A wide range of portable chlorophyll meters are increasingly being used to measure leaf chlorophyll content as an indicator of plant performance, providing reference data for remote sensing studies. We tested the effect of leaf anatomy on the relationship between optical assessments of chlorophyll (Chl) against biochemically determined Chl content as a reference. Optical Chl assessments included measurements taken by four chlorophyll meters: three transmittance-based (SPAD-502, Dualex-4 Scientific, and MultispeQ 2.0), one fluorescence-based (CCM-300), and vegetation indices calculated from the 400-2500 nm leaf reflectance acquired using an ASD FieldSpec and a contact plant probe. Three leaf types with different anatomy were included: dorsiventral laminar leaves, grass leaves, and needles. On laminar leaves, all instruments performed well for chlorophyll content estimation (R2 > 0.80, nRMSE < 15%), regardless of the variation in their specific internal structure (mesomorphic, scleromorphic, or scleromorphic with hypodermis), similarly to the performance of four reflectance indices (R2 > 0.90, nRMSE < 16%). For grasses, the model to predict chlorophyll content across multiple species had low performance with CCM-300 (R2 = 0.45, nRMSE = 11%) and failed for SPAD. For Norway spruce needles, the relation of CCM-300 values to chlorophyll content was also weak (R2 = 0.45, nRMSE = 11%). To improve the accuracy of data used for remote sensing algorithm development, we recommend calibration of chlorophyll meter measurements with biochemical assessments, especially for species with anatomy other than laminar dicot leaves. The take-home message is that portable chlorophyll meters perform well for laminar leaves and grasses with wider leaves, however, their accuracy is limited for conifer needles and narrow grass leaves. Species-specific calibrations are necessary to account for anatomical variations, and adjustments in sampling protocols may be required to improve measurement reliability.

Department of Applied Geoinformatics and Cartography Faculty of Science Charles University Albertov 6 12800 Prague Czech Republic

Department of Plant Experimental Biology Faculty of Science Charles University Viničná 5 12800 Prague Czech Republic

Global Change Research Institute of the Czech Academy of Sciences Bělidla 986 4a 60300 Brno Czech Republic

University of Maryland Baltimore County and NASA Goddard Space Flight Center Code 618 Greenbelt MD 20771 USA

Zobrazit více v PubMed

Richardson, A. D., Duigan, S. P. & Berlyn, G. P. An evaluation of noninvasive methods to estimate foliar chlorophyll content. DOI

Houborg, R., Anderson, M. & Daughtry, C. Utility of an image-based canopy reflectance modeling tool for remote estimation of LAI and leaf chlorophyll content at the field scale. DOI

Zhang, Y., He, N., Li, M., Yan, P. & Yu, G. Community chlorophyll quantity determines the spatial variation of grassland productivity. PubMed DOI

Brewster, C., Fenner, N. & Hayes, F. Chronic ozone exposure affects nitrogen remobilization in wheat at key growth stages. PubMed DOI

Gräf, M. et al. Application of leaf analysis in addition to growth assessment to evaluate the suitability of greywater for irrigation of PubMed DOI

Hong, H. et al. Warming delays but grazing advances leaf senescence of five plant species in an alpine meadow. PubMed DOI

Chi, D., Van Meerbeek, K., Yu, K., Degerickx, J. & Somers, B. Foliar optical traits capture physiological and phenological leaf plasticity in PubMed DOI

Lhotáková, Z. et al. Detection of multiple stresses in scots pine growing at post-mining sites using visible to near-infrared spectroscopy. PubMed DOI

Kandpal, K. C. & Kumar, A. Migrating from invasive to noninvasive techniques for enhanced leaf chlorophyll content estimations efficiency. PubMed DOI

Beamish, A. et al. Recent trends and remaining challenges for optical remote sensing of DOI

Burnett, A. C. et al. A best-practice guide to predicting plant traits from leaf-level hyperspectral data using partial least squares regression. PubMed DOI

Angel, Y. & McCabe, M. F. Machine learning strategies for the retrieval of leaf-chlorophyll dynamics: Model choice, sequential versus retraining learning, and hyperspectral predictors. PubMed DOI PMC

Gates, D. M., Keegan, H. J., Schleter, J. C. & Weidner, V. R. Spectral properties of plants. DOI

Lichtenthaler, H. K. [34] Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. in (ed. Enzymology, B.-M. in) vol. 148 350–382 (Academic Press, 1987).

Lichtenthaler, H. K., Buschmann, C., Rinderle, U. & Schmuck, G. Application of chlorophyll fluorescence in ecophysiology. PubMed DOI

Gitelson, A. A. & Solovchenko, A. Non-invasive quantification of foliar pigments: Possibilities and limitations of reflectance- and absorbance-based approaches. PubMed DOI

Jacquemoud, S. & Ustin, S.

Mershon, J., Becker, M. & Bickford, C. Linkage between trichome morphology and leaf optical properties in New Zealand alpine DOI

Neuwirthová, E., Lhotáková, Z., Lukeš, P. & Albrechtová, J. Leaf surface reflectance does not affect biophysical traits modelling from VIS-NIR spectra in plants with sparsely distributed trichomes. DOI

Gitelson, A. A., Buschmann, C. & Lichtenthaler, H. K. Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements. DOI

Gitelson, A. A., Chivkunova, O. B. & Merzlyak, M. N. Nondestructive estimation of anthocyanins and chlorophylls in anthocyanic leaves. PubMed DOI

Junker, L. V. & Ensminger, I. Relationship between leaf optical properties, chlorophyll fluorescence and pigment changes in senescing PubMed DOI

Hoch, W. A., Singsaas, E. L. & McCown, B. H. Resorption protection. Anthocyanins facilitate nutrient recovery in autumn by shielding leaves from potentially damaging light levels. PubMed DOI PMC

Tattini, M. et al. Epidermal coumaroyl anthocyanins protect sweet basil against excess light stress: Multiple consequences of light attenuation. PubMed DOI

Gould, K. S., Jay-Allemand, C., Logan, B. A., Baissac, Y. & Bidel, L. P. R. When are foliar anthocyanins useful to plants? Re-evaluation of the photoprotection hypothesis using DOI

Jordheim, M. et al. High concentrations of aromatic acylated anthocyanins found in cauline hairs in PubMed DOI

Merzlyak, M. N., Chivkunova, O. B., Solovchenko, A. E. & Naqvi, K. R. Light absorption by anthocyanins in juvenile, stressed, and senescing leaves. PubMed DOI PMC

Donnelly, A., Yu, R., Rehberg, C., Meyer, G. & Young, E. B. Leaf chlorophyll estimates of temperate deciduous shrubs during autumn senescence using a SPAD-502 meter and calibration with extracted chlorophyll. DOI

Kuhlgert, S. et al. MultispeQ Beta: A tool for large-scale plant phenotyping connected to the open PhotosynQ network. PubMed DOI PMC

Parry, C., Blonquist, J. M. & Bugbee, B. In situ measurement of leaf chlorophyll concentration: Analysis of the optical/absolute relationship: The optical/absolute chlorophyll relationship. PubMed DOI

Brown, L. A., Williams, O. & Dash, J. Calibration and characterisation of four chlorophyll meters and transmittance spectroscopy for non-destructive estimation of forest leaf chlorophyll concentration. DOI

Cerovic, Z. G., Masdoumier, G., Ghozlen, N. B. & Latouche, G. A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll and epidermal flavonoids. PubMed DOI PMC

Goulas, Y., Cerovic, Z. G., Cartelat, A. & Moya, I. Dualex: A new instrument for field measurements of epidermal ultraviolet absorbance by chlorophyll fluorescence. PubMed DOI

Buschmann, C. Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves. PubMed DOI

Gitelson, A. A., Buschmann, C. & Lichtenthaler, H. K. The chlorophyll fluorescence ratio F735/F700 as an accurate measure of the chlorophyll content in plants. DOI

Verrelst, J. et al. Quantifying vegetation biophysical variables from imaging spectroscopy data: A review on retrieval methods. PubMed DOI PMC

Féret, J.-B. et al. Estimating leaf mass per area and equivalent water thickness based on leaf optical properties: Potential and limitations of physical modeling and machine learning. DOI

Serrano, L. Effects of leaf structure on reflectance estimates of chlorophyll content. DOI

Slaton, M. R., Hunt, E. R. & Smith, W. K. Estimating near-infrared leaf reflectance from leaf structural characteristics. PubMed DOI

Ustin, S. & Jacquemoud, S.

Baránková, B., Lazár, D. & Nauš, J. Analysis of the effect of chloroplast arrangement on optical properties of green tobacco leaves. DOI

Fukushima, K. & Hasebe, M. Adaxial-abaxial polarity: The developmental basis of leaf shape diversity: Development and evolution of leaf types. PubMed DOI

Conklin, P. A., Strable, J., Li, S. & Scanlon, M. J. On the mechanisms of development in monocot and eudicot leaves. PubMed DOI

Marenco, R. A., Antezana-Vera, S. A. & Nascimento, H. C. S. Relationship between specific leaf area, leaf thickness, leaf water content and SPAD-502 readings in six Amazonian tree species. DOI

Coste, S. et al. Assessing foliar chlorophyll contents with the SPAD-502 chlorophyll meter: A calibration test with thirteen tree species of tropical rainforest in French Guiana. DOI

Evans, J., Caemmerer, S., Setchell, B. & Hudson, G. The relationship between CO2 transfer conductance and leaf anatomy in transgenic tobacco with a reduced content of rubisco. DOI

Aasamaa, K., Niinemets, Ü. & Sõber, A. Leaf hydraulic conductance in relation to anatomical and functional traits during PubMed DOI

Jifon, J. L., Syvertsen, J. P. & Whaley, E. Growth environment and leaf anatomy affect nondestructive estimates of chlorophyll and nitrogen in Citrus sp. Leaves. DOI

Nauš, J., Prokopová, J., Řebíček, J. & Špundová, M. SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement. PubMed DOI

McClendon, J. H. & Fukshansky, L. On the interpretation of absorption spectra of leaves–II. The non-absorbed ray of the sieve effect and the mean optical pathlength in the remainder of the leaf. DOI

Davis, P. A., Caylor, S., Whippo, C. W. & Hangarter, R. P. Changes in leaf optical properties associated with light-dependent chloroplast movements: Chloroplast movement and leaf optics. PubMed DOI

McClendon, J. H. & Fukshansky, L. On the interpretation of absorption spectra of leaves–I. Introduction and the correction of leaf spectra for surface reflection. DOI

Uddling, J., Gelang-Alfredsson, J., Piikki, K. & Pleijel, H. Evaluating the relationship between leaf chlorophyll concentration and SPAD-502 chlorophyll meter readings. PubMed DOI

Dong, T. et al. Assessment of portable chlorophyll meters for measuring crop leaf chlorophyll concentration. DOI

Lukeš, P., Neuwirthová, E., Lhotáková, Z., Janoutová, R. & Albrechtová, J. Upscaling seasonal phenological course of leaf dorsiventral reflectance in radiative transfer model. DOI

Bercu, R. Anatomical aspects of Ficus lyrata Warb. (Moraceae) leaf.

Bercu, R. Some general anatomical aspects of

Falcioni, R. et al. High resolution leaf spectral signature as a tool for foliar pigment estimation displaying potential for species differentiation. PubMed DOI

Zar, J. H.

Dong, R. et al. Estimating plant nitrogen concentration of maize using a leaf fluorescence sensor across growth stages. DOI

Ghosh, M., Swain, D. K., Jha, M. K., Tewari, V. K. & Bohra, A. Optimizing chlorophyll meter (SPAD) reading to allow efficient nitrogen use in rice and wheat under rice-wheat cropping system in eastern India. DOI

Padilla, F. M. et al. Influence of time of day on measurement with chlorophyll meters and canopy reflectance sensors of different crop N status. DOI

Arellano, P., Tansey, K., Balzter, H. & Boyd, D. S. Field spectroscopy and radiative transfer modelling to assess impacts of petroleum pollution on biophysical and biochemical parameters of the Amazon rainforest. DOI

Colzi, I. et al. Impact of microplastics on growth, photosynthesis and essential elements in PubMed DOI

Zhen, J. et al. Mapping leaf chlorophyll content of mangrove forests with Sentinel-2 images of four periods.

Cary, K. L. & Pittermann, J. Small trees, big problems: Comparative leaf function under extreme edaphic stress. PubMed DOI

Huemmrich, K. F. et al. Leaf-level chlorophyll fluorescence and reflectance spectra of high latitude plants. DOI

Darvishzadeh, R. et al. Mapping leaf chlorophyll content from Sentinel-2 and RapidEye data in spruce stands using the invertible forest reflectance model.

Fiorentini, M. et al. Nitrogen and chlorophyll status determination in durum wheat as influenced by fertilization and soil management: Preliminary results. PubMed DOI PMC

Červená, L. et al. Determination of chlorophyll content in selected grass communities of Krkonoše Mts. tundra based on laboratory spectroscopy and aerial hyperspectral data.. DOI

Ludwig, A. D., Doktor, D., Goss, R., Sasso, S. & Feilhauer, H. The leaf is always greener on the other side of the lab: Optical in-situ indicators for leaf chlorophyll content need improvement for semi-natural grassland areas. DOI

McClendon, J. H. The micro-optics of leaves. I. Patterns of reflection from the epidermis.

Liu, S. Comparison of two noninvasive methods for measuring the pigment content in foliose macrolichens. PubMed DOI

Bachofen, C., D’Odorico, P. & Buchmann, N. Light and VPD gradients drive foliar nitrogen partitioning and photosynthesis in the canopy of European beech and silver fir. PubMed DOI

Hoeppner, J. M. et al. Mapping canopy chlorophyll content in a temperate forest using airborne hyperspectral data. DOI

Lai, Y. et al. Bidirectional reflectance factor measurement of conifer needles with microscopic spectroscopy imaging. DOI

Ren, J. et al. Tree growth response to soil nutrients and neighborhood crowding varies between mycorrhizal types in an old-growth temperate forest. PubMed DOI

Olascoaga, B., Mac Arthur, A., Atherton, J. & Porcar-Castell, A. A comparison of methods to estimate photosynthetic light absorption in leaves with contrasting morphology. PubMed DOI PMC

Malenovský, Z. et al. Applicability of the PROSPECT model for Norway spruce needles. DOI

Mesarch, M. A., Walter-Shea, E. A., Asner, G. P., Middleton, E. M. & Chan, S. S. A revised measurement methodology for conifer needles spectral optical properties: Evaluating the influence of gaps between elements.

Yáñez-Rausell, L., Malenovsky, Z., Clevers, J. G. P. W. & Schaepman, M. E. Minimizing measurement uncertainties of coniferous needle-leaf optical properties. Part II: Experimental setup and error analysis. DOI

Čepl, J. et al. Heritable variation in needle spectral reflectance of Scots pine ( DOI

Hejtmánek, J. et al. Revealing the complex relationship among hyperspectral reflectance, photosynthetic pigments, and growth in Norway spruce ecotypes. PubMed DOI PMC

Einzmann, K. et al. Early detection of spruce vitality loss with hyperspectral data: Results of an experimental study in Bavaria Germany. DOI

Zhang, Y., Wang, A., Li, J. & Wu, J. Water content estimation of conifer needles using leaf-level hyperspectral data. PubMed DOI PMC

Neuwirthová, E. et al. Leaf age matters in remote sensing: Taking ground truth for spectroscopic studies in hemiboreal deciduous trees with continuous leaf formation. DOI

Borsuk, A. M. & Brodersen, C. R. The spatial distribution of chlorophyll in leaves. PubMed DOI PMC

Jacquemoud, S. & Baret, F. PROSPECT: A model of leaf optical properties spectra. DOI

Jiang, J., Comar, A., Weiss, M. & Baret, F. FASPECT: A model of leaf optical properties accounting for the differences between upper and lower faces. DOI

Shi, H., Jiang, J., Jacquemoud, S., Xiao, Z. & Ma, M. Estimating leaf mass per area with leaf radiative transfer model. DOI

Kallel, A. FluLCVRT: Reflectance and fluorescence of leaf and canopy modeling based on Monte Carlo vector radiative transfer simulation. DOI

Kallel, A. Leaf polarized BRDF simulation based on Monte Carlo 3-D vector RT modeling. DOI

Théroux-Rancourt, G. et al. Digitally deconstructing leaves in 3D using X-ray microcomputed tomography and machine learning. PubMed DOI PMC

Borsuk, A. M., Roddy, A. B., Théroux-Rancourt, G. & Brodersen, C. R. Structural organization of the spongy mesophyll. PubMed DOI PMC

Casa, R., Castaldi, F., Pascucci, S. & Pignatti, S. Chlorophyll estimation in field crops: An assessment of handheld leaf meters and spectral reflectance measurements. DOI

Neuwirthová, E. et al. Asymmetry of leaf internal structure affects PLSR modelling of anatomical traits using VIS-NIR leaf level spectra. DOI

Švik, M. et al. Retrieving plant functional traits through time series analysis of satellite observations using machine learning methods. DOI

Hunt, L. et al. Leaf functional traits in relation to species composition in an arctic-alpine tundra grassland. PubMed DOI PMC

Lhotáková, Z. et al. Foliage biophysical trait prediction from laboratory spectra in norway spruce is more affected by needle age than by site soil conditions. DOI

Markwell, J., Osterman, J. C. & Mitchell, J. L. Calibration of the Minolta SPAD-502 leaf chlorophyll meter. PubMed DOI

Wellburn, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. DOI

Mancinelli, A. L., Yang, C.-P.H., Lindquist, P., Anderson, O. R. & Rabino, I. Photocontrol of anthocyanin synthesis. PubMed DOI PMC

R Core Team. R: The R Project for Statistical Computing,R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. https://www.r-project.org/ (2021).

Tang, Y., Horikoshi, M. & Li, W. ggfortify: Unified interface to visualize statistical results of popular R packages. DOI