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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.

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Fig. 1: Diagram of the saturation front moving deeper within the leaf as Δw increases.
Fig. 2: Uncorrected gas exchange measurements of a cotton leaf using a double-sided, clamp-on chamber.
Fig. 3: Series resistances to the diffusion of water vapour across a cotton leaf.
Fig. 4: Mesophyll conductance to the diffusion of CO2 (gm) as a function of Δw.

Data Availability

All generated and analysed data from this study are included in the published article and its Supplementary Information.


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We thank P. Groeneveld for his assistance in building the leaf chamber and parts for the gas exchange system. G.D.F. and L.A.C. acknowledge ARC support in the form of a Discovery Grant (no. DP210103186). We thank W. Stiller, CSIRO Agriculture and Food, Narrabri, Australia, for providing the cotton seeds.

Author information

Authors and Affiliations



S.C.W. conducted the gas exchange measurements and developed the concept. The late M.J.C. conceived the initial concept. M.H.-P. worked on the oxygen isotopes and analysis. H.S.-W. worked on the noble gases and isotopes. L.A.C. worked on the oxygen isotopes and analysis. D.A.M. measured the water potentials. G.D.F. and D.A.M. worked on the theory and modelling. All authors contributed to concept development and writing the paper.

Corresponding author

Correspondence to Graham D. Farquhar.

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The authors declare no competing interests.

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Nature Plants thanks Michael Blatt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Uncorrected gas exchange measurements in a sunflower leaf using a double-sided clamp-on chamber.

(a) CO2 assimilation rate, A, transpiration rate, E, and leaf conductance to water vapour, g, as functions of increasing Δw = wsat - wa. (b) The internal CO2 concentration, ci, of upper and lower substomatal cavities, and the ci difference (upper minus lower) between them are plotted as functions of Δw = wsat - wa. Dotted line denotes zero difference between upper (adaxial) ci and lower (abaxial) ci. Photosynthetically active radiation was fixed at 1000 µmol m−2 s−1.

Extended Data Fig. 2 Relative humidity inside (a) two cotton leaves and (b) five sunflower leaves.

Relative humidity inside (a) two cotton leaves and (b) five sunflower leaves as estimated by the ci difference technique and simultaneously (within minutes) by the oxygen isotope method.

Extended Data Fig. 3 Resistances to water diffusion.

Resistances to water diffusion estimated using routine gas exchange calculations (\(R_{{{{\mathrm{H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\)), corrected values (\(cR_{{{{\mathrm{H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\)) and using noble gases (\(R_{{{{\mathrm{argon - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\),\(R_{{{{\mathrm{neon - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\) and \(R_{{{{\mathrm{helium - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\)). Estimations of the unsaturated mesophyll air space resistance as \(R_{{{{\mathrm{unsat}}}}} = R_{{{{\mathrm{H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}} - cR_{{{{\mathrm{H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\) and the intercellular air space resistance to water as \(R_{{{{\mathrm{ias - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}} = R_{{{{\mathrm{x - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}} - cR_{{{{\mathrm{H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\). Three leaves are presented as examples for the noble gases experiments, cotton leaf using argon (a and b), sunflower using neon (c and d) and cotton using helium (e and f).

Extended Data Fig. 4 Comparison of \(R_{{{{\mathrm{ias - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\)derived from noble gas measurements.

Comparison of \(R_{{{{\mathrm{ias - H}}}}_{{{\mathrm{2}}}}{{{\mathrm{O}}}}}\)derived from noble gas measurements, with that derived more crudely but simply as 2(ciu-cil)/A.

Supplementary information

Supplementary Information

Supplementary Sections 1–3.

Reporting Summary

Supplementary Data 1

Measurements for the ten species.

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Wong, S.C., Canny, M.J., Holloway-Phillips, M. et al. Humidity gradients in the air spaces of leaves. Nat. Plants 8, 971–978 (2022).

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