Introduction
C4 photosynthesis has evolved independently in multiple
grass lineages (Grass Phylogeny workshop 2012) thus leading to
remarkable structural, anatomical and physiological trait diversity
(Christin et al. , 2013). Studies suggest that this trait
diversity among the C4 species could be attributed to
their adaptation to different environmental variables like temperature,
fire frequency and precipitation (Edwards & Smith, 2010; Visseret al. , 2012; Zhou et al. , 2018). In general,
C4 species mostly occupy the lower latitudes where light
availability and temperature likely do not strongly limit photosynthesis
and growth (Pearcy & Ehleringer, 1984). Instead, precipitation may be
an important factor affecting trait diversity in C4species; particularly, in traits associated with photosynthetic C-gain
and transpirational water-loss (Edwards & Still, 2008; Osborne & Sack,
2012; Zhou et al. , 2018). During the adaption to habitats with
low water availability, a fundamental challenge for plants will be to
maintain photosynthetic C-gain while minimizing transpirational
water-loss associated with high evaporative demand. This tradeoff could
be achieved partly through coordination of leaf-level photosynthetic and
hydraulic traits (Brodribb et al. , 2007; Nardini & Luglio, 2014;
de Boer et al. , 2016). However, the extent of variation and
coordination among these traits, particularly those associated with
internal CO2-diffusion conductance (gm)
and leaf hydraulic conductance (Kleaf), has not been
well studied in C4 species adapted to habitats with
varying water availabilities (Osborne & Sack, 2012; Liu & Osborne,
2015; Taylor et al. , 2018). Although this type of trait variation
and coordination has been studied in C3 plants there
could be significant differences in C4 plants due to
their unique anatomy and physiology (Kocacinar & Sage, 2003; Osborne &
Sack, 2012; Ocheltree et al. , 2016; Zhou et al. , 2018).
During adaptation to drier habitats, species can exhibit several
leaf-level structural and anatomical traits that can help maximize
photosynthetic C-gain at a given water-loss (Wright et al. , 2001;
Galmés et al. , 2012; Ivanova et al. , 2018b). For instance,
presence of greater stomatal densities and amphistomaty (stomata on both
leaf surfaces) in drier habitats, is beneficial as it reduces the role
of boundary layer conductance in constraining leaf gas exchange, helps
optimize leaf interior water status for CO2 transport by
reducing temperature gradients, and helps reduce the effective leaf
thickness by decreasing the CO2-diffusion pathlength
(Galmés et al. , 2012; Muir, 2018; Drake et al. , 2019;
Muir, 2019; Pathare et al. , 2020). Greater stomatal densities and
smaller stomatal size in drier habitats may increase theoretical maximum
stomatal conductance (gmax) which in turn could help
plants maximize C-gain particularly during intermittent periods of water
availability (Hetherington & Woodward, 2003; Franks & Beerling, 2009).
Furthermore, mesophyll traits like Smes and
Sc - the parameters that characterize exchange surfaces
for CO2, negatively correlated with water availability
in the C3 species of European steppe plant communities
and were suggested as indicators of increasingly drought adapted steppe
plants (Ivanova et al. , 2018a; Ivanova et al. , 2018b).
These structural and anatomical adaptations could help maximize internal
CO2-diffusion conductance (gm), at a
given stomatal conductance (gsw), thus leading to
greater photosynthetic rates (Anet) as well as
leaf-level water-use efficiency (WUE) in species adapted to drier
habitats (Flexas et al. , 2008; Flexas et al. , 2013;
Ivanova et al. , 2018a; Ivanova et al. , 2018b). However,
very few studies, mostly based on C3 species, have
investigated the leaf-level structural and anatomical traits associated
with gm that could be a characteristic of plant
adaptation to drier habitats (Ivanova et al. , 2018a; Ivanovaet al. , 2018b). Alternatively, even though C4species can successfully occupy drier and warmer habitats and form
grasslands over vast areas globally, there is a little information about
leaf-level structural and anatomical traits that influence
photosynthetic C-gain and water-use in these species. Specifically, we
are unaware of any studies that have investigated the relationship of
gm and associated anatomical traits in diverse
C4 species from habitats with different water
availability. In a previous study (Pathare et al. , 2020) we
investigated the structural and anatomical determinants of
gm in diverse C4 grasses and found that,
leaf thickness, adaxial stomatal densities (SDada),
stomatal ratio (SR) and Smes had a positive effect on
gm. In the current study, our aim is to determine if the
variation in above traits among the C4 species could be
related to adaptation to habitats with different water
availabilities.
Our first hypothesis (H1) is that,
C4 grasses adapted to lower MAP will show leaf
anatomical traits associated with
greater gm in order to maximize photosynthetic C-gain.
Though we hypothesized a greater gm in
C4 grasses adapted to low MAP (H1), one would expect an
increase in water cost relative to photosynthetic C-gain, because
gm and associated traits have been shown to scale
positively with leaf hydraulic conductance (Kleaf) in
C3 species (Flexas et al. , 2013; Xiong et
al. , 2015; Xiong et al. , 2017; Drake et al. , 2019).
Kleaf is an important trait associated with leaf water
transport and represents the conductance to flow of water from the leaf
petiole through the xylem, then through the bundle sheath and finally
through the mesophyll to the site of evaporation (Sack & Holbrook,
2006; Noblin et al. , 2008; Buckley, 2015; Buckley et al. ,
2015). Though Kleaf is partitioned between the xylem
(Kx) and the outside xylem pathways
(Kox), changes to Kox are expected to
have the largest effects on Kleaf (Buckley et
al. , 2015; Scoffoni et al. , 2017; Xiong & Nadal, 2019).
Consequently, leaf-level anatomical traits that influence
Kox such as leaf thickness, vein-to-epidermis distance
(VED), vein length per unit of leaf area (VLA) and bundle sheath and
mesophyll traits are expected to have a significant effect on
Kleaf (Griffiths et al. , 2013; Sack et
al. , 2013; Buckley et al. , 2015). For instance, greater leaf
thickness and VED, if associated with low VLA, may increase the length
of post-venous water path thus leading to lower Kleaf.
Alternatively, greater VLA in thinner leaves may increase
Kleaf by providing additional parallel flow paths
through the vein system and decreasing the horizontal pathlength from
veins to sites of evaporation (Brodribb et al. , 2007; Sack &
Scoffoni, 2013; Buckley et al. , 2015; Drake et al. , 2019).
Furthermore, greater bundle sheath (BS) surface area ratio, lower BS
cell wall thickness (BSCW) and BS exposed to
intercellular air spaces (BSias) and greater
Smes may all enhance Kleaf (Buckleyet al. , 2015; Caringella et al. , 2015; Xiong et
al. , 2017; Scoffoni et al. , 2018). Investigating the
coordination of above traits related to water-use with traits related to
C-gain will provide insights into the water cost associated with
photosynthetic C-gain in C4 species adapted to habitats
with varying water availabilities.
Previous studies on C3 species have shown a strong
positive linkage of Kleaf with gsw and
hence Anet (Brodribb et al. , 2007; Brodribb &
Feild, 2010; Flexas et al. , 2013; Scoffoni et al. , 2016).
Additionally, the few studies that address the coordination of
Kleaf and gm show that these two traits
scale positively with each other in C3 species as they
share some structural and anatomical traits that form the mechanistic
basis for their coordination independent of gsw (Flexaset al. , 2013; Xiong et al. , 2015; Xiong et al. ,
2017) but see (Théroux-Rancourt et al. , 2014; Loucos et
al. , 2017; Wang et al. , 2018). For example, Smespositively correlates with both gm and
Kleaf (Flexas et al. , 2013; Xiong et al. ,
2015; Xiong et al. , 2017) , since greater Smesincreases the number of parallel pathways for
CO2-diffusion inside mesophyll cells (Evans et
al. , 2009) as well as the evaporating surface area for water thus
increasing gm and Kleaf respectively
(Sack & Scoffoni, 2013; Xiong et al. , 2017). In summary, the
positive correlation of Kleaf with gmimplies a greater water cost associated with greater C-gain, which could
be detrimental in drier conditions where using water efficiently will be
important for plant growth and fitness. Hence, a safer strategy for
plants is to maintain lower Kleaf in drier conditions at
the cost of Anet and growth rates (Sinclair et
al. , 2008; Nardini & Luglio, 2014; Scoffoni et al. , 2016).
However, these generalizations are mostly based on studies of
C3 species. It is unclear if these results would apply
to C4 grasses that are adapted to relatively drier
habitats and may show different coordination between the traits
associated with photosynthetic C-gain and transpirational water loss
(Kocacinar & Sage, 2003; Ocheltree et al. , 2016; Zhou et
al. , 2018). Increased bundle sheath size and vein densities are the
anatomical precursors for evolution of C4 species from
their C3 ancestors that led to higher
Kleaf in the C4 species compared to
C3 species. (Osborne & Sack, 2012; Christin et
al. , 2013; Griffiths et al. , 2013). At the same time, evolution
of carbon concentrating mechanism in C4 species allows
maintenance of lower gs and higher leaf water potential.
Hence, it has been proposed that once the C4 species
evolved, subsequent selection for traits leading to greater
Kleaf would be lessened particularly during adaptation
to drier habitats and there could be a decoupling between
Kleaf and Anet within the
C4 lineages (Zhou et al. , 2018). Consequently, in
contrast to C3 species, maintaining greater
Kleaf in order to achieve higher Anetmay not be necessary in C4 species and
Kleaf may be uncoupled from gsw,
Anet (Kocacinar & Sage, 2003; Ocheltree et al. ,
2016) and potentially gm. However, to our knowledge,
there have been no previous studies on the correlation of
gm with Kleaf or traits associated with
Kleaf in C4 species adapted to habitats
with diverse MAP. Building knowledge upon previous evidences, we
hypothesized (H2) that C4 species adapted to habitats
with low MAP will show traits associated with lower
Kleaf that will maximize photosynthetic C-gain at a
given water loss.
To test the above hypotheses, we selected 18 C4 grasses
that varied significantly in structural and anatomical traits (Pathareet al. , 2020). The grasses were grown under common growth
conditions and abundant water and nutrient supply which avoids the
influence of environmental conditions on traits and thus helps identify
the differences that could be a result of species adaptation to their
habitat of evolution or common occurrence (Reich et al. , 2003).
We measured important leaf-level structural and anatomical traits
associated with photosynthetic C-gain and gm and
transpirational water-loss and Kleaf in 18 diverse
C4 grasses. There is a significant knowledge gap about
how C4-gm variability relates with
habitat climate variables like MAP largely because of the lack of
techniques to estimate C4-gm in field as
well as laboratory conditions. However, the recent developments provide
the opportunity to estimate C4-gm under
laboratory conditions and thus investigate the relationship of habitat
climate variables with gm in diverse C4species. Here, we use a recently developed method, based on modeling of
leaf oxygen isotope discrimination during photosynthesis, to estimate
gm in 18 diverse C4 grasses (Barbouret al. , 2016; Ubierna et al. , 2017; Ogee et al. ,
2018). Kleaf was estimated from anatomical traits like
leaf thickness, vein-to-epidermis distance (VED) and vein-to-vein
distance (IVD) as recently described by de Boer et al ., (2016)
using the semi-empirical model of Brodribb et al ., (2007).