Mind the leaf anatomy while taking ground truth with portable chlorophyll meters.

Measurements of four chlorophyll meters — three transmittance-based (SPAD-502, Dualex-4 Scientific, and MultispeQ 2.0) and one fluorescence-based (CCM-300), were calibrated against biochemically assessed chlorophyll content (Chl) on three distinctive common leaf types differing in leaf anatomy: laminar (i.e., broadleaved woody species with different anthocyanin content verified by biochemical assay) dorsiventral leaves, narrow grass leaves, and conifer needles. Reflectance in the 400-2500 nm range was measured on the laminar leaf samples using a contact probe.

Methods

In the present study we investigated three distinctive leaf anatomical types: laminar (i.e., deciduous woody species) leaves, grass leaves, and needles. The three groups are defined as follows: 1) laminar leaves of woody angiosperms dorsiventrally flattened (i.e., bifacial) leaves with differentiated mesophyll to palisade and spongy parenchyma and reticulate anastomosing vasculature (Laminar leaves). We further distinguished three anatomical subtypes of laminar leaves 1a) mesomorphic leaves of deciduous tree species, 1b) scleromorphic leaves of evergreen trees and shrubs, and 1c) scleromorphic leaves with pronounced hypodermis represented by Ficus species 2) The second group included C3 grasses with bifacial strap-like leaves with undifferentiated mesophyll with longitudinally arranged vasculature (Grass leaves), and 3) gymnosperm equilateral needle-like leaves without differentiated mesophyll and vascular bundle in the central cylinder (Needles) represented only by Norway spruce (Picea abies) though with irradiance induced differentiation into sun and shaded ecotypes.

Collection of leaf samples

Leaves were collected at four different locations in the Czech Republic during the growing seasons 2019, 2020, and 2021. The dates (as DOY - day of the year are indicated in particular datasets). Woody plants with laminar bifacial leaves with differentiated mesophyll were collected in the Botanical Garden of Charles University in Prague (50.072N, 14.424E). Plants were selected to correspond to one of the following leaf subtypes: 1) mesomorphic leaves of deciduous species, 2) scleromorphic leaves of evergreen trees and shrubs and 3) scleromorphic leaves with pronounced hypodermis represented by indoor grown Ficus species. Usually, shaded leaves were sampled from the ground.

For independent verification of the relationship of Chl content to chlorophyll meter reading, leaves were sampled in the floodplain forest at the confluence of the rivers Morava and Dyje, near the town of Lanžhot (48.682N, 16.946E) using deciduous woody plants with laminar bifacial leaves and differentiated mesophyll. Sunlit and shaded branches were cut by a tree climber from mature trees of Acer campestre L., Carpinus betulus L., Fraxinus angustifolia Vahl., Populus alba L., Quercus cerris L., Quercus robur L. and Tilia cordata Mill. 

Grass leaves were represented by four coexisting wild species from Poaceae family (Calamagrostis villosa (Chaix) J.F.Gmel., Deschampsia cespitosa (L.) P.Beauv., Molinia caerulea (L.) Moench and Nardus stricta L.) and were collected in relict alpine-arctic grass tundra in the Krkonoše (Giant Mountains) (50.734N, 15.696E). For each species, six plots with homogeneous canopy cover of the species were sampled. 

Needle leaves were represented by mature trees of Norway spruce (Picea abies (L.) H. Karst.) collected at the experimental station Bílý Kříž, Beskydy Mountains, Czech Republic (49.503N, 18.539E). Sunlit and shaded branches were cut by a tree climber, and samples were taken from the current year's needles, the previous year's needles, and four-year-old needles. 

Leaf sampling

Laminar leaves: leaves were measured immediately after being detached from the branch or stored in a refrigerator for no more than 30 minutes before processing. First, the reflectance of the leaves was measured using a spectroradiometer and a contact probe. Second, readings from all portable chlorophyll meters were recorded. Third, one disk (area = 68 mm²) was cut from each leaf for Chl and anthocyanin extraction. Finally, a square segment of the leaf was cut out and immersed in fixative solution for anatomical analysis. A second leaf of similar size, colour, position in the canopy, and developmental stage was removed from the branch, weighed, scanned, and later dried and weighed again. This "twin" was used to assess leaf mass per area (LMA), equivalent water thickness (EWT).

Grass leaves: chlorophyll meter readings were taken on grass leaves attached to the plant using a chlorophyll meter (CCM_CFR,) then leaves were collected immediately in the field for Chl extraction. A 2 cm long leaf segment was cut, flattened under a microscope glass, photographed for area assessment, and stored in plastic vials in a refrigerator before freezing. A subsample was weighed fresh, scanned, and dried for calculation of LMA and EWT.

Needles: Shoots were separated from the branch, sorted by age, and stored in a refrigerator for no longer than 24 hours before processing. First, CCM_CFR were taken from the middle part of three needles and the same needles were used for Chl extraction. A second parallel set of needles was immersed in fixative solution for anatomical analysis. The third set of needles was weighed fresh, scanned, and dried for calculation of LMA and EWT.

Optical assessment of Chl content using portable chlorophyll meters

Three transmittance-based chlorophyll meters: SPAD-502 SPAD), Dualex-4 Scientific (Dx) and MultispeQ (MSPQ), and one fluorescence chlorophyll meter: CCM-300 (CCM), were used for optical assessment of Chl content in leaves. For laminar leaves, three readings were taken on each leaf with each instrument from the adaxial leaf side. Measurements were taken in the central part of the leaf, avoiding the midrib and main veins. The three measurements were averaged, and the average was used as a representative value for the leaf. Measurements with all four chlorophyll meters (SPAD_values, Dx_values, MSPQ_values, CCM_CFR) were obtained for laminar leaves. For grass leaves, Chl values were measured at a single location in the apical third of the leaf blade. All four grass species were measured by CCM (CCM_CFR), and three species with a wide enough lamina to cover the SPAD measurement area (Calamagrostis villosa, Deschampsia cespitosa, and Molinia careulea) were also measured by SPAD and SPAD_values detected. For the spruce needles, three needles were measured only once with the CCM, always taking a reading in the central part of the needle. The average of these three needle measurements was used to relate to Chl.

Reflectance measurements and spectral processing

Reflectance was measured for laminar leaves collected in Botanical Garden of Charles University in Prague and deciduous trees from floodplain forest. Leaf reflectance from the adaxial side of the leaves was measured with an ASD FieldSpec 4 Wide-Res spectroradiometer with attached contact probe (ASD Inc., Boulder, CO, USA). Three measurements per leaf were always taken, when leaf size allowed. Measurements were placed at the same locations where chlorophyll meter readings were taken. Leaf reflectance spectra ranging from 350 to 2500 nm were normalized against a white reference spectrum (99% Spectralon white panel) to obtain relative reflectance spectra. The median of the spectral curve from three measurements was used as a representative value for the leaf.