the chloroplasts has been measured to be 76%

Next…the density / packaging

an arrangement of mesophyll cells that are more loosely packed
together may allow for a more homogenous distribution of light
throughout the mesophyll. This would not only increase Sm and Sc
but also the light level for chloroplasts at lower layers in the
mesophyll that would enhance the gm

Diffusion through the cell wall is the first step in the liquid phase of
CO2 diffusion through the leaf.
The walls contain pores filled with apoplastic fluid; these pores form
the interface between the gas and liquid phases of diffusion and
provide routes for CO2 to move from the intercellular airspaces into
mesophyll cells.

Cell wall structure.
(A) Fresh, unfixed onion cell wall
(B) 11% of image A set as an apparent pore.
(C) Diagram of a cell wall in plan view
showing a microfibril array spaced one
radius apart. This results in a structure with
50% water content and direct pores
occupying 11% of the plan area.
(D) Cross-sectional view of the wall showing
the changing size of each pore and the
tortuosity with depth through the matrix.

If cell walls represent a significant proportion of the liquid phase resistance, then leaves
with thicker cell walls would have lower mesophyll conductance per unit of exposed
chloroplast surface area.

(A) Relationship between mesophyll resistance per unit of exposed chloroplast surface area and mesophyll cell
wall thickness. Solid lines are shown for various effective porosities.
(B) CO2 assimilation rate per unit of exposed chloroplast surface area in relation to mesophyll cell wall thickness.
(C) Draw-down between Ci and Cc in relation to cell wall thickness

Leaves with thin mesophyll cell walls have much greater
mesophyll conductance for a given exposed chloroplast surface
area than leaves with thick mesophyll cell walls

However, one must consider how thin a mesophyll cell wall can be
made without jeopardising its structural integrity
The ideal solution will likely be complicated and depend on factors
such as the specific plant in question, and its mesophyll cell size and
cell wall composition

Cellular membranes and CO2 diffusion

Cellular membranes are composed of a lipid bilayer and function to
modulate movement of molecules in and out of the cell.
CO2 is hydrophobic and nonpolar
initial studies on membrane permeability in artificial lecithin-
cholesterol bilayers concluded that biological lipid membranes
offered little to no resistance to CO2

These membrane models, however, did not account for the dense
packing of proteins within the membrane and differing lipid
composition. Unlike animal cells, plant membranes do not contain
cholesterol but instead have a suite of related sterols

Cellular membranes impose up to 50% of the resistance to CO2
diffusion within mesophyll cells
These membranes include the plasma membrane (PM) and the dual
membranes of the chloroplast envelope (CE). The CO2 permeability
of the CE is assumed to be half that of the PM, as the envelope is
composed of dual membranes, thus increasing the resistance

the PM and CE contain channels and transporters that facilitate
CO2 transfer across the membrane and manipulation of these has
become a target for understanding and improving mesophyll
conductance in plants

The functional aquaporin unit is composed of four monomer
channels coming together to form a tetramer, which creates a fifth
central pore

Expression of a tobacco AQP1 homologue, NtAQP1 (also known as
NtPIP1) in oocytes also conferred increased CO2 permeability

plant aquaporin NtAQP1, when expressed in Xenopus oocytes, has
CO2 permeability
Xenopus oocytes were injected with carbonic anhydrase, an enzyme
that accelerates the conversion of CO2 to HCO3.

CO2 transport into the cells caused a decrease in intracellular pH in
controls injected with carbonic anhydrase only, and also in NtAQP1-
expressing carbonic anhydrase oocytes

From the rates of pHi decrease, it was found that the CO2 uptake
rates of controls (injected with carbonic anhydrase) and the initial
CO2 uptake rates of oocytes overexpressing NtAQP1 were,
respectively, 45% higher

Now that we saw about aquaporins indirectly involving
carbonic anhydrase
why don’t we directly try increasing this enzyme?

CA activity in the stroma is likely to have a large impact on gm, with
models predicting that removal of stromal CAs would
decrease gm by up to 44% and photosynthetic assimilation by 7%
https://doi.org/10.1104/pp.111.172346

By coupling CA to a CO2 channel, diffusing CO2 is instantaneously
converted to bicarbonate, ensuring a strong pull down effect for
CO2 into the cytosol

This relationship could potentially be further exploited through an
engineered complex (metabolon) of aquaporin and CA at the plasma
membrane
This ensures their interaction remains constant and optimised.

Concluding mesophyll conductance improvement
❖ The Path of Carbon Dioxide Within the Leaf
❖ Gas Phase
❖ Liquid Phase
■ Entering the Medium: Carbonic Anhydrases
■ Cell Wall
■ Diffusion Through Membranes: The Role of Aquaporins
■ Cytosol
■ Chloroplast
doi:10.1016/b978-0-12-813164-0.00017-x

❖ The Path of Carbon Dioxide Within the Leaf
CO2 diffuses from the intercellular air spaces to the mesophyll cells,
then overcomes the resistance of the polymer matrix that constitutes the
cell wall, passes through the lipid bilayers (both plasmalemma and
chloroplast envelopes) and, finally, reaches the sites of carboxylation of
the enzyme Rubisco inside the chloroplast stroma.
doi:10.1016/b978-0-12-813164-0.00017-x

❖ Gas Phase
Once CO2 has entered the leaf via stomata, it has to diffuse through the
intercellular air space fraction of the leaf or porosity, which, together
with the leaf thickness, determines the gas-phase conductance
doi:10.1016/b978-0-12-813164-0.00017-x

❖ Liquid Phase
Entering the Medium: Carbonic Anhydrases
CO2 has to enter the liquid phase to reach the chloroplast stroma. Liquid
phase conductance (gias) involves different steps: from the cell wall pores, the
plasmalemma, cytosol, and chloroplast envelopes to the stroma. A role for
carbonic anhydrases (CA), which catalyze the conversion of CO2 to HCO3, has
been proposed as an enhancer of carbon diffusion through the liquid phase
doi:10.1016/b978-0-12-813164-0.00017-x

❖ Liquid Phase
Cellwall
Once CO2 is in the liquid phase, the cell wall constitutes one of the main
obstacles for its path. In fact, low gm values have been reported in
angiosperm species with thick mesophyll cell walls and cell wall thickness is
key to explain the lower photosynthesis capacity of ferns compared to higher
plants
doi:10.1016/b978-0-12-813164-0.00017-x

❖ Liquid Phase
Diffusion Through Membranes: The Role of Aquaporins
Aquaporins have been proposed as the nexus of the gm and hydraulic
conductance relationship; in fact, the proportion of tetramers that constitute
the whole protein may determine its relative contribution to H2O and CO2
transport
doi:10.1016/b978-0-12-813164-0.00017-x

❖ Liquid Phase
Cytosol
The spacing between the cell wall and chloroplasts is difficult to determine, as
the uncoupling of chloroplasts from the cell’s edge observed in some cases
may be due to fixation artifacts
Tobacco plants with altered chloroplast arrangement, resulting in increased
cytosol path, showed reduced assimilation and gm
doi:10.1016/b978-0-12-813164-0.00017-x

❖ Liquid Phase
Chloroplast
The last major step of CO2 diffusion is its entrance within the chloroplasts. In
this regard, the surface of chloroplasts exposed to the intercellular air spaces
(Sc/S) has been revealed as a strong determinant of mesophyll conductance
as it reduces the path length of CO2 diffusion, thus enhancing its arrival at the
sites of carboxylation in the stroma. Chloroplast number, size, and
arrangement determine Sc/S and even chloroplast movements can induce
short-term variations of gm
doi:10.1016/b978-0-12-813164-0.00017-x

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