Unsaturation in the air spaces of leaves and its implications.

Modern plant physiological theory stipulates that the resistance to water movement from plants to the atmosphere is overwhelmingly dominated by stomata. This conception necessitates a corollary assumption-that the air spaces in leaves must be nearly saturated with water vapour; that is, with a relative humidity that does not decline materially below unity. As this idea became progressively engrained in scientific discourse and textbooks over the last century, observations inconsistent with this corollary assumption were occasionally reported. Yet, evidence of unsaturation gained little traction, with acceptance of the prevailing framework motivated by three considerations: (1) leaf water potentials measured by either thermocouple psychrometry or the Scholander pressure chamber are largely consistent with the framework; (2) being able to assume near saturation of intercellular air spaces was transformational to leaf gas exchange analysis; and (3) there has been no obvious mechanism to explain a variable, liquid-phase resistance in the leaf mesophyll. Here, we review the evidence that refutes the assumption of universal, near saturation of air spaces in leaves. Refining the prevailing paradigm with respect to this assumption provides opportunities for identifying and developing mechanisms for increased plant productivity in the face of increasing evaporative demand imposed by global climate change.

Humidity gradients in the air spaces of leaves.

Stomata are orifices that connect the drier atmosphere with the interconnected network of more humid air spaces that surround the cells within a leaf. Accurate values of the humidities inside the substomatal cavity, wi, and in the air, wa, are needed to estimate stomatal conductance and the CO2 concentration in the internal air spaces of leaves. Both are vital factors in the understanding of plant physiology and climate, ecological and crop systems. However, there is no easy way to measure wi directly. Out of necessity, wi has been taken as the saturation water vapour concentration at leaf temperature, wsat, and applied to the whole leaf intercellular air spaces. We explored the occurrence of unsaturation by examining gas exchange of leaves exposed to various magnitudes of wsat - wa, or Δw, using a double-sided, clamp-on chamber, and estimated degrees of unsaturation from the gradient of CO2 across the leaf that was required to sustain the rate of CO2 assimilation through the upper surface. The relative humidity in the substomatal cavities dropped to about 97% under mild Δw and as dry as around 80% when Δw was large. Measurements of the diffusion of noble gases across the leaf indicated that there were still regions of near 100% humidity distal from the stomatal pores. We suggest that as Δw increases, the saturation edge retreats into the intercellular air spaces, accompanied by the progressive closure of mesophyll aquaporins to maintain the cytosolic water potential.

Unsaturation of vapour pressure inside leaves of two conifer species.

Stomatal conductance (gs) impacts both photosynthesis and transpiration, and is therefore fundamental to the global carbon and water cycles, food production, and ecosystem services. Mathematical models provide the primary means of analysing this important leaf gas exchange parameter. A nearly universal assumption in such models is that the vapour pressure inside leaves (ei) remains saturated under all conditions. The validity of this assumption has not been well tested, because so far ei cannot be measured directly. Here, we test this assumption using a novel technique, based on coupled measurements of leaf gas exchange and the stable isotope compositions of CO2 and water vapour passing over the leaf. We applied this technique to mature individuals of two semiarid conifer species. In both species, ei routinely dropped below saturation when leaves were exposed to moderate to high air vapour pressure deficits. Typical values of relative humidity in the intercellular air spaces were as low 0.9 in Juniperus monosperma and 0.8 in Pinus edulis. These departures of ei from saturation caused significant biases in calculations of gs and the intercellular CO2 concentration. Our results refute the longstanding assumption of saturated vapour pressure in plant leaves under all conditions.

Differential shrinkage of mesophyll cells in transpiring cotton leaves: implications for static and dynamic pools of water, and for water transport pathways.

Shrinkage of palisade cells during transpiration, previously measured for sclerophyllous leaves of Eucalyptus where cells shrank equally, was compared with shrinkage in thin mesophytic leaves of cotton (Gossypium hirsutum L.). Selected vapour pressure differences (Δe) from 0.6 to 2.7kPa were imposed during steady-state photosynthesis and transpiration. Leaves were then cryo-fixed and cryo-planed paradermally, and images obtained with a cryo-scanning electron microscope (CSEM). Diameters of palisade 'cavity cells' within sub-stomatal cavities, and surrounding palisade 'matrix cells' were measured on CSEM images. Cavity and spongy mesophyll cells shrank progressively down to Δe=2.7kPa, while matrix cells remained at the same diameter at all Δe. Diameters were also measured of cavity and matrix cells quasi-equilibrated with relative humidities (RHs) from 100% to 86%. In leaves quasi-equilibrated with 95% RH, the cavity cells shrank so much as to be almost unmeasurable, while matrix cells shrank by only 6%. These data suggest that there are two distinct pools of water in cotton leaves: cavity plus spongy mesophyll cells (two-thirds of leaf volume) which easily lose water; and matrix cells (one-third of leaf volume), which retain turgor down to relative water loss=0.4, providing structural rigidity to prevent wilting. This phenomenon is probably widespread among mesophytic leaves.

Environmental effects on oxygen isotope enrichment of leaf water in cotton leaves.

The oxygen isotope enrichment of bulk leaf water (Delta(b)) was measured in cotton (Gossypium hirsutum) leaves to test the Craig-Gordon and Farquhar-Gan models under different environmental conditions. Delta(b) increased with increasing leaf-to-air vapor pressure difference (VPd) as an overall result of the responses to the ratio of ambient to intercellular vapor pressures (e(a)/e(i)) and to stomatal conductance (g(s)). The oxygen isotope enrichment of lamina water relative to source water (Delta(1)), which increased with increasing VPd, was estimated by mass balance between less enriched water in primary veins and enriched water in the leaf. The Craig-Gordon model overestimated Delta(b) (and Delta(1)), as expected. Such discrepancies increased with increase in transpiration rate (E), supporting the Farquhar-Gan model, which gave reasonable predictions of Delta(b) and Delta(1) with an L of 7.9 mm, much less than the total radial effective length L(r) of 43 mm. The fitted values of L for Delta(1) of individual leaves showed little dependence on VPd and temperature, supporting the assumption that the Farquhar-Gan formulation is relevant and useful in describing leaf water isotopic enrichment.

(18)O spatial patterns of vein xylem water, leaf water, and dry matter in cotton leaves.

Three leaf water models (two-pool model, Péclet effect, and string-of-lakes) were assessed for their robustness in predicting leaf water enrichment and its spatial heterogeneity. This was achieved by studying the (18)O spatial patterns of vein xylem water, leaf water, and dry matter in cotton (Gossypium hirsutum) leaves grown at different humidities using new experimental approaches. Vein xylem water was collected from intact transpiring cotton leaves by pressurizing the roots in a pressure chamber, whereas the isotopic content of leaf water was determined without extracting it from fresh leaves with the aid of a purpose-designed leaf punch. Our results indicate that veins have a significant degree of lateral exchange with highly enriched leaf water. Vein xylem water is thus slightly, but progressively enriched in the direction of water flow. Leaf water enrichment is dependent on the relative distances from major veins, with water from the marginal and intercostal regions more enriched and that next to veins and near the leaf base more depleted than the Craig-Gordon modeled enrichment of water at the sites of evaporation. The spatial pattern of leaf water enrichment varies with humidity, as expected from the string-of-lakes model. This pattern is also reflected in leaf dry matter. All three models are realistic, but none could fully account for all of the facets of leaf water enrichment. Our findings acknowledge the presence of capacitance in the ground tissues of vein ribs and highlight the essential need to incorporate Péclet effects into the string-of-lakes model when applying it to leaves.