Simple SummaryThis study investigates the spatial analysis of morphological and chemical changes and how reflectance hyperspectroscopy and fluorescence kinetics spectroscopy can enhance our understanding of biophysical, biochemical, and photochemical changes inCodiaeum variegatum(L.) A. Juss, a plant with variegated leaves and different pigments. The analysis included pigment profiling, hyperspectral curves, chlorophyll a fluorescence induction kinetics, and multivariate analyses associated with 23 JIP test parameters and 34 vegetation indexes. The results show that the analysis of chemical composition combined with vegetation indexes, such as PRI, PSSRc, ARI1, RARS, and SIPI, are highly correlated with pigment concentration and photochemical components of photosystems in leaves. Furthermore, decreased damage to energy transfer in the electron transport chain is associated with the accumulation of carotenoids, anthocyanins, flavonoids, and phenolic compounds linked with specific wavelengths. Our results reveal the potential of optical spectroscopy techniques and multivariate data analyses to enhance the management and monitoring of the leaf color status of plants. This is the first study and report on the monitoring of nonuniform leaves, particularly in the alteration of photosystem changes in variegated leaves together with high-throughput analyses.AbstractThe adjustments that occur during photosynthesis are correlated with morphological, biochemical, and photochemical changes during leaf development. Therefore, monitoring leaves, especially when pigment accumulation occurs, is crucial for monitoring organelles, cells, tissue, and whole-plant levels. However, accurately measuring these changes can be challenging. Thus, this study tests three hypotheses, whereby reflectance hyperspectroscopy and chlorophyll a fluorescence kinetics analyses can improve our understanding of the photosynthetic process inCodiaeum variegatum(L.) A. Juss, a plant with variegated leaves and different pigments. The analyses include morphological and pigment profiling, hyperspectral data, chlorophyll a fluorescence curves, and multivariate analyses using 23 JIP test parameters and 34 different vegetation indexes. The results show that photochemical reflectance index (PRI) is a useful vegetation index (VI) for monitoring biochemical and photochemical changes in leaves, as it strongly correlates with chlorophyll and nonphotochemical dissipation (Kn) parameters in chloroplasts. In addition, some vegetation indexes, such as the pigment-specific simple ratio (PSSRc), anthocyanin reflectance index (ARI1), ratio analysis of reflectance spectra (RARS), and structurally insensitive pigment index (SIPI), are highly correlated with morphological parameters and pigment levels, while PRI, moisture stress index (MSI), normalized difference photosynthetic (PVR), fluorescence ratio (FR), and normalized difference vegetation index (NDVI) are associated with photochemical components of photosynthesis. Combined with the JIP test analysis, our results showed that decreased damage to energy transfer in the electron transport chain is correlated with the accumulation of carotenoids, anthocyanins, flavonoids, and phenolic compounds in the leaves. Phenomenological energy flux modelling shows the highest changes in the photosynthetic apparatus based on PRI and SIPI when analyzed with Pearson’s correlation, the hyperspectral vegetation index (HVI) algorithm, and the partial least squares (PLS) to select the most responsive wavelengths. These findings are significant for monitoring nonuniform leaves, particularly when leaves display high variation in pigment profiling in variegated and colorful leaves. This is the first study on the rapid and precise detection of morphological, biochemical, and photochemical changes combined with vegetation indexes for different optical spectroscopy techniques.