Hyperspectral imaging reveals early drought stress and associated molecular responses in lettuce for space agriculture.

In NASA's controlled-environment plant growth systems, early and autonomous detection of crop stress is critical for sustaining food production during long-duration space missions. Hyperspectral imaging (HSI) has proven effective for early stress detection, yet the molecular processes underlying diagnostically informative spectral signals remain poorly defined. Here, we present a two-stage phenomics-to-molecular framework to evaluate whether hyperspectral signatures associated with early drought detection correspond to coordinated molecular stress responses in lettuce. In the first stage, reflectance and fluorescence HSI were used to identify early drought detection windows in 'Dragoon' lettuce subjected to controlled water limitation over a 15-day treatment period with daily imaging. Classification models integrating reflectance and fluorescence outperformed single-modality models and achieved high accuracy as early as day after treatment (DAT) 4, reaching up to 97% at DAT 5. Partial least squares discriminant analysis (PLS-DA) identified predictive wavelengths concentrated in blue-green, red, and red-edge regions associated with chlorophyll absorption and photosystem II activity. In the second stage, independent transcriptomic and untargeted metabolomic profiles were integrated with hyperspectral signatures using MOFA2 to establish biological context. This analysis revealed a dominant drought axis characterized by early activation of ABA signaling, osmotic adjustment, phenylpropanoid metabolism, and lipid and membrane remodeling, with maximal molecular divergence at DAT 5, coinciding with peak hyperspectral classification performance. Notably, wavelengths optimized for early stress discrimination were systematically shifted toward shorter, optically efficient regions relative to those most strongly associated with downstream metabolic abundance, indicating that HSI primarily captures early structural and energetic consequences of molecular stress responses rather than direct biochemical composition. Together, these results demonstrate that hyperspectral imaging can function as a non-destructive, biologically interpretable molecular proxy for drought stress, providing a foundation for compact, hands-free sensing systems capable of distinguishing stress-specific plant states in space agriculture.