Mesophyll conductance to CO₂ (g_m ) may respond to light either through regulated dynamic mechanisms or due to anatomical and structural factors. At low light, some layers of cells in the leaf cross-section approach photocompensation and contribute minimally to bulk leaf photosynthesis and little to whole leaf g_m (g_m,leaf ). Thus, the bulk g_m,leaf will appear to respond to light despite being based upon cells having an anatomically fixed mesophyll conductance. Such behaviour was observed in species with contrasting leaf structure using the variable J or stable isotope method of measuring g_m,leaf . A species with bifacial structure, Arbutus × 'Marina', and an isobilateral species, Triticum durum L., had contrasting responses of g_m,leaf upon varying adaxial or abaxial illumination. Anatomical observations, when coupled with the proposed model of g_m,leaf to photosynthetic photon flux density (PPFD) response, successfully represented the observed gas exchange data. The theoretical and observed evidence that g_m,leaf apparently responds to light has large implications for how g_m,leaf values are interpreted, particularly limitation analyses, and indicates the importance of measuring g_m under full light saturation. Responses of g_m,leaf to the environment should be treated as an emergent property of a distributed 3D structure, and not solely a leaf area-based phenomenon.

Trees experience two distinct environments: thermally-variable air and thermally-buffered soil. This generates intra-tree temperature gradients, which can affect carbon metabolism and water transport. In this study, we investigated whether carbohydrate allocation within trees is assisted by temperature gradients. We studied pistachio (Pistacia integerrima) to determine: (1) temperature-induced variation in xylem sugar concentration in excised branches; (2) changes in carbon allocation in young trees under simulated spring and fall conditions; and (3) seasonal variability of starch levels in mature orchard trees under field conditions. We found that warm branches had less sugar in perfused sap than cold branches due to increasing parenchyma storage. Simulated spring conditions promoted allocation of carbohydrates from cold roots to warm canopy and explained why starch levels surged in canopies of orchard trees during early spring. This driving force of sugar transport is interrupted in fall when canopies are colder than roots and carbohydrate redistribution is compartmentalized. On the basis of these findings, we propose a new mechanistic model of temperature-assisted carbohydrate allocation that links environmental cues and tree phenology. This data-enabled model provides insights into thermal "fine-tuning" of carbohydrate metabolism and a warning that the physiological performance of trees might be impaired by climatic changes.

In agricultural and natural systems, diffuse light can enhance plant primary productivity due to deeper penetration into and greater irradiance of the entire canopy. However, for individual sun-grown leaves from three species, photosynthesis is actually less efficient under diffuse compared with direct light. Despite its potential impact on canopy-level productivity, the mechanism for this leaf-level diffuse light photosynthetic depression effect is unknown. Here, we investigate if the spatial distribution of light absorption relative to electron transport capacity in sun- and shade-grown sunflower (Helianthus annuus) leaves underlies its previously observed diffuse light photosynthetic depression. Using a new one-dimensional porous medium finite element gas-exchange model parameterized with light absorption profiles, we found that weaker penetration of diffuse versus direct light into the mesophyll of sun-grown sunflower leaves led to a more heterogenous saturation of electron transport capacity and lowered its CO₂ concentration drawdown capacity in the intercellular airspace and chloroplast stroma. This decoupling of light availability from photosynthetic capacity under diffuse light is sufficient to generate an 11% decline in photosynthesis in sun-grown but not shade-grown leaves, primarily because thin shade-grown leaves similarly distribute diffuse and direct light throughout the mesophyll. Finally, we illustrate how diffuse light photosynthetic depression could overcome enhancement in canopies with low light extinction coefficients and/or leaf area, pointing toward a novel direction for future research.

Premise

X-ray microcomputed tomography (microCT) can be used to measure 3D leaf internal anatomy, providing a holistic view of tissue organization. Previously, the substantial time needed for segmenting multiple tissues limited this technique to small data sets, restricting its utility for phenotyping experiments and limiting our confidence in the inferences of these studies due to low replication numbers.

Methods and results

We present a Python codebase for random forest machine learning segmentation and 3D leaf anatomical trait quantification that dramatically reduces the time required to process single-leaf microCT scans into detailed segmentations. By training the model on each scan using six hand-segmented image slices out of >1500 in the full leaf scan, it achieves >90% accuracy in background and tissue segmentation.

Conclusions

Overall, this 3D segmentation and quantification pipeline can reduce one of the major barriers to using microCT imaging in high-throughput plant phenotyping.

The mesophyll surface area exposed to intercellular air space per leaf area (S_m ) is closely associated with CO₂ diffusion and photosynthetic rates. S_m is typically estimated from two-dimensional (2D) leaf sections and corrected for the three-dimensional (3D) geometry of mesophyll cells, leading to potential differences between the estimated and actual cell surface area. Here, we examined how 2D methods used for estimating S_m compare with 3D values obtained from high-resolution X-ray microcomputed tomography (microCT) for 23 plant species, with broad phylogenetic and anatomical coverage. Relative to 3D, uncorrected 2D S_m estimates were, on average, 15-30% lower. Two of the four 2D S_m methods typically fell within 10% of 3D values. For most species, only a few 2D slices were needed to accurately estimate S_m within 10% of the whole leaf sample median. However, leaves with reticulate vein networks required more sections because of a more heterogeneous vein coverage across slices. These results provide the first comparison of the accuracy of 2D methods in estimating the complex 3D geometry of internal leaf surfaces. Because microCT is not readily available, we provide guidance for using standard light microscopy techniques, as well as recommending standardization of reporting S_m values.

Leaves balance CO₂ and radiative absorption while maintaining water transport to maximise photosynthesis. Related species with contrasting leaf anatomy can provide insights into inherent and stress-induced links between structure and function for commonly measured leaf traits for important crops. We used two walnut species with contrasting mesophyll anatomy to evaluate these integrated exchange processes under non-stressed and drought conditions using a combination of light microscopy, X-ray microCT, gas exchange, hydraulic conductance, and chlorophyll distribution profiles through leaves. Juglans regia had thicker palisade mesophyll, higher fluorescence in the palisade, and greater low-mesophyll porosity that were associated with greater gas-phase diffusion (g_IAS ), stomatal and mesophyll (g_m ) conductances and carboxylation capacity. More and highly-packed mesophyll cells and bundle sheath extensions (BSEs) in Juglans microcarpa led to higher fluorescence in the spongy and in proximity to the BSEs. Both species exhibited drought-induced reductions in mesophyll cell volume, yet the associated increases in porosity and g_IAS were obscured by declines in biochemical activity that decreased g_m . Inherent differences in leaf anatomy between the species were linked to differences in gas exchange, light absorption and photosynthetic capacity, and drought-induced changes in leaf structure impacted performance via imposing species-specific limitations to light absorption, gas exchange and hydraulics.

Maintaining high rates of photosynthesis in leaves requires efficient movement of CO₂ from the atmosphere to the mesophyll cells inside the leaf where CO₂ is converted into sugar. CO₂ diffusion inside the leaf depends directly on the structure of the mesophyll cells and their surrounding airspace, which have been difficult to characterize because of their inherently three-dimensional organization. Yet faster CO₂ diffusion inside the leaf was probably critical in elevating rates of photosynthesis that occurred among angiosperm lineages. Here we characterize the three-dimensional surface area of the leaf mesophyll across vascular plants. We show that genome size determines the sizes and packing densities of cells in all leaf tissues and that smaller cells enable more mesophyll surface area to be packed into the leaf volume, facilitating higher CO₂ diffusion. Measurements and modelling revealed that the spongy mesophyll layer better facilitates gaseous phase diffusion while the palisade mesophyll layer better facilitates liquid-phase diffusion. Our results demonstrate that genome downsizing among the angiosperms was critical to restructuring the entire pathway of CO₂ diffusion into and through the leaf, maintaining high rates of CO₂ supply to the leaf mesophyll despite declining atmospheric CO₂ levels during the Cretaceous.