Connecting leaf structure with function in amphistomatic leaves

While different distributions of stomata between the surfaces of laminar leaves have long been recognised and correlated with environmental conditions, there remain significant gaps in our understanding of how the stomatal distribution impacts key functional processes such as gas exchange, the movement of water inside leaves and the hydraulic triggers for stomatal closure. Amphistomaty (where leaves have stomata present on both leaf surfaces) has potential agricultural applications and may be a useful paleo-proxy for open vegetation types. These applications have driven a renewed interest in understanding the benefits and disadvantages of amphistomatic leaves compared with the more common, 'hypostomatic' leaves, which have all stomata limited to the lower leaf surface. In this thesis, I examine amphistomatic leaves from a functional or mechanistic viewpoint to understand potential advantages, disadvantages and insights into the evolutionary driver (s) of this mode of stomatal distribution. I consider both leaf anatomy and morphology (leaf form) and how stomata on both leaf surfaces instead of on a single surface can change the critical physiological processes of gas exchange and leaf water transport. When each surface of an amphistomatic leaf experiences a different evaporative demand, maintaining an optimal balance between carbon uptake and transpirational water loss would require both surfaces to regulate their stomata independently of one another. My results indicate that there is a degree of independence between the leaf surfaces such that the hydraulic conditions experienced in the tissues outside the shared xylem network for one leaf surface can differ from the hydraulic conditions experienced simultaneously by the opposite leaf surface. This means that temperature gradients through the leaf generated by natural conditions can, in fact, result in differences in stomatal operation between surfaces. The extent to which stomata on different leaf surfaces can operate independently is influenced by the overall leaf water content, as water loss via one surface can decrease the water available to the opposite surface. My results demonstrate that when there is less water available within a leaf, the independent stomatal regulation of surfaces is compromised. Thus, when the water available within a leaf is limited, the reliance on a shared vascular network may reduce the ability of each surface to optimise gas exchange independently. I found no evidence of an amphistomatic advantage relating to construction efficiency; vein density to stomatal density ratio was consistent with that of hypostomatic species. For dorsiventral Helianthus annuus leaves with differentiated cell types in the upper and lower portions of the leaf, differences in the length of the hydraulic pathway were associated with differences in hydraulic conductance when water loss was driven through the upper leaf surface as opposed to the lower leaf surface. These results imply that a reduced capacity to supply water to the upper surface, combined with consistently higher radiant heat load (horizontally orientated leaves), would result in the upper stomata closing earlier and more frequently than lower stomata. This could reduce the benefits associated with amphistomaty and potentially lead to fewer upper stomata. Vapour pressure deficit (VPD) is a principal determinant of the transpirational water loss from within a leaf to the surrounding air. Consequently, the stomatal response to VPD is critical for both large-scale modelling applications as well as a smaller-scale understanding of mechanistic leaf function. In this thesis, I investigated several poorly understood aspects of this response using amphistomatic, Eucalyptus globulus leaves. These include whether the trigger for stomatal response is localised or regional and the extent to which abscisic acid (ABA) initiates and maintains stomatal closure. Hysteretic reopening of stomata following VPD driven closure suggests that ABA plays a role in the localised, surface-specific stomatal closure. Comparisons between the equilibrated leaf water potential at which significant ABA increases occur, and the calculated xylem water potential at which stomatal closure commences, indicates that for localised stomatal closure, biosynthesis of foliar ABA occurs outside of the vasculature within the mesophyll tissue due to the calculated xylem water potential being less negative than that required to trigger ABA production. This suggests a large gradient in water potential exists through the mesophyll of a transpiring leaf and is contrary to previous research that suggests ABA biosynthesis is triggered within the vascular tissue. A detailed, mechanistic understanding of how leaves with stomata on two leaf surfaces operate underpins our ability to confidently utilise the stomatal ratio for agricultural improvement or as a paleo-proxy. Throughout this thesis, I demonstrate that while the advantage of increased CO2 uptake appears to be a central driver toward amphistomaty, leaf hydraulic function dictates the extent to which the gas exchange efficiencies can be realised under different environmental settings.