Volume 233, Issue 2 p. 851-861
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The three-dimensional construction of leaves is coordinated with water use efficiency in conifers

Santiago Trueba

Corresponding Author

Santiago Trueba

School of the Environment, Yale University, New Haven, CT, 06511 USA

University of Bordeaux, INRAE, UMR BIOGECO, Pessac, 33615 France

Author for correspondence:

Santiago Trueba

Email:[email protected];

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Guillaume Théroux-Rancourt

Guillaume Théroux-Rancourt

University of Natural Resources and Life Sciences, Vienna, Department of Integrative Biology and Biodiversity Research, Institute of Botany, Vienna, 1180 Austria

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J. Mason Earles

J. Mason Earles

Department of Viticulture and Enology, University of California, Davis, CA, 95616 USA

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Thomas N. Buckley

Thomas N. Buckley

Department of Plant Sciences, University of California, Davis, CA, 95916 USA

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David Love

David Love

Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA, 30602 USA

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Daniel M. Johnson

Daniel M. Johnson

Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA, 30602 USA

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Craig Brodersen

Craig Brodersen

School of the Environment, Yale University, New Haven, CT, 06511 USA

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First published: 06 October 2021
Citations: 4

Summary

  • Conifers prevail in the canopies of many terrestrial biomes, holding a great ecological and economic importance globally. Current increases in temperature and aridity are imposing high transpirational demands and resulting in conifer mortality. Therefore, identifying leaf structural determinants of water use efficiency is essential for predicting physiological impacts due to environmental variation.
  • Using synchrotron-generated microtomography imaging, we extracted leaf volumetric anatomy and stomatal traits in 34 species across conifers with a special focus on Pinus, the richest conifer genus.
  • We show that intrinsic water use efficiency (WUEi) is positively driven by leaf vein volume. Needle-like leaves of Pinus, as opposed to flat leaves or flattened needles of other genera, showed lower mesophyll porosity, decreasing the relative mesophyll volume. This led to increased ratios of stomatal pore number per mesophyll or intercellular airspace volume, which emerged as powerful explanatory variables, predicting both stomatal conductance and WUEi.
  • Our results clarify how the three-dimensional organisation of tissues within the leaf has a direct impact on plant water use and carbon uptake. By identifying a suite of structural traits that influence important physiological functions, our findings can help to understand how conifers may respond to the pressures exerted by climate change.

Introduction

Conifer forests thrived on the Earth surface during the Mesozoic period until the radiation and diversification of angiosperms during the Cretaceous, which was followed by angiosperm ecological dominance attributed to increased physiological performance and reduced generation times (Bond, 1989; Boyce et al., 2009; Crepet & Niklas, 2009; de Boer et al., 2012). Yet, after 100 Myr of competition with the angiosperms, conifers remain prominent in the canopy of many biomes (Brodribb et al., 2012). Conifers are found in ecosystems from high latitudes to the equator, and they have major economic importance in the wood and paper industries (McFarlane & Sands, 2013). Conifer-dominated forests are not exempt from the impacts of drought and aridity resulting from ongoing global climatic changes (Dai, 2011; Brodribb et al., 2020; Kharuk et al., 2021). This is particularly alarming given that 48% of the 722 conifer taxa of the world are currently under threat (Blackmore et al., 2011). Global increases in temperature coupled with rising vapour pressure deficit (VPD) place increased strain on plant hydraulic and photosynthetic systems (Choat et al., 2018; Grossiord et al., 2020). There is strong evidence that tree water use efficiency (WUE) has increased in recent decades, most probably the result of rising atmospheric CO2, allowing plants to open their stomata less frequently, thereby conserving water (Keenan et al., 2013; Mathias & Thomas, 2021). However, the underlying physiological mechanisms behind this trend need to be further elucidated (Guerrieri et al., 2019). Furthermore, there is still a lack of knowledge on how leaf structural organisation influences key functions such as photosynthetic carbon acquisition, stomatal conductance, and the interplay between both driving intrinsic water use efficiency (WUEi).

Leaf-level photosynthetic metabolism has an important role in maintaining global ecological processes (Hetherington & Woodward, 2003). Therefore, exploring tissue organisation inside the leaf, and specifically the mesophyll cells where water and gas diffusion occur, is important for understanding carbon, water and energy fluxes at whole ecosystem levels. The leaf mesophyll consists of photosynthetic parenchyma cells located between the epidermis and the endoderm and transfusion tissue layers that surround the veins. Once inside the leaf, diffusion of CO2 from the intercellular airspace (IAS) to the chloroplast stroma (i.e. mesophyll conductance) is a major constraint on photosynthetic performance (Flexas et al., 2012; Gago et al., 2020). This pathway includes the IAS, but also the diffusion of CO2 across mesophyll cell walls, cell membranes and the chloroplast envelope, which can significantly limit photosynthesis in gymnosperms (Carriquí et al., 2020). Previous work has shown that mesophyll surface area per leaf area (Sm; µm2 µm−2) has a strong influence on maximum photosynthesis (Nobel et al., 1975; Smith & Nobel, 1977). More recently, it has been suggested that the surface area of the mesophyll exposed to the IAS per unit of mesophyll volume (SAmes/Vmes; µm2 µm−3) can influence plant photosynthetic performance given that the mesophyll-IAS boundary is the primary interface between the atmosphere and the photosynthetic cells (Earles et al., 2018; Théroux-Rancourt et al., 2021). Other volumetric anatomical traits such as mesophyll porosity (i.e. IAS as a fraction of total mesophyll volume; µm3 µm−3) might be relevant in promoting diffusion through the IAS, and it has been suggested that mesophyll palisade porosity is correlated with stomatal conductance across four different Arabidopsis mutants (Lundgren et al., 2019). The relevance of such anatomical traits arises from the hypothesis that photosynthetic capacity could be enhanced by increasing surface area of mesophyll exposed to the IAS, allowing for more potential surface for CO2 diffusion across mesophyll cell walls, and chloroplast envelopes (Evans et al., 2009; Earles et al., 2018). However, the correct estimation of such anatomical traits using standard two-dimensional (2D) techniques is difficult as it relies on 2D approximations of the complex, three-dimensional (3D) shape of the mesophyll (Théroux-Rancourt et al., 2017; Earles et al., 2019).

Given the elementary vascular architecture of the conifer leaf, which typically consists of a single vein without further branching (Zwieniecki et al., 2004; Brodribb et al., 2010), and the large variation in conifer leaf shape (Fig. 1), area-based traits might not allow for an accurate comparison of strategies to optimise leaf structure with function within conifers (Earles et al., 2019; Théroux-Rancourt et al., 2021). Furthermore, given the structural relevance of the central vasculature, traits other than those related to the mesophyll might also influence CO2 and water diffusion in the conifer leaf. Therefore, a central question is whether nonvascular tissues are tightly coupled to vein volume with proportional relationships, or whether they show greater variability to compensate for reduced hydraulic efficiency. We predict that higher volumes of vascular tissue per unit leaf volume (Vvein/Vleaf; µm3 µm−3) should be a good predictor of gas exchange and WUEi because of the mechanistic link between hydraulic conductance and maximum photosynthetic rates in conifers (Brodribb & Feild, 2000). We also predict a positive relationship between gas exchange efficiency and the ratio of mesophyll surface area exposed to the IAS and vein volume (SAmes/Vvein; µm2 µm−3), where a greater investment in vein volume per unit area of bulk tissue surface should increase the hydraulic capacity to replace water lost to transpiration. In conifer leaves, water moves across the transfusion tissue and a ring of parenchymatous sheath cells or an endoderm before reaching the mesophyll (Hu & Yao, 1981). Therefore, transfusion tissue and endoderm volume relative to total leaf volume (VTT+en/Vleaf; µm3 µm−3), should also influence the efficiency of water movement within the leaf. Along with the previously described features, many conifer species also possess resin ducts, which play a major role in chemical and physical defence (Gaylord et al., 2013; Breshears et al., 2018), but necessarily displace vascular or photosynthetic tissue, incurring both maintenance and construction costs, but also lost opportunity costs for net carbon gain. Finally, water movement inside the leaf ends at the stomatal pores, which play a major role in regulating water loss and maintaining water status in conifers (Brodribb et al., 2014). Within this context, it is possible to determine the proportionality of different anatomical traits, the coordination between supply and demand for CO2 and H2O, and the physical constraints of leaf construction. To probe these relationships, we describe how conifers build their elementary, yet diverse leaves, and how structural features relate to key physiological traits.

Details are in the caption following the image
Proportion of different tissue volumes relative to total leaf volume inside the 3D leaf space in conifers (a). Average values of Pinus species belonging to the Pinus (21 spp.) and Strobus (5 spp.) subgenera are included. Microtomography 3D images of needle-leaved Pinus pungens (b; Pinus subgenus) and Pinus monticola (c; Strobus subgenus) and flat-leaved Wollemia nobilis (d). Segmented tissues are indicated with different colours and stomatal apertures are indicated with arrows. Bars, 250 µm. A plot of the relative tissue volume of all 34 species is available in Supporting Information Fig. S2.

This study presents a survey of the three-dimensional organisation of the conifer leaf using microtomography (microCT) imaging (Supporting Information Table S1). Our study includes 34 conifer species (Fig. S1), with a special focus on the genus Pinus, the largest extant genus of conifers (Gernandt et al., 2005). Pinus species can be found in a broad range of environmental conditions, suggesting wide structural and functional diversity (Martínez-Vilalta et al., 2004). Pinus subgenera Pinus and Strobus can be distinguished by having two or one vascular bundles respectively, centrally located inside the needle-like leaf (Gernandt et al., 2005) (Fig. 1a–c). Despite their rather simple anatomical organisation, conifer leaves have extensive morphological diversity ranging from flat leaves to needle-like leaves with different degrees of transversal flattening. Leaf morphological diversity in conifers results in different physiological performances, with flat-leaved species having lower photosynthetic assimilation and respiration rates than needle-leaved species (Brodribb & Feild, 2008; Schmiege et al., 2020, 2021). Yet, the differences in 3D anatomical structure across conifers with different leaf morphologies, which could explain their contrasting photosynthetic performance, need to be elucidated. We hypothesise that features enhancing mesophyll surface area for gas diffusion, will be positively correlated with the light-saturated assimilation rate of CO2 (Asat) and stomatal conductance (gs). We also provide a volume-based stomatal density estimation, a trait we expect better captures the interplay between the evaporative surfaces invested in the nonlaminar mesophyll volume and the number of stomata needed to provide CO2, with the expectation that leaves with higher number of stomata per mesophyll volume would have both higher rates of gas exchange and WUEi due to an enhancement of the epidermal pores serving as evaporative surface relative to the photosynthetic tissue.

Materials and Methods

Plant material

Sampling included 34 conifer species from various biomes, physiologies and leaf morphologies. Sampling included taxa from four different families of conifers: Araucariaceae (2 spp.), Pinaceae (30 spp.), Podocarpaceae (1 sp.), and Taxaceae (1 sp.). Our sampling particularly focused on the genus Pinus with 26 species including representatives from the two subgenera: Pinus (21 spp.) and Strobus (5 spp.), which differ in the number of vascular bundles per leaf (Gernandt et al., 2005). Sampling also represents three distinct conifer leaf morphologies: flat leaves (Araucaria, Retrophyllum, Taxus and Wollemia; Fig. 1d), flattened needles (Abies, Larix, Picea and Tsuga), and needle-like leaves (Pinus; Fig. 1b,c). Flattened needles, such as those commonly found in non-Pinus Pinaceae, are generally shorter and flattened in cross-section compared with Pinus needle-like leaves, which have almost equal width and height (Fig. 1b,c) and are generally longer. Fully expanded leaves from adult plants were collected in the Berkeley Arboretum of the University of California Botanical Garden, and the University of Georgia’s Thompson Mills Arboretum. Samples from both locations were used for microCT scanning and gas exchange measurements. Whole shoots were cut, wrapped in moist paper towels, and transported in dark plastic bags to avoid desiccation before scanning.

X-ray microtomography scanning and image segmentation

Microtomography imaging was performed at the Lawrence Berkeley National Laboratory Advanced Light Source, beamline 8.3.2. Leaves were scanned at the mid-section within 24 h of excision. Two replicates were scanned for 18 species, while a single leaf was scanned for the remaining 16 species (Dataset S1). Samples were wrapped with polyimide tape, which allows X-ray transmittance while preventing sample desiccation. Wrapped leaf samples were placed in a plastic 1000 µl pipette tip with the lower end submerged in water and centred in the microCT X-ray beam. Scans were completed in c. 15 min in continuous tomography mode at 21 keV capturing 1025 projection images of 200–250 ms each. Images were captured using alternatively ×5 or ×10 objective lenses depending on leaf diameter, yielding final pixel resolutions of 1.27 and 0.625 μm. Images were reconstructed using TomoPy (Gürsoy et al., 2014). Raw tomographic datasets were reconstructed using both gridrec and phase retrieval methods, both of which yield complementary results being efficient in segmenting cell boundaries and larger air voids, respectively (Théroux-Rancourt et al., 2017). Image stacks of c. 2600 8-bit greyscale images were generated from the reconstruction process. Airspace was segmented in both gridrec and phase reconstruction methods by visually defining a range of pixel intensity values and the binary image stacks from both reconstruction methods were combined. Boundaries delimiting the areas occupied by the endoderm + transfusion tissue, epidermis, mesophyll, resin ducts, and veins were manually drawn using a graphic tablet (Wacom Cintiq Pro 16; Wacom Co, Saitama, Japan) using ImageJ software (Schneider et al., 2012). Leaf veins were depicted here as the vascular bundle comprising both xylem and phloem tissues. Leaf tissue boundaries were drawn as regions of interest in six to eight images randomly distributed across the full stack. The combination of the binary image derived from both reconstruction methods, along with the tissue boundaries, resulted in a composite image stack where each leaf tissue was classified. Leaf segmentation, which allowed us to automatically delimit different tissues across the full stack using a limited set of hand-segmented composite slices was done using random-forest classification (Théroux-Rancourt et al., 2020).

Three-dimensional anatomy and stomatal traits

We extracted the volume and surface area of leaf anatomical traits from the full segmented stacks (Théroux-Rancourt et al., 2020). We estimated the volume of the epidermis and hypodermis (Vep), which were segmented together when hypodermal cell layers were present, due to the proximity of both peripheral tissues. Mesophyll cells (Vcell) and mesophyll IAS (VIAS) were estimated independently, and mesophyll volume (Vmes) was estimated as the sum of Vcell and VIAS. Conifer leaves have an endoderm (Lersten, 1997) or a uniseriate ring of parenchymatous sheath cells that surrounds the transfusion tissue. Transfusion tissue, a common feature of gymnosperm leaves, is located between the sheath cells and the veins, and it is composed of transfusion tracheids, transfusion parenchyma, and albuminous cells (Hu & Yao, 1981). We estimated the volume of the ensemble conformed by the transfusion tissue and the adjacent endoderm (VTT+en). Furthermore, we estimated the volume of resin ducts (Vresin), and veins (Vvein). All volume metrics are reported in μm3. Relative volumes for each tissue (in μm3 μm−3) were estimated as a fraction of tissue per Vleaf, total leaf volume, which corresponds to the sum of the surface of the leaf cross-section on each slice multiplied by slice depth across the full stack. We calculated mesophyll porosity (μm3 μm−3) as VIAS/Vmes. The mesophyll surface area exposed to the IAS (SAmes) was used to estimate the mesophyll surface area per mesophyll volume (SAmes/Vmes; μm2 μm−3). Additionally, we estimated the exposed mesophyll surface area per transfusion tissue + endoderm volume (SAmes/VTT+en; μm2 μm−3) and vein volume (SAmes/Vvein; μm2 μm−3). Total leaf area Aleaf (μm2) was measured by summing up the perimeter of each slice and multiplying it by slice depth. We used the ratio SAmes/Aleaf to calculate the mesophyll surface area per total leaf area (Sm; μm2 μm−2). Stomatal estimations were performed by counting all visible stomata on the leaf surface of each scan using Avizo 9.4 software (FEI Co., Hillsboro, OR, USA). Absolute stomatal counts were used in relation to mesophyll volumetric anatomy to estimate traits accounting for the interaction of stomata pore number and VIAS, Vmes and SAmes units. Mesophyll volumetric features were assessed in mm3 for stomatal pore density estimations. We also accounted for stomatal number per leaf surface to assess potential differences in stomatal density estimations based on surface vs volume fractions. Furthermore, we performed a comparison of surface- and volume-based anatomical estimations using 2D slices and the full 3D stack. Methods are further explained in Notes S1. Average trait values for each species are available in Dataset S1. A list of measured anatomical variables, including abbreviations and units, is available in Table S1.

Gas exchange and WUE measurements

Stomatal conductance (gs; mmol m−2 s−1) and light-saturated CO2 assimilation rate (Asat; µmol m−2 s−1) were measured on a subset of 18 species (Dataset S1) and used to estimate leaf-level intrinsic water use efficiency (WUEi = Asat/gs; µmol mol−1). Asat data for Pinus strobus were removed before analyses due to potential measurement inaccuracies. Gas exchange measurements were performed on 3–5 individuals for each species with a Li-Cor-6800 gas exchange system (Li-Cor Biosciences, Lincoln, NE, USA) between 09:00 h and 14:00 h on fully sunlit outer canopy foliage. Chamber temperature was set to 25°C, light source was set to 1500 µmol m−2 s−1 and chamber CO2 was set to 400 ppm. Following the gas exchange measurements, the leaf area contained within the chamber was marked using a permanent marker, collected, and the projected leaf area was measured using a leaf area meter (LI3100C; Li-Cor Biosciences).

Data analysis

All statistical analyses and data treatment were performed using R v.3.6.3 (R Core Team, 2020). Anatomical traits were assembled and averaged for each species for data analysis. Assumptions for residual homogeneity and normality were tested before data analyses. Phylogenetic relationships, including branch length calibrations and divergence times, were obtained from published data (Magallón et al., 2015; Smith & Brown, 2018). To predict leaf physiological traits based on volumetric anatomical variables we used phylogenetic generalised least-squares analyses (PGLS) with a lambda (λ) maximum likelihood optimisation to control for phylogenetic nonindependence between related species (Felsenstein, 1985; Freckleton et al., 2002). A two-parameter exponential function, y = a(1 − exp(−bx)), was additionally used to describe the relationship of WUEi and Vvein/Vleaf (a = 130, b = 109, r2 = 0.50). The assemblage of the composite phylogenetic tree of studied conifers (Dataset S2) was carried out using the package ape (Paradis & Schliep, 2019) and PGLS models were fit using the package caper (Orme et al., 2018). Standard major axes (SMA) were implemented with the package smatr (Warton et al., 2012) to test allometric scaling between tissue volumes. A principal component analysis (PCA) was used to explore the covariation of selected traits and the distribution of leaf morphologies and conifer groups as explained by the physiological and anatomical traits measured. Given the marked differences in leaf anatomical structure across conifer leaf morphological types, and between Pinus subgenera Pinus and Strobus, we explored potential variation in 3D anatomical traits by plotting each group within the PCA. We further explored differences across leaf morphologies and between conifer clades by performing a one-way ANOVA on measured structural traits. Physiological features were not compared between leaf morphological types due to insufficient data availability. A similar variance meta-analysis, including post hoc Tukey’s honest significant differences, was used to compare the studied conifer species with other gymnosperms, along with angiosperms and ferns. Data for comparisons across plant groups were obtained from a recently published study (Théroux-Rancourt et al., 2021) and are available in Dataset S3.

Results

The mesophyll (Vmes/Vleaf), including cells and airspace, represents the dominant leaf volume fraction for all 34 measured conifer species in this study, occupying an average of 60% of the total leaf volume (Figs 1, S2). The second largest volume fraction was either the combined transfusion tissue and endoderm (VTT+en/Vleaf) that surrounds the vascular cylinder, or the epidermis (Vep/Vleaf), which represented an average of 22% and 16% of the leaf volume, respectively (Fig. 1a). Veins (Vvein/Vleaf) and resin ducts (Vresin/Vleaf) represented the smallest fraction of the total leaf volume (Fig. 1a), and resin ducts were completely absent in six measured species (Dataset S1). We found weak structural coordination amongst tissue volumes within the conifer leaf (SMA; Table S2). However, a negative allometric scaling between Vep/Vleaf and Vmes/Vleaf was observed (r2 = 0.27, P < 0.01), indicating that increasing the relative allocation to the mesophyll was done in conjunction with a decrease in the relative allocation to the epidermis. Average values of all measured volumetric traits for each species are included in Dataset S1.

A multivariate analysis of trait covariation defined two major axes explaining 48% of inertia (Fig. 2). Inertia of the first axis was mainly explained by VIAS/stomate, the amount of air volume connected to a stomate (18.27%), stomata/Vmes, the stomatal density per mesophyll volume (15.04%), Vmes/Vleaf (14.74%) and gs (14.08%). Increasing gs was associated on the first axis with increases in stomata/Vmes and Asat, and decreases in mesophyll porosity, Vmes/Vleaf, and VIAS/stomate (Fig. 2). The second axis was largely explained by Vvein/Vleaf (18.04%), SAmes/Vvein (14.83%) and Sm (14.45%). Increasing WUEi was associated on the second axis with increases in Sm and Vvein/Vleaf, and decreases in stomata/Vmes (Fig. 2). Leaf morphological types were dispersed along both major axes (Fig. 2). Needle-like leaves were isolated due to their higher Vep/Vleaf and stomata/Vmes. Flat leaves and flattened needles largely overlapped due to their porous and voluminous mesophylls as opposed to needle-like leaves (Fig. 3a,b). Flat leaves and flattened needles also converged in having lower stomata/Vmes (Fig. 3c) and higher VIAS/stomate (Fig. 3d) than needle-like leaves. However, flattened needles were differentiated due to some species having significantly higher SAmes/Vvein (Fig. 2; Table S3). Conifer groups were also effectively segregated based on their volumetric anatomy. Non-Pinus conifer species were segregated on the first axis (Fig. 2) due to their higher porosity (Fig. 1d), Vmes/Vleaf, and VIAS/stomate values, while species from the Pinus subgenus were grouped (Fig. 2) due to higher Vep/Vleaf (Fig. 1b), along with a relatively larger SAmes/Vmes. Species from the Strobus subgenera were located between the previously described conifer groups (Fig. 2). Structural divergences of leaf morphologies, distinguishing three distinct functional groups, along with the segregation of different conifer clades in the PCA analysis were further supported with one-way ANOVA analyses on 3D leaf anatomical traits and stomatal density (Tables S3, S4). Relative vein volume and SAmes/Vmes were highly conserved (Fig. S3; Tables S3, S4), whereas SAmes/Vvein, along with traits related to the ratio of stomatal pore number and mesophyll tissue volumes had significant differences across both leaf morphologies and conifer groups (Figs 3c,d, S3; Tables S3, S4).

Details are in the caption following the image
Principal component analysis (PCA) of physiological and volumetric anatomy traits of conifer species. Arrows and trait colour gradients indicate the contribution of each variable to the axes. Species from the two Pinus subgenera, along with other studied conifer species are indicated in different colours, and 95% confidence ellipses are included. Confidence ellipses for different leaf morphologies are also included. Species bearing flat leaves (squares, dotted line), flattened needle leaves (triangles, narrow dotted line), and needle-like leaves (circles, dashed line) are identified. Asat, light-saturated photosynthetic assimilation rate; gs, stomatal conductance; Porosity, mesophyll porosity; SAmes/Vmes, mesophyll surface area per unit mesophyll volume; SAmes/Vvein, mesophyll surface area per unit vein volume; Sm, mesophyll surface area per total leaf area; stom/Vmes, stomatal pore number per unit mesophyll volume; Vep/Vleaf, epidermis volume relative to total leaf volume; VIAS/stom, intercellular airspace volume per stomate; Vmes/Vleaf, mesophyll volume relative to total leaf volume; Vresin/Vleaf, resin duct volume relative to total leaf volume; VTT+en/Vleaf, transfusion tissue and endoderm volume relative to total leaf volume; Vvein/Vleaf, vein volume relative to total leaf volume; WUEi, intrinsic water use efficiency.
Details are in the caption following the image
Differences in mesophyll volumetric traits (a, b) and stomatal features (c, d) across flat (squares), flattened needle (triangles) and needle-like (circles) leaves in the conifer species studied. Horizontal lines, boxes and bars show the median, quartiles and extreme values, respectively. Grey dots are species data points. P-value notations represent results of one-way ANOVAs between groups. *, P ≤ 0.05; ***, P ≤ 0.001. Letters indicate significant differences between leaf morphological types. Furthermore information and other comparisons of anatomical traits across leaf morphology types are available in Supporting Information Table S3. Porosity, mesophyll porosity; stomata/Vmes, stomatal pore number per unit mesophyll volume; VIAS/stomate, intercellular airspace volume per stomate; Vmes/Vleaf, mesophyll volume relative to total leaf volume.

To explore the functional implications of 3D tissue content, we determined their relationships to gas exchange parameters such as Asat, gs and WUEi. WUEi was best predicted by Vvein/Vleaf (Fig. 4a), where higher Vvein/Vleaf enhances leaf WUEi. Furthermore, 2D anatomical estimations of the ratio Avein/Aleaf were comparable with Vvein/Vleaf, extracted using a 3D approach (Notes S1; Fig. S4). The mesophyll surface area exposed to vein volume (SAmes/Vvein) was also an accurate predictor and showed a negative relationship with WUEi (Fig. 4b). A positive relationship of WUEi with Sm was also found using generalised least-square models corrected for phylogenetic relatedness (PGLS) analyses (Table S5). Asat was the physiological trait showing the least linkage with 3D structural traits. However, we found a negative relationship between Asat and mesophyll porosity along with Vmes/Vleaf (Fig. S5a), suggesting that conifer species with lower relative mesophyll volumes have greater photosynthetic assimilation rates. Additionally, VIAS/stomate was also negatively related to Asat (Fig. S5b). The number of stomata per unit mesophyll tissue volume predicted gs and WUEi (Fig. 5). For instance, high stomatal densities relative to mesophyll volume (stomata/Vmes) enhanced gs (Fig. 5a) while decreasing WUEi (Fig. 5b). Evolutionary coordination between leaf volumetric anatomy and physiological traits was supported by PGLS analyses (Table S5). Stomatal density measured on a 2D leaf surface area basis (stomata/Aleaf), included here as a reference of a more standard approach, did not relate to any physiological trait (Table S5). Therefore, the interaction of mesophyll volume and stomatal pore numbers emerged as a key trait to explain leaf physiological performance (Fig. 5). A comparison of the studied conifers with published data for other gymnosperm species, along with angiosperms and ferns, showed that conifers had fewer stomata per unit mesophyll volume than angiosperms sensu largo (Fig. S6; Dataset S3). However, this difference was less important when considering evergreen angiosperms alone. Conifers showed a similar stomata/Vmes ratio as other gymnosperm species and ferns (Fig. S6).

Details are in the caption following the image
Water use efficiency (WUEi) as a function of vein volumetric traits of conifer leaves. Relationships of WUEi with relative vein volume (a) and the surface area of the mesophyll exposed to the intercellular airspace per unit of vein volume (b) across 18 conifer species. Solid regression lines and 95% confidence intervals (grey shaded areas) are included. Pinus species from the subgenera Pinus (green) and Strobus (yellow), along with other conifer species (purple) are indicated. Species bearing flat leaves (square), flattened needle leaves (triangles), and needle-like leaves (circles) are also identified. Phylogenetic generalised least-squares coefficients of determination are included. **, P < 0.01; ***, P < 0.001. Additional information is available in Supporting Information Table S5.
Details are in the caption following the image
Physiological traits explained by volumetric stomatal density in conifer leaves. Relationships of stomatal conductance (a) and water use efficiency (b) with the number of stomata per unit of mesophyll volume across 18 conifer species. Solid regression lines and 95% confidence intervals (grey shaded areas) are included. Pinus species from the subgenera Pinus (green) and Strobus (yellow), along with other conifer species (purple) are indicated. Species bearing flat leaves (square), flattened needle leaves (triangles), and needle-like leaves (circles) are also identified. Phylogenetic generalised least-squares coefficients of determination are included. *, P < 0.05; **, P < 0.01. Additional information available in Supporting Information Table S5.

Discussion

Conifers, with their long-lasting and anatomically elementary leaves, must rely on leaf construction to enable sufficient carbon assimilation to survive and reproduce while limiting water loss. However, the simple design of coniferous leaves has been considered to have a poor, if not absent, hydraulic connection between the vein xylem and the bulk leaf tissue (Zwieniecki et al., 2007). This is unsurprising as conifer leaves are not as fully vascularised as those of angiosperms and present a single cohort of relatively inefficient leaves (Bond, 1989). The likely consequence of this poor hydraulic connection is a larger difference in water potential between the veins and the mesophyll and epidermis in transpiring leaves, which may force stomata to close even when water potential is relatively high in the veins (Zwieniecki et al., 2007). Our results show that, despite occupying a small fraction (c. 2%) of the leaf volumetric matrix, vein tissue volume has a great impact on the leaf WUEi (Fig. 4a). As the relative vein volume expands, the space between the vascular tissues and the bulk leaf tissues become smaller, potentially reducing the hydraulic resistance for water transport from the vasculature to the bulk leaf. Our study provides an estimation of the full volumetric space occupied by the vein tissue relative to the photosynthetic cells. While we consider volumetric estimates to be more accurate given that they integrate traits over a larger leaf fraction than 2D estimates, we found that standard 2D anatomical estimations of the relative surface covered by veins over a few leaf cross sections could be accurately used to predict the vein volumetric fraction and, in turn, WUE in conifer leaves (Notes S1; Fig. S4). Stomatal and venation densities covary in angiosperms, directly affecting gas exchange and water use capacities (Carins Murphy et al., 2014). However, conifers do not provide a dense network of veins within the mesophyll to irrigate the photosynthesising cells in water. Consequently, conifers might be constrained to increase the relative volume of veins as a single way to provide more water to the leaf.

Previous work has suggested that narrow, needle-like leaves in conifers, which is a common feature in the Pinaceae (Brodribb & Feild, 2008; Brodribb et al., 2010), would be a response to alleviate their lack of hydraulic redundancy. With the photosynthetic tissue encircling the single vascular cylinder, the distance from the vein to the epidermis largely sets hydraulic conductance outside the xylem, and mesophyll volume becomes a major limiting factor in cylindrical, narrow leaves. Increasing the radial pathlength for water transport should effectively limit leaf hydraulic conductance, in turn limiting photosynthetic rates (Brodribb et al., 2010). Our data support this hypothesis, with a negative relationship between mesophyll volume and Asat (Fig. S5a). Therefore, within our dataset we found evidence for a significant photosynthetic penalty for increasing leaf width and mesophyll volume in flat and flattened needle leaves (Fig. 3b). With a limited ability to maximise photosynthetic capacity through hydraulic ramification, the cylindrical needle-like leaf may offer opportunities for other structural elements that allow improved hydraulic performance. Accessory transfusion tissue is one of them, where specialised cells connect the veins to the mesophyll tissue, providing more water to the photosynthesising cells and improving hydraulic contact to the epidermis (Brodribb et al., 2007). This might lead to increased photosynthetic rates in needle-like leaves, which presented the most voluminous transfusion tissues (Table S3). Our results support this linkage, with a positive relationship of the combined volumes of transfusion tissue and endoderm with photosynthetic assimilation (Table S5). It has been stated that reaching high photosynthetic rates requires high leaf porosity values, which might increase CO2 diffusion (Brodribb et al., 2020). Yet, we observed a different trend with a negative relationship between mesophyll porosity and Asat (Table S5). Moreover, Asat was strongly negatively related with Vmes/Vleaf in our dataset (Fig. S5a). Illumination-induced fluorescence has been shown to be more evenly distributed in needle-like leaves lacking palisade mesophyll layers (Johnson et al., 2005). Therefore, the decrease of Asat in flat leaves with more voluminous mesophylls and often presenting a more differentiated palisade layer might be explained by a limitation of light propagation across the mesophyll. Interestingly, we found conspicuous differences in leaf design between both Pinus subgenera and other conifers (Figs 1, 2; Table S4), with narrow needle-leaved Pinus possessing less voluminous and porous mesophylls. Such differences in mesophyll construction, which enhance the exchange surface by condensing the mesophyll cells in the more compact mesophyll of needle-like leaves, could explain why Asat is greatest in Pinus species.

SAmes/Vvein is another feature that has diverged across leaf morphologies and conifer clades (Tables S3, S4). We propose SAmes/Vvein as another anatomical trait involved in regulating the control over the loss of water (Fig. 4b). Minimising this ratio would mean that less surface is available for evaporation for a given vein water volume, increasing the time before this capacitor is depleted, and thereby lowering the ‘safety margin’ between stomatal closure and xylem cavitation (Zwieniecki et al., 2007). Also, in the context of poorly connected hydraulic design, decreasing SAmes/Vvein would minimise the apoplastic surface for water to travel from the vein to the epidermis, therefore by proxy decreasing the water path length and increasing connectivity to the epidermis to allow stomata to stay open longer and photosynthesis to continue. In our dataset, decreasing SAmes/Vvein was achieved mainly by increasing the relative volume of veins, that is investing more in vascular tissue (Fig. 4). The relative benefit of investing in vein volume to increase the efficiency in water use seems to plateau above c. 3% of leaf volume, and most species invest in veins close to that relative volume (Fig. 4a). Pinus species, which bear needle-like leaves have, in addition, decreased the relative volume of the mesophyll by decreasing airspace volume (Figs 1, 3a,b, S3a,b) with plicate mesophyll cells (Esau, 1977). Decreasing porosity leads to more cells being in contact with each other, thereby decreasing SAmes, that is the surface of cells exposed to the IAS. Increasing IAS had a positive effect on WUE in six angiosperm species (Mediavilla et al., 2001). Although positive, we could not find a significant relationship between mesophyll porosity and WUEi in conifer leaves (Table S5). Beyond considering mesophyll features alone, mesophyll volumetrics in interaction with stomatal pore number emerged here as key traits to explain conifer gas exchange and WUEi (Fig. 5). Using Arabidopsis and wheat as model plants, it has been suggested that stomatal differentiation during leaf development might induce mesophyll airspace formation (Lundgren et al., 2019). Our study shows that this coordination between VIAS, along with Vmes, and stomatal number have a significant impact on carbon assimilation and gas exchange on conifers (Fig. 5; Table S5), further supporting this crucial linkage. Considered in a wider context, our observations might provide a novel structural basis to explain the lower photosynthetic rates of ferns and gymnosperms compared with angiosperms, as we show that they have greater mesophyll volume per stoma, acting as a bottleneck that limits their evaporative capacities (Fig. S6).

Current increases in temperature and atmospheric CO2 concentrations might impact the structure and function of conifer forests worldwide, and it has been proposed that improved WUE could alleviate the temperature effect (Brodribb et al., 2020). Previous studies have shown higher WUEi under increasing CO2 in conifer species, having stronger WUEi responses than angiosperms (Dalling et al., 2016; Klein et al., 2016; Adams et al., 2020). Additionally, it has been recently shown that higher plasticity in the vascular tissue of the needles of Pinus pinaster enhances their WUEi (Bert et al., 2021). Using an experimental approach, needles of Larix kaempferi growing under higher CO2 showed increased mesophyll surface area per leaf area, coupled with higher photosynthetic rates (Eguchi et al., 2004). Moreover, it was shown that elevated CO2 increased mesophyll surface and decreased stomatal density in Pinus sylvestris needles (Lin et al., 2001). Therefore, under such elevated CO2 conditions, we might expect to observe a lower stomata/Vmes ratio, which would enhance WUEi according to our predictions (Fig. 5b). Furthermore, given the recently demonstrated link between enhanced WUEi and vascular tissue plasticity in conifer needles (Bert et al., 2021), we expect that coordinated changes in vascular and mesophyll tissue volumetrics, along with shifts in stomatal pore number in conifer leaves, may allow conifer species to cope and adapt to the pressure exerted by increasing VPD in many global biomes (Grossiord et al., 2020) by maintaining similar carbon assimilation levels with lower water consumption.

Acknowledgements

This work was supported by the NSF grants IOS-1626966, IOS-1852976 and IOS-1146746, the Austrian Science Fund (FWF), project M2245, and the Vienna Science and Technology Fund (WWTF), project LS19-013. We thank the Berkeley Arboretum of the University of California Botanical Garden, and the University of Georgia’s Thompson Mills Arboretum for providing plant material. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Science, of the US Department of Energy under contract no. DE-AC02-05CH11231. TNB acknowledges support from the USDA National Institute of Food and Agriculture (Hatch Award 1016439 and Award 2020-67013-30913) and the NSF (IOS-1951244, IOS-1557906).

    Author contributions

    CB, DMJ, GT-R, ST and TNB designed research; CB, DL, DMJ, GT-R, JME and ST performed measurements and collected data; ST analysed the data and wrote the first draft of the manuscript with major contributions from CB and GT-R. All authors contributed to manuscript revisions.

    Data availability

    The datasets that support the findings of this study are available as supplementary files.