Method-Dependent Variation in Stomatal Trait Estimation and Its Functional Implications in Oryza Species

Reliable quantification of stomatal traits is essential for understanding plant gas exchange and its responses to environmental changes. However, grasses present methodological challenges due to their complex leaf surface topography and the distinctive dumbbell-shaped stomata arranged in longitudinal rows. Here, we combined a literature-based synthesis with comparative experiments to assess how measurement methods influence stomatal trait estimates and to characterize interspecific variation and environmental plasticity across Oryza species. Literature-based synthesis revealed large cross-study variations in reported stomatal density, with apparent differences of up to one order of magnitude among studies using SEM and impression-based methods, although this variation may also reflect differences in genotypes, environments, developmental stages, and sampling protocols. Experimental comparisons confirmed consistent differences between SEM and imprint measurements, although the differences were smaller in magnitude. Across species, stomatal density, guard-cell length, and guard-cell width exhibited substantial diversity and conserved rankings across environments, while guard-cell length showed greater environmental sensitivity. Moreover, method-dependent differences significantly affected estimates of anatomical maximum stomatal conductance. Our results demonstrate that measurement methodology is an important source of uncertainty in stomatal trait estimation, even within the same leaf, and that Oryza species display broad interspecific morphological variations but comparatively constrained environmental responses. These findings highlight the need to standardize imaging protocols and to account for method-dependent differences for accurate interpretation of relationships between stomatal morphology and functions in rice.