tri-PRI: A three band reflectance index tracking dynamic photoprotective mechanisms in a mature eucalypt forest

Plants actively regulate excess absorbed energy to protect photosynthetic machinery through heat dissipation in a process known as non-photochemical quenching (NPQ), a process useful for quantifying plant health and productivity. NPQ can be indirectly measured in the visible wavelengths between 500 nm and 560 nm, most commonly through the Photochemical Reflectance Index (PRI). However, there remains a lack of consensus regarding the optimal functional form and band selection to calculate PRI for the purpose of measuring NPQ mechanisms. Here, we quantitatively evaluate the effectiveness of leaf-level parametric and non-parametric spectral formulations, band locations, and number of bands to track the xanthophyll pigment cycle in a tall mature Eucalypt forest. Subsequently, our recommended approach is the new ‘tri-PRI’ index robust to constitutive pigment pool sizes across the canopy profile. tri-PRI is a Triangular Vegetation Index (TVI) (tri-PRI=0.5[(520 - 490)(R545nm - R490nm) - (545 – 490) R520nm - R490nm)]) using three reflectance bands around 490 nm, 520 nm and 545 nm, and has a physiological photosynthetic basis. We found that tri-PRI significantly outperformed PRI and other two band combinations for quantifying the xanthophyll EPoxidation State ‘EPS’ (tri- PRI R2=0.75 versus PRI R2=0.23), as well as the NPQ and PSII active chlorophyll fluorescence quenching yields. The new band placement enhanced the dynamic EPS absorption peak, while the third band provided an additional normalisation to minimise the confounding effects of pigments with overlapping spectral features. tri-PRI also performed comparably to parametric and non- parametric hyperspectral techniques and formulations using continuous spectral regions, highlighting the utility of targeted multispectral indices over hyperspectral approaches. This leaf-level study represents a foundational step toward indirectly measuring dynamic photosynthetic activity across the canopy profile in a tall mature Eucalypt forest to inform upscaling efforts from above-canopy remote sensing platforms. The application of tri-PRI and other top- performing multi-band TVI formulations for predicting EPS presented here should be explored across different canopy types, temporal-, and spatial scales