*: These authors contributed equally 24 25One-sentence summary: 26The gas phase of mesophyll conductance is impacted by the 3D traits tortuosity, path 27 lengthening and airspace connectivity, in addition to porosity. 2 JME, GTR, and CRB conceived the study and developed the methods, with 31 contributions from MEG and AJM; JME and GTR acquired and analyzed the data; ABR 32 performed the phylogenetic analyses; JME and GTR wrote the manuscript; ABR, MEG, 33 AJM, and CRB complemented the writing.
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ABSTRACT 42The leaf intercellular airspace (IAS) is generally considered to have high conductance to 43 CO 2 diffusion relative to the liquid phase. While previous studies accounted for leaf-level 44 variation in porosity and mesophyll thickness, they omitted 3D IAS traits that potentially 45 influence IAS conductance (g IAS ). Here we re-evaluated the standard equation for g IAS 46 by incorporating tortuosity, lateral path lengthening, and IAS connectivity. We measured 47 and spatially mapped these geometric IAS traits for 19 Bromeliaceae species with CAM 48 or C3 photosynthetic pathways using X-ray microCT imaging and a novel computational 49 approach. We found substantial variation in porosity (0.04-0.73 m 3 m -3 ), tortuosity 50 -2 s -1 bar -1 ) plants due to a coordinated decline in these IAS traits. Our re-evaluated 54 equation also generally predicted lower g IAS values than the former one. Moreover, we 55 observed high spatial heterogeneity in these IAS geometric traits throughout the 56 mesophyll, especially within CAM leaves. Our data show that IAS traits that better 57 capture the 3D complexity of leaves strongly influence g IAS and that the impact of the 58 IAS on mesophyll conductance should be carefully considered with respect to leaf 59 anatomy. We provide a simple function to estimate tortuosity and lateral path 60 lengthening in the absence of access to imaging tools such as X-ray microCT or other 61 novel 3D image-processing techniques.
INTRODUCTION 63By volume, as little as 3% and up to 73% (this study) of 64 the inside of a leaf is composed of air. Such a wide range of values results from the 65 multiple roles that mesophyll cells play in leaf function, the degree of reticulation of the 66 embedded vein network, and cell size and shape, all reflecting the various adaptations 67 plants have made in colonizing nearly every terrestrial habitat on Earth. From an 68 evolutionary perspective, the transition from oceans to land exposed plant tissues to air, 69 which dramatically lowered the resistance for CO 2 diffusion to chloroplasts by ~10,000-70fold. Evolutionary development of the leaf intercellular airspace (IAS) is therefore 71 considered a key innovation to profit from that lowered diffusion resistance (Ligrone et 72 al., 2012). Yet, terrestrial inhabitation also exposed leaves to the risk of desiccation. 73Plants presumably navigated this trade-off by developing a complex spatial cellular 74 arrangement in order to produce a more or less tortuous 3D IAS network that rapidly 75 delivered CO 2 t...