# Modifying mesophyll conductance to optimise photosynthesis in crops

Coralie E. Salesse-Smith, University of Illinois at Urbana-Champaign, USA; Steven M. Driever, Wageningen University and Research, The Netherlands; and Victoria C. Clarke, The Australian National University, Australia

## **Modifying mesophyll conductance to optimise photosynthesis in crops**

*Coralie E. Salesse-Smith, University of Illinois at Urbana-Champaign, USA; Steven M. Driever, Wageningen University and Research, The Netherlands; and Victoria C. Clarke, The Australian National University, Australia*

1 Introduction

Modifying mesophyll conductance to optimise

photosynthesis in crops


#### **1 Introduction**

The need to feed a growing global population, combined with a changing climate and diminishing resources, has driven researchers towards improving crop productivity through a variety of approaches (Bailey-Serres et al., 2019; Ray et al., 2013). Recently, this effort has begun to focus on improving photosynthesis as a key driver of crop yields. The process of photosynthesis is dependent upon acquisition of carbon dioxide (CO2) from the atmosphere, which is converted into carbohydrates by the enzyme Rubisco within the chloroplast. In C3 plants, Modifying mesophyll conductance to optimise

photosynthesis in crops

© The Authors 2023. This is an open access chapter distributed under a Creative Commons Attribution 4.0 License (CC BY)

http://dx.doi.org/10.19103/AS.2022.0119.10

the diffusion of atmospheric CO2 into leaf chloroplasts is a limiting step in this process, and improving the conductance to CO2 diffusion within leaves, termed mesophyll conductance (*gm*), is predicted to increase photosynthetic rates and improve water use efficiency (Long et al., 2015; Lundgren and Fleming, 2020).

Modelling has suggested that improving the conductance of CO2 into chloroplasts would increase photosynthetic assimilation rates due to several mechanisms (Wu et al., 2019). Rubisco efficiency would be improved as higher concentration of available CO2 increases the carboxylation rates of Rubisco, resulting in less photorespiration. With more CO2 available for fixation, the nitrogen cost of synthesising and maintaining photosynthetic machinery is also reduced due to increased efficiency in the system (Evans and Clarke, 2019), allowing for grain protein content to be maintained under higher assimilation rates without additional fertilisation.

An increase in *gm* would also allow higher photosynthetic rates to be achieved without greater water loss through an adjustment of stomatal conductance. At the surface of the leaf, the boundary layer and stomatal properties influence both water vapour transpiration and gas diffusion into the sub-stomatal cavity. Thus, a trade-off exists between preventing water loss through transpiration and maintaining CO2 diffusion to allow for photosynthesis, which is regulated dynamically by stomatal conductance. With higher *g*m, stomatal conductance could be reduced while still maintaining the same chloroplastic CO2 concentration, reducing water losses due to transpiration through the stomata.

The partial pressure of CO2 within the chloroplast is highly variable due to the rate of CO2 assimilation by Rubisco and the total conductance to CO2. Higher assimilation rates consume more CO2, tending to decrease CO2 concentration in the chloroplast, while increases in *gm* tend to increase it. Therefore, the combination of improved *gm* with other complementary photosynthetic improvements will be essential to offset reductions in chloroplast CO2 partial pressure, further enhancing the impact of increased *gm*.

The path of CO2 to the chloroplast is different in C3 and C4 photosynthesis plants, with the specialised anatomy of C4 plants creating a spatial separation that concentrates CO2. This impacts the way in which mesophyll conductance is estimated and interpreted. In this chapter, we will focus on strategies to improve mesophyll conductance in C3 plants only. For a comprehensive discussion of recent advances in mesophyll conductance in C4 (and CAM photosynthesis) plants see Cousins et al. (2020).

On its journey from the atmosphere into chloroplasts in C3 plants, CO2 faces several barriers that impose resistance to diffusion, including the cell wall, plasma membrane, cytosol, chloroplast envelopes and chloroplast stroma (Fig. 1, Clarke et al., 2021). These resistances together determine the overall mesophyll conductance. This book chapter will describe each factor contributing

**Figure 1** A Star Wars-inspired cartoon depicting the CO2 diffusion journey inside the leaf with Princess Leia representing a CO2 molecule (a) and the corresponding electron micrographs illustrating the actual CO2 diffusion pathway within a tobacco leaf (b and c). For photosynthetic CO2 assimilation to occur, CO2 has to diffuse through stomata (entrance), intercellular airspace, cell wall, membranes (plasma membrane and chloroplast envelope) and the liquid phase in the cytosol and chloroplast stroma. Once inside the chloroplast, CO2 is then fixed by Rubisco into energy-rich sugars as part of the photosynthesis process. Bar in *b* = 10 µm, bar in *c* = 1 µm. Adapted from Clarke et al. (2021).

to the resistance to CO2 diffusion within C3 leaves and discuss research efforts to overcome these resistances and improve the CO2 concentration at the site of carboxylation.

## **2 Points of resistance to diffusion of CO2**

The CO2 diffusion path within the leaf and its encompassing sequence of barriers directly determines mesophyll conductance. These barriers to CO2 movement can be described as a series of resistances in the leaf, referred to as mesophyll resistance (*rm*), which is inverse to *gm* (*rm* = 1/*gm*). Although existing experimental methods cannot measure the individual components of *rm* or *gm*, these components can be studied separately using computational models, and understanding each component is critical to understand *gm* as a whole.

Mesophyll resistance is generally divided as follows (Lundgren and Fleming, 2020; Ren et al., 2019):


Gas phase resistance encompasses CO2 movement through intercellular airspaces of the leaf, while liquid phase resistance is made up of the following series of resistances (Cousins et al., 2020; Evans et al., 2009):


Liquid phase resistance accounts for up to 90% of mesophyll resistance to CO2 diffusion through the leaf (Lu et al., 2016; Tosens et al., 2012) and is the primary focus of the following sections which discuss the roles of cell anatomy and biochemical processes in determining *gm*.

#### **3 The interaction between mesophyll cell anatomy, light and** *g<sup>m</sup>*

The constraints on CO2 diffusion within the mesophyll are determined to a large extent by the shape, size and density (packing) of mesophyll cells and the position and orientation of the chloroplasts within, since these factors determine the liquid path length that CO2 must travel from the intercellular air spaces to the chloroplasts. First, the arrangements of cells within the mesophyll in relation to CO2 diffusion and light are discussed. In particular, the 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 of the cells exposed to the intercellular airspace (*Sm*), the surface area of the chloroplasts exposed to the intercellular airspace (Sc ) and the ratio *Sc* /*Sm*. *Sc* /*Sm* expresses the relative fraction of available area that is occupied by chloroplasts through which CO2 can diffuse towards the site of Rubisco.

The most straightforward improvement of *gm* would be through an increase of *Sm* and *Sc* . The presence of many small cells within the mesophyll increases both *Sm* and *Sc* , which has been proposed as a potential target for improving *gm*. Indeed, several studies have shown that this strategy increases *gm*, with a positive effect on photosynthetic capacity. In *Arabidopsis*, increased cell number and packing increased *gm* (Lehmeier et al., 2017) and in rice, leaves with lobed cells had an increased *Sm*, increased *gm* and photosynthetic capacity (Sage and Sage, 2009). 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. Cell shape as well as cell number and packing are therefore both important to consider when improving *gm* and the effects on *Sm* and *Sc* are reviewed below.

Increasing mesophyll cell number and packing does increase *Sm* but cannot be increased indefinitely, as it comes at a higher construction cost (cell wall material) and subsequently smaller leaves (Lundgren and Fleming, 2020; Ren et al., 2019). Increasing cell number and packing would logically increase *Sm*, but there is a trade-off between the number, packing and size of cells, since cell size is negatively related to cell number within a given space. Following the simplified model of Ren et al. (2019), *Sm* is related to the circumference of the cell when considered as a spherical object. If a single large cell has an *Sm* of π, then nine smaller cells within the same space would have an *Sm* of 3π. However, the nine cells have more areas where chloroplasts overlap and this would decrease *Sc* . Therefore, an increase in *Sm* by higher cell density is only beneficial for increasing *gm* if *Sc* is not reduced proportionally due to cell surface overlapping.

*Sc* is also influenced by chloroplast number, movement and distribution in the mesophyll. A larger number of chloroplasts per cell can enhance *Sc* (Miyazawa and Terashima, 2001). But, as for a larger cell number, this cannot be increased without limit. Some species have a ratio of *Sc* to *Sm* (*Sc* /*Sm*) that is far below 1, where there could be benefit in an increased *Sc* . Still, a larger effect is expected from greater *Sm* to allow for further increases of *Sc* . Therefore *Sc* and *Sm* should be increased simultaneously to realise the greatest enhancement of *gm* (Ren et al., 2019). The re-distribution and movement of chloroplasts is also known to change *Sc* (Hanba et al., 2002; Oguchi et al., 2003). Under high light intensity, the chloroplasts can be arranged vertically along the anticlinal cell walls (profile position) to avoid damage by excess light intensity. Under low light intensity, they can move in a perpendicular position (face position) to maximise light capture. The possibility of chloroplasts to move and still face the mesophyll airspace is limited when chloroplast number is high. For example, in rice, the chloroplasts are very tightly packed in the lobed mesophyll cells (Sage and Sage, 2009). However, under high light intensity, tightly packed chloroplasts and limited chloroplast movement can increase the risk of high light damage and may be more disadvantageous than gains in *gm*.

The distribution of light strongly depends on the 3D anatomy and arrangement of mesophyll cells and has an influence on the so-called 'apparent' *g*m: the effective *gm* at leaf level that can vary with light intensity and CO2 concentration, even though the underlying conductance at cell level remains the same (Evans, 2021). This is because within the mesophyll, the assimilation of CO2 is distributed among many chloroplasts. These chloroplasts are all spatially distributed at different depths and consequently experience different light environments depending on the vertical attenuation of light in the mesophyll (Earles et al., 2019). Light attenuation is affected by the colour and intensity of the incoming light as well as the shape of the mesophyll cells. For instance, vertical columnar pallisade cells allow light to penetrate deeper into the leaf (Terashima and Saeki, 1983; Vogelmann and Evans, 2002; Vogelmann and Martin, 1993). Irregularly shaped spongy mesophyll cells and air spaces increase reflection and refraction of light, causing optical path lengthening (Terashima et al., 2009; Vogelmann and Martin, 1993). This is also known as the detour effect and, apart from cell shape, is due to the higher refractive index of cells versus air. Thus, the spatial distribution and shape of cells and chloroplasts influence light attenuation within the mesophyll. This becomes evident at low light where the chloroplasts in the lower layers of mesophyll cells may operate at the light compensation point and do not contribute much to the total *gm*, while at high light, this contribution becomes substantial (Théroux-Rancourt and Gilbert, 2017). Indeed, 'apparent' *gm* has been shown to respond to light intensity at both steady state (Busch et al., 2020; Xiong et al., 2018; Yin et al., 2020) and under dynamic conditions (Carriquí et al., 2019; Liu et al., 2022; Sakoda et al., 2021). So although cell wall and membrane properties like *Sm* and *Sc* are important, the 3D structure and arrangement of mesophyll cells determine to a large extent how the 'apparent' *gm* reacts to environmental changes such as light.

#### **4 Leaf age and** *g<sup>m</sup>*

An example of the link between leaf anatomy and *gm* can be observed in leaves of different age and canopy position. Several studies have examined the modulation of *gm* by the mesophyll cell architecture in relation to leaf age, canopy position and light intensity. During the development of a leaf, it has been shown that *gm* can limit photosynthesis considerably (Carriquí et al., 2021; Clarke et al., 2021; Hanba et al., 2001; Hoshika et al., 2020; Marchi et al., 2008; Miyazawa et al., 2003; Miyazawa and Terashima, 2001; Niinemets et al., 2012; Tosens et al., 2012). In most species, *gm* increases with leaf expansion and reaches a maximum with full expansion and then declines with leaf age (Loreto et al., 1994; Niinemets et al., 2012). The increase in *gm* during expansion is largely due to the growth and expansion of mesophyll cells and the increase of leaf thickness both of which increase *Sm* and *Sc* . The subsequent decline in *gm* after expansion has been attributed to the increase in cell wall thickness (Miyazawa et al., 2003; Miyazawa and Terashima, 2001; Sugiura et al., 2020; Tosens et al., 2012) and reduction of cell wall effective porosity (Niinemets et al., 2012). In tobacco, *gm* reductions with leaf age were accompanied by both cell wall thickening and a decrease in *Sc* /*Sm* (Clarke et al., 2021).

Another observed effect of leaf age was that of reduction of the thickness of chloroplasts. The thickness of the chloroplast determines the diffusion path length of CO2 inside the chloroplast stroma and, in theory, can influence the conductance of CO2 in the liquid phase; an important component of *gm*. A decrease in chloroplast stroma thickness with leaf age has been observed in *Arabidopsis* (Carriquí et al., 2021), while other studies have not seen a decrease with age (Clarke et al., 2021) or have not found a correlation between chloroplast stroma thickness and *gm* (Tosens et al., 2012). It is noteworthy that chloroplast stroma thickness was negatively affected by the growth light intensity in some species (Tosens et al., 2012) but not in others (Oguchi et al., 2003; Vidal et al., 1990). Still, chloroplast stroma thickness can potentially be an important factor for *gm*. This was best illustrated in a study on transgenic tobacco with reduced Rubisco content that had lower chloroplast stroma thickness compared with the wild type and was accompanied by a lower CO2 drawdown (Evans et al., 1994).

The observed changes in *gm* with leaf age highlight that an increase in *gm* can be realised by:


However, this will need to be accomplished at a cell density that allows maximal exposure of cell walls to the intercellular airspace. In that regard, 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 'apparent *gm*' (Fig. 2).

The effects of leaf age on *gm* illustrate that mesophyll anatomy influences *gm* to a large extent and that it can be exploited. There is a variation in the extent to which these leaf age effects are observed not only between species, but also in response to growth light intensity. Especially in canopies with steep light gradients, the effect of light intensity may potentially be stronger than that of leaf age but has not been extensively studied in relation to *gm* in C3 plants.

**Figure 2** A diagram of mesophyll tissue shown in cross section through the leaf. The arrangement of mesophyll cells can be more closely packed (high cell density) or more loosely packed (low cell density), influencing both the gas diffusion pathways (red lines) and light distribution throughout the mesophyll (black lines).

#### **5 Cell wall diffusion**

Diffusion through the cell wall is the first step in the liquid phase of CO<sup>2</sup> diffusion through the leaf. Cell walls are structurally complex barriers that function to protect plants from biotic and abiotic stresses, maintain structural integrity and support cell division and plant growth (Cosgrove, 2005; Houston et al., 2016). 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. Several model studies of *g*m have suggested that the cell wall is one of the main constraints on mesophyll conductance (Evans et al., 1994; Gago et al., 2020; Terashima et al., 2011; Tomas et al., 2013; Xiao and Zhu, 2017; Yin and Struik, 2017).

The path through the cell wall is complicated, and the overall resistance depends on the number of channels or pores, the effective length of each channel and the rate of diffusion along each channel (see Fig. 3 in Evans et al., 2009). These considerations can be expressed mathematically using four components: cell wall thickness, tortuosity of the path through cell wall pores, cell wall porosity and diffusivity of CO2 through the apoplastic fluid (Evans, 2021). Although one cannot definitively calculate cell wall resistance and each of its components, it can be estimated with the help of plant models. The impact of cell wall thickness on *g*m is relatively well studied being that it is the only component affecting cell wall conductance to CO2 that can be easily and reliably measured. Cell wall thickness has been measured in hundreds of species using transmission electron microscopy (Gago et al., 2020). Several studies have reported that cell wall thickness is negatively correlated with mesophyll conductance, suggesting selection or genetic modification for thinner cell walls could be a good target to enhance mesophyll conductance (Clarke et al., 2021; Onoda et al., 2017; Ren et al., 2019; Terashima et al., 2011; Tosens et al., 2012). However, one must consider how thin a mesophyll cell wall can be made without jeopardising its structural integrity (Cosgrove, 2018). 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 (Lundgren and Fleming, 2020). Additionally, the lack of data on the regulation of cell wall thickness and genes that may be involved make targeted changes quite challenging. Moreover, much uncertainty exists with the other three components of cell wall resistance partly due to the fact that there are currently no good methods available to experimentally measure these parameters (Evans, 2021).

The composition of the cell wall is another element which may influence *g*m by modifying CO2 diffusion. Primary cell walls are composed of a complex carbohydrate matrix of cellulose, hemicellulose and pectin (Cosgrove and Jarvis, 2012; Cosgrove, 2005; Tenhaken, 2015). It is not difficult to envision that altering the absolute amounts or ratios of these components will affect CO2 diffusion through the cell wall. These changes could potentially modify cell wall thickness or alter porosity and the liquid path length through the cell wall (Ellsworth et al., 2018). However, how changes in cell wall composition affect tortuosity and porosity remains relatively unexplored (Carriquí et al., 2020). Pectins are known to have a role in regulating cell wall porosity but little is known about their direct effect on CO2 diffusion (Fleischer et al., 1999; Rondeau-Mouro et al., 2008). The ability to directly measure these parameters is essential in order to answer these questions.

A small number of recently published studies have started to examine the relationships between cell wall composition and mesophyll conductance using cell wall mutants, stress treatments and interspecific variation. Ellsworth et al. (2018) observed that rice mutants unable to synthesise mixed-linkage glucans (a cell wall hemicellulose) had reduced photosynthetic rates, *g*m, *S*<sup>c</sup> and leaf mass per area. In addition, the authors calculated the effective porosity of the mesophyll cell walls, which was estimated to be significantly reduced in the mutants relative to the control. From this it was concluded that mixed-linkage glucans in cell walls improve effective porosity and CO2 diffusivity in rice (Ellsworth et al., 2018). Another study looking at pectin methylesterase and pectin acetylesterase mutants in Arabidopsis reported reductions in CO2 assimilation and *g*m associated with reduced galacturonic acid content (the main component of pectin; Roig-Oliver et al., 2021). Two other studies looking at water stress in grapevines and tobacco found that decreases in photosynthesis and *g*m were associated with changes in cellulose and pectin content in the cell wall (Clemente-Moreno et al., 2019; Roig Oliver et al., 2019). Furthermore, to assess whether cell wall composition plays a role in existing differences in *g*m between species from common environments, Carriquí et al. (2020) measured cell wall composition, CO<sup>2</sup> assimilation and *g*<sup>m</sup> in seven gymnosperm species. The study found *g*m to be correlated with cell wall composition, likely signifying a change in the diffusivity of the cell wall. The authors conclude that higher ratios of pectin to hemicellulose and/or pectin to cellulose was correlated with increased *g*m, suggesting pectin has a role in regulating CO2 diffusivity in the cell wall of gymnosperms.

These novel studies have started to provide critical information about the relationship between *gm* and cell wall properties. However, additional work is needed to explore new variations on cell wall composition such as increasing or decreasing the amounts of individual primary cell wall components. Analyses comparing species in other land plant groups besides gymnosperms will also be helpful since differences in main cell wall components exist between plant types (Popper et al., 2011). The information gained from such studies can be used to directly target changes in cell wall composition through genetic engineering or selection. Based off our current knowledge, we propose some potential targets for modification with the goals of gleaning a better understanding of and enhancing CO2 diffusivity across the cell wall:


Overall, altering cell wall properties such as composition and thickness is a promising route for reducing CO2 diffusion resistance across the cell wall and increasing mesophyll conductance. However, true engineering remains elusive because many fundamental questions about the genetic control of cell wall properties and the relationship between those properties and *gm* remain unanswered. Future exploratory work, as well as the development of new measurement techniques for measuring diffusion and relevant cell wall parameters, will be crucial for successful genetic engineering of mesophyll conductance.

After crossing the mesophyll cell wall, a series of liquid phase resistances remains between CO2 and the site of carboxylation. These are the plasma membrane, cell cytosol, chloroplast envelopes and the liquid stroma inside the chloroplasts. The resistance imposed by the membrane components (plasma and chloroplast envelope) are discussed together, followed by liquid cytosol and stroma components.

#### **6 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, and initial studies on membrane permeability in artificial lecithincholesterol bilayers concluded that biological lipid membranes offered little to no resistance to CO2 (Gutknecht et al., 1977). 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 (Reszczyńska and Hanaka, 2020).

Alteration of lipid content in plant membranes could be a target for improving *gm*; however, as the membranes are very protein-dense, the available free lipid membrane for CO2 diffusion is likely quite limited. The lipid and protein composition of membranes can nevertheless be significantly remodelled in response to environmental cues, suggesting optimisation of this process could potentially improve overall *gm* (Uemura et al., 1995).

Cellular membranes impose up to 50% of the resistance to CO2 diffusion within mesophyll cells (Endeward et al., 2017; Evans, 2021). 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 (Evans, 2021). While some argue the Meyer–Overton rule (also known as the solubility-diffusion model) accounts for all gaseous CO2 transfer across membranes (Missner and Pohl, 2009), 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.

## **7 Improving** *gm* **using aquaporins as CO2 channels**

Aquaporins are a prominent gene family involved in CO2 transfer across membranes. While initially named for their water transfer capabilities, further studies have reported aquaporins to be multifunctional proteins, capable of facilitating the transfer of various solutes, ions and gases, including CO2 (Groszmann et al., 2017). The functional aquaporin unit is composed of four monomer channels coming together to form a tetramer, which creates a fifth central pore. Solute specificity is influenced by individual monomer properties and their combinations in the tetramer (Otto et al., 2010). Aquaporins are dynamic proteins that can move in and out of the membrane in response to environmental stimuli, and the pores within the tetramer can also be gated, allowing close regulation of channel activity (see Groszmann et al., 2017 for a detailed discussion of plant aquaporin structure, function and regulation).

Early work in mammalian systems first identified aquaporin proteins as CO2 channels. The human red blood cell aquaporin, AQP1, when expressed in *Xenopus* oocytes, increased membrane permeability to CO2 (Cooper and Boron, 1998; Nakhoul et al., 1998). In plants, members of the PIP aquaporin family are homologous to mammalian AQP1, and generally localise to the plasma membrane, with some isoforms also detected in chloroplast envelopes by Western blot and proteomic analysis (Beebo et al., 2013; Uehlein et al., 2008).

Expression of a tobacco AQP1 homologue, NtAQP1 (also known as NtPIP1;5s, De Rosa et al., 2020), in oocytes also conferred increased CO2 permeability (Uehlein et al., 2003). A function in mesophyll conductance was predicted and *in vivo* ectopic or antisense expression study of NtAQP1 in tobacco altered mesophyll conductance, with ectopic expression increasing *gm* by up to 20% (Flexas et al., 2006). Additional studies targeting aquaporins from other species and across environmental conditions have had mixed effects on mesophyll conductance, with some studies reporting effects on *gm* (Ermakova et al., 2021; Hanba et al., 2004; Heckwolf et al., 2011; Mori et al., 2014; Uehlein et al., 2008; Xu et al., 2019), while in others studies, no effects were observed (Clarke et al., 2022; Kromdijk et al., 2020).

Questions remain about why manipulation of aquaporins does not produce consistent effects on mesophyll conductance, and several factors have been suggested that may be impacting the *gm* phenotype in these studies. The underlying *gm* capacity of a species may affect the ability to detect improvements in *gm*. Many crop species already have relatively high *gm*, driven in part by their thin cell walls (see 'Cell Walls' above). Modelling has suggested that when basal *gm* is higher, increases will be subtle and harder to detect (Clarke et al., 2022). A study in tomato reported that ectopic expression of *NtAPQ1* did not increase *gm* until basal *gm* levels had been sufficiently reduced through simultaneous ectopic expression of hexokinase (Kelly et al., 2014). Recent studies have also highlighted the dynamic effects of environmental factors on *gm*. Conditions such as photoperiod, light intensity, day/night temperature, water availability, humidity and nutrient supply can all alter anatomical and biochemical components that influence *gm.* The conditional effects of growth environment and growth stage between aquaporin lossof-function mutants and *gm* have been reported in rice (Huang et al., 2021), and in tomato, an aquaporin knockout mutant was only shown to reduce *g*<sup>m</sup> when mutants were grown under high CO2 levels (Zhang et al., 2021). Further studies to better understand these complex relationships will be essential to ensure manipulation of aquaporins can provide a consistent impact on *gm*, and aquaporins remain a strong engineering target for enhancing *gm*.

To further efforts to improve the membrane conductivity to CO2, it may also be beneficial to look beyond aquaporins to find candidates from other systems where CO2 transfer across membranes occurs. Red blood cells (RBCs), for example, require the ability to rapidly uptake CO2 from the blood stream, and indeed AQP1 is present on these membranes (see Michenkova et al., 2021 for a comprehensive review). However, another protein, the Rhesus-AG protein (part of the Rhesus complex) is also present on the RBC membrane and also acts as a CO2 channel, contributing half of CO2 uptake in these cells (Endeward et al., 2008; Ripoche et al., 2006). Plants do not contain Rhesus proteins but do contain the somewhat homologous AmtB protein which is an ammonium channel (Marini et al., 1997). Rhesus and AmtB proteins are bidirectional channels, moving CO2 or NH3 respectively along a concentration gradient. Expression of a mammalian protein such as Rhesus-AG into plant membranes could be a strong target for increasing mesophyll conductance if the protein can be assembled and targeted correctly. No studies have reported success in expressing the related mammalian AQP1 *in planta* however, so this may not be a feasible approach. Identification of further CO2 channels or transporters from plants or more closely related non-plant systems would provide new targets for engineering improved *gm*. Expression of genes from non-plant photosynthesising organisms, such as green or red algae, may have more compatibility. Beyond CO2 channels, several bicarbonate transporters have been identified in plants that may also support increased *gm* if targeted correctly (Poschenrieder et al., 2018).

## **8 CO2 solubility in liquids**

On its journey to Rubisco, CO2 must transverse two liquid compartments:


CO2 gas can dissolve into liquids, but it can also be reversibly converted to bicarbonate which has a much higher solubility (Badger and Price, 1994). This reaction is catalysed by the enzyme carbonic anhydrase (CA), making the interconversion almost instantaneous. CAs are the second most abundant enzyme in plants (after Rubisco) and most of this activity is thought to support the diffusion of CO2 through the cytosol and chloroplast stroma (Ogée et al., 2018). In plants, CAs have three forms, α, β and γ, that differ in their activity, expression and localisation patterns (Momayyezi et al., 2020). β-CA is the dominant isoform in plants and is found either free in the cytosol and stroma (Fabre et al., 2007) or bound to organelle membranes via interaction with aquaporin proteins (Wang et al., 2016).

The localisation of CA within the cell is critical for facilitating carbon concentrating mechanisms such as cyanobacterial carboxysomes or in C4 photosynthesis, where CA localisation determines what form of CO2 exists within each compartment. Here we discuss the role of CA in C3 plants with respect to its impact on mesophyll conductance, and how it may be engineered for improvement.

## **9 Improving** *gm* **with carbonic anhydrases**

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% (Tholen and Zhu, 2011). Experimental work using anti-sense CAs confirmed carbon discrimination is altered (Price et al., 1994; Williams et al., 1996), or that photosynthesis was reduced (Sasaki et al., 1998), though *gm* itself was not altered. More recent studies have shown evidence that manipulation of CA can alter *gm* (Gillon and Yakir, 2001; Perez-Martin et al., 2014). With many forms of CA present within plants, it is possible that functional redundancy is compensating for CA knock-down effects, and more research is needed to determine what levels of CA are optimal in various compartments.

A key development in our understanding of the role CAs have in mesophyll conductance has been the discovery of CA interaction with aquaporin proteins (Perez-Martin et al., 2014; Wang et al., 2016). 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 that ensures their interaction remains constant and optimised. A similar metabolon of a CA coupled to a bicarbonate transporter at either the apoplast/plasma membrane boundary or chloroplast envelope/stroma boundary could also improve CO2 delivery through the mesophyll cell to the stroma. Overall, more research is needed to better understand the relationships between CA isoforms, localisation and mesophyll conductance to help guide targeted improvements in CO2 diffusion within mesophyll cells.

#### **10 Estimating** *g<sup>m</sup>*

Although mesophyll conductance cannot be measured directly, a handful of techniques have been developed to indirectly estimate *g*m and thorough reviews are available (Cousins et al., 2020; Pons et al., 2009). Two techniques have proven to be most reliable and are based on gas exchange measurements either (i) in conjunction with carbon isotope discrimination (Δ13C); or (ii) in combination with chlorophyll fluorescence (Evans et al., 1986; Harley et al., 1992).

Carbon isotope discrimination methods are based on measuring the 13C/12C ratio of CO2 remaining after photosynthetic CO2 fixation. The heavier isotope of carbon, 13C is discriminated against during diffusion through the leaf and the biochemical processes of photosynthesis (Farquhar et al., 1982). This is because 13CO2 has lower diffusivity in air and liquids relative to the lighter isotope (12CO2) and the carboxylating enzyme Rubisco preferentially binds 12CO2. Correspondingly, photosynthetic products are enriched in 12C, while gas flowing over a photosynthesizing leaf is enriched in 13C and exhibits an increased Δ13C compared to the ambient air. Carbon isotope discrimination has generally been measured one of two ways: by the cryogenic trapping of CO2 and subsequent measurement of Δ13C using isotope ratio mass spectrometry (Evans et al., 1986) or by using tunable diode laser (TDL) absorption spectrometry (Barbour et al., 2007) which can obtain simultaneous measurements of carbon isotope discrimination and gas exchange.

Estimating mesophyll conductance from gas exchange combined with chlorophyll fluorescence relies on the modelled relationship between CO<sup>2</sup> assimilation (*A*), the electron transport rate (*J*) and the concentration of CO2 at Rubisco (*C*<sup>c</sup> ; FvCB model Farquhar et al., 1980). Two main modelling methods are used to estimate *g*m: the constant *J* method and variable *J* method (Harley et al., 1992). In the constant *J* method, gas exchange measurements are made across a range of *C*<sup>i</sup> values where *J* is assumed to be constant. In this method, fluorescence measurements are used solely to verify the assumption that *J* is constant under the measured range. Alternatively, the variable *J* method uses chlorophyll fluorescence to estimate *J* and then *g*m (Harley et al., 1992). This method can be applied for any value of *C*<sup>i</sup> , even when *J* is not constant.

Each of the aforementioned methods relies on different models and assumptions for the calculation of *g*m and has corresponding limitations. They are all considered suitable for the determination of *g*m values; however, several factors can and should be considered when determining which method is best suited for the planned experiment (Table 1). It is also common for values of *g*m to vary depending on the estimation method applied, with the Δ13C method often yielding higher estimates then chlorophyll fluorescence methods (Kromdijk et al., 2020). When possible, more than one method should be used to estimate *g*m to increase confidence in the results due to uncertainties associated with each method.


#### **11 Strategies for altering** *g<sup>m</sup>*

Strategies aimed at understanding and improving crop performance have changed over the years. During the Green Revolution, yields were increased by optimising light interception and carbon partitioning; however, these traditional methods for crop improvements are reaching a plateau, highlighting the need for novel approaches to ensure food production can keep up with the growing population (Ort et al., 2015). One promising target is the process of photosynthesis, which has not been fully optimised in food crops. Here, we discuss the use of selective breeding and synthetic biology via genetic engineering as possible approaches to enhancing photosynthesis by increasing mesophyll conductance.

Selective breeding has successfully been used in agriculture to produce crops with desirable traits, such as disease resistance and larger grains, fruits and vegetables. For artificial selection of a trait to be successful, the trait must exhibit genetic variation and that variation must be heritable. Several studies have been published looking at existing interspecific and intraspecific natural variation of mesophyll conductance (Carriquí et al., 2015, 2020; Faralli and Lawson, 2020). A summary of published studies reveals significant variation of mesophyll conductance in some of the world's most important crops, where the measured values vary by as much as a factor of ten within a species (Table 2). These studies indicate a useful source of unexploited genetic diversity that could be used for selective breeding to improve *gm* and potentially the photosynthetic efficiency of these crops.

Synthetic biology has also been used successfully in agriculture to produce disease-resistant and nutrient-enriched crops (Kumar et al., 2020). Genetic engineering of plants via the introduction of one or multiple transgenes can be used to express genes from other plants or organisms that may facilitate enhancement of mesophyll conductance, whether it is through altered cell morphology, cell wall structure, membrane composition or other biochemical processes. This strategy can also be used to stack multiple favourable traits



\*Values were converted to units of mol m−2 s−1 assuming pressure of 1 standard atmosphere.

together, for example, *gm* traits with other photosynthetic traits. Additionally genome-editing technology such as CRISPR can be used to knockdown, knockout or upregulate target genes of interest within the plant genome. Photosynthetic improvements via genetic engineering are a promising avenue that is currently underutilised partially due to insufficient knowledge and understanding about several aspects of mesophyll conductance.

At present, there are relatively few studies that have used genetic engineering approaches to alter mesophyll conductance. Studies utilizing RNAi technology to knockdown carbonic anhydrase activity in tobacco have shown little effect on mesophyll conductance to CO2 (Price et al., 1994; Williams et al., 1996). Increases in mesophyll conductance and net photosynthesis were observed in rice through combined ectopic expression of cyanobacteria ICTB, a possible bicarbonate transporter and FBP/Sbpase, which plays an important role in ribulose-1,5-bisphosphate (RuBP) regeneration (Gong et al., 2015). This study illustrates the potential utility of using multigene constructs to engineer improved mesophyll conductance. Most success with engineering enhanced *gm* has been limited to changes in aquaporin expression. Transgenic overexpression of tobacco aquaporin 1 (NtAQP1) increased mesophyll conductance along with photosynthetic rates and CO2 concentrations in the chloroplasts of tobacco plants (Flexas et al., 2006), while RNAi knockdown of NtAQP1 decreased photosynthesis and CO2 conductance within tobacco leaves (Uehlein et al., 2008). Ectopic expression of the barley aquaporin PIP2;1 and overexpression of the rice aquaporin PIP1;2 both resulted in increased mesophyll conductance in rice, with the latter also increasing plant biomass and grain yield (Hanba et al., 2004; Xu et al., 2019). Together these studies highlight the importance of aquaporins as key targets for improving CO2 conductance within the leaf. However, in some species, stacking aquaporin overexpression with other CO2 diffusion traits will be necessary to see measurable differences in *gm*.

#### **12 Conclusion and future trends**

Mesophyll conductance to CO2 is influenced by a complex combination of anatomical and biochemical traits within leaves. Engineering improvements to mesophyll conductance presents an appealing target for boosting photosynthesis, especially in limited water environments where improved CO2 supply to Rubisco would allow stomatal conductance (and therefore water loss) to be reduced without a photosynthetic penalty. The stacking of improved *gm* traits with improvements to Rubisco or RUBP regeneration efficiency should also deliver further enhancements to photosynthesis as CO2 concentration at the site of fixation becomes limiting. Manipulation of individual resistances to CO<sup>2</sup> within leaves and cells can also be stacked together for further improvements to the overall mesophyll conductance.

In order to deliver this, further comprehensive studies are needed to facilitate engineering enhanced mesophyll conductance and photosynthetic efficiency, such as large-scale mutant screens to identify suitable genes for additional study and manipulation. Specifically, improvements in the fundamental knowledge of genetic controls in the following areas are required:


## **13 Where to look for further information**

Further useful information about mesophyll conductance and efforts to improve it to benefit plant productivity can be found in the following resources:


## **14 Acknowledgements**

This work was supported by a sub-award from the University of Illinois as part of the Realizing Increased Photosynthetic Efficiency (RIPE) project, funded by the Bill & Melinda Gates Foundation to V. C. C. and C. E.-S. S.

## **15 Author contributions**

C. E.-S. S., S. M. D. and V. C. C. all wrote and edited the manuscript and are equal co-authors.

## **16 References**

Badger, M. R. and Price, G. D. 1994. The role of carbonic anhydrase in photosynthesis. *Annual Review of Plant Physiology and Plant Molecular Biology* 45(1), 369–392.

Bailey-Serres, J., Parker, J. E., Ainsworth, E. A., Oldroyd, G. E. D. and Schroeder, J. I. 2019. Genetic strategies for improving crop yields. *Nature* 575(7781), 109–118.


Carriquí, M., Nadal, M., Clemente-Moreno, M. J., Gago, J., Miedes, E. and Flexas, J. 2020. Cell wall composition strongly influences mesophyll conductance in gymnosperms. *The Plant Journal: For Cell and Molecular Biology* 103(4), 1372–1385.

