The aims of the study were: (i) to compare leaf reflectance in visible (VIS) (400–700 nm), near-infrared (NIR) (740–1140 nm) and short-wave infrared (SWIR) (2000–2400 nm) spectral ranges measured monthly by a contact probe on a single leaf and a stack of five leaves (measurement setup (MS)) of two broadleaved tree species during the vegetative season; and (ii) to test if and how selected vegetation indices differ under these two MS. In VIS, the pigment-related spectral region, the effect of MS on reflectance was negligible. The major influence of MS on reflectance was detected in NIR (up to 25%), the structure-related spectral range; and weaker effect in SWIR, the water-related spectral range. Vegetation indices involving VIS wavelengths were independent of MS while indices combining wavelengths from both VIS and NIR were MS-affected throughout the season. The effect of leaf stacking contributed to weakening the correlation between the leaf chlorophyll content and selected vegetation indices due to a higher leaf mass per area of the leaf sample. The majority of MS-affected indices were better correlated with chlorophyll content in both species in comparison with MS-unaffected indices. Therefore, in terms of monitoring leaf chlorophyll content using the contact probe reflectance measurement, these MS-affected indices should be used with caution, as discussed in the paper. If the vegetation indices are used for assessment of plant physiological status in various times of the vegetative season, then it is essential to take into consideration their possible changes induced by the particular contact probe measurement setup regarding the leaf stacking.

Hyperspectral reflectance contains valuable information about leaf functional traits, which can indicate a plant’s physiological status. Therefore, using hyperspectral reflectance for high-throughput phenotyping of foliar traits could be a powerful tool for tree breeders and nursery practitioners to distinguish and select seedlings with desired adaptation potential to local environments. We evaluated the use of 2 nondestructive methods (i.e., leaf and proximal/canopy) measuring hyperspectral reflectance in the 350- to 2,500-nm range for phenotyping on 1,788 individual Scots pine seedlings belonging to lowland and upland ecotypes of 3 different local populations from the Czech Republic. Leaf-level measurements were collected using a spectroradiometer and a contact probe with an internal light source to measure the biconical reflectance factor of a sample of needles placed on a black background in the contact probe field of view. The proximal canopy measurements were collected under natural solar light, using the same spectroradiometer with fiber optical cable to collect data on individual seedlings’ hemispherical conical reflectance factor. The latter method was highly susceptible to changes in incoming radiation. Both spectral datasets showed statistically significant differences among Scots pine populations in the whole spectral range. Moreover, using random forest and support vector machine learning algorithms, the proximal data obtained from the top of the seedlings offered up to 83% accuracy in predicting 3 different Scots pine populations. We conclude that both approaches are viable for hyperspectral phenotyping to disentangle the phenotypic and the underlying genetic variation within Scots pine seedlings.

We examined the seasonal changes in biophysical, anatomical, and optical traits of young leaves, formed throughout the vegetative season due to sylleptic growth, and mature leaves formed by proleptic growth in spring. Leaf developmental categories contribute to the top-of-canopy reflectance and should be considered when taking ground truth for remote sensing studies (RS). Deciduous tree species, Betula pendula, Populus tremula, and Alnus incana, were sampled from May to October 2018 in an Estonian hemiboreal forest. Chlorophyll and carotenoid content were detected biochemically; leaf anatomical traits (leaf, palisade, and spongy mesophyll thickness) were measured on leaf cross-sections; leaf reflectance was measured by a spectroradiometer with an integrating sphere (350–2500 nm). Biophysical and anatomical leaf traits were related to 64 vegetation indices (VIs). Linear models based on VIs for all tested leaf traits were more robust if both juvenile and mature leaves were included. This study provides information on which VIs are interchangeable or independent. Pigment and leaf thickness sensitive indices formed PC1; water and structural trait related VIs formed an independent group associated with PC3. Type of growth and leaf age could affect the validation of biophysical and anatomical leaf trait retrieval from the optical signal. It is, therefore, necessary to sample both leaf developmental categories—young and mature—in RS, especially if sampling is only once within the vegetation season.

Scaling leaf-level optical signals to the canopy level is essential for airborne and satellite-based forest monitoring. In evergreen trees, biophysical and optical traits may change as foliage ages. This study aims to evaluate the effect of age in Norway spruce needle on biophysical trait-prediction based on laboratory leaf-level spectra. Mature Norway spruce trees were sampled at forest stands in ten headwater catchments with different soil properties. Foliage biophysical traits (pigments, phenolics, lignin, cellulose, leaf mass per area, water, and nitrogen content) were assessed for three needle-age classes. Complementary samples for needle reflectance and transmittance were measured using an integrating sphere. Partial least square regression (PLSR) models were constructed for predicting needle biophysical traits from reflectance—separating needle age classes and assessing all age classes together. The ten study sites differed in soil properties rather than in needle biophysical traits. Optical properties consistently varied among age classes; however, variation related to the soil conditions was less pronounced. The predictive power of PLSR models was needle-age dependent for all studied traits. The following traits were predicted with moderate accuracy: needle pigments, phenolics, leaf mass per area and water content. PLSR models always performed better if all needle age classes were included (rather than individual age classes separately). This also applied to needle-age independent traits (water and lignin). Thus, we recommend including not only current but also older needle traits as a ground truth for evergreen conifers with long needle lifespan.

Scots pine ( Pinus sylvestris L.) is characterized by considerable intraspecific adaptive variability in response to environmental stress factors due to its wide geographical range. Adaptability is key for forestry, promising resilience against upcoming Europe's climate‐driven droughts. We studied three provenances of pedigreed Scots pine seedlings from distinct upland and lowland habitats in the Czech Republic. A water deficit was induced in 2‐year‐old, potted seedlings in a greenhouse. Their physiological responses to drought were investigated at the beginning of growing season during the development of new shoots, and after subsequent summer rewatering. (1) We analyzed several physiological traits to assess their effectiveness in detecting treatment effects: steady‐state quantum yield of PSII (QY Lss), maximum quantum yield of PSII (QY max), steady‐state non‐photochemical quenching (NPQ Lss), needle chlorophyll fluorescence ratio (SFR_R), and needle temperature normalized to ambient temperature (∆T), using a high‐throughput phenotyping unit. The divergence in SFR_R, QY max, QY Lss, NPQ Lss, and ΔT suggests that drought stress significantly impacts photosynthetic efficiency and heat dissipation, with recovery occurring after rewatering. (2) We detected differences within and among provenances utilizing a single nucleotide polymorphism genotyping array and linear mixed models integrating estimated genomic relationships to investigate genetic variation in needle functional traits in time. Throughout the experiment, heritability ( h ² ) varied widely among traits—with QY max and QY Lss showing the greatest variability (from 0 to 0.37), NPQ Lss exhibiting a narrower range aside from two outlier peaks, and SFR_R and ∆T displaying lower variability and lower h ² values (0–0.24). The photosynthesis‐related traits (QY max, QY Lss) showed the highest genetic variation, underscoring their potential for early‐age phenotyping and selection of drought‐tolerant genotypes. These findings address practical problems in forest management, particularly in light of changing weather patterns and climate variability, and provide a foundation for advanced optically based, early‐age phenotyping to enhance forest resilience.