Transcriptomic and predictive modeling reveal photosynthetic and stress responses associated with ALAD silencing in...

Suppression of δ-aminolevulinic acid dehydratase in citrus disrupts chloroplast development and photosynthetic stability, triggering broad transcriptional reprogramming across metabolic, stress, and hormone signaling pathways. Chlorosis is often the earliest visible indicator of plant stress, whether caused by abiotic or biotic factors. Complex stress networks can ultimately lead to the loss of photosynthetic organelles and pigments, resulting in reduced cell viability and eventually, cell death. However, plants possess coordinated mechanisms to maintain homeostasis under adverse conditions. δ-Aminolevulinic acid dehydratase (ALAD), a central enzyme in tetrapyrrole biosynthesis, is essential for chlorophyll production and photosynthetic function. Here, we examined the physiological and transcriptomic consequences of ALAD suppression in Citrus macrophylla using virus-induced gene silencing. At the cellular level, ALAD-deficient plants exhibited a reduced number of plastids and altered plastid morphology. Transcriptomic profiling revealed major alterations in genes associated with photosynthesis, Fe and Zn nutrient uptake, and stress- and defense-related pathways, including reactive oxygen species (ROS) detoxification, hormone signaling, and secondary metabolism. Integrating these transcriptional and physiological changes into predictive models for photosynthesis- and redox-related genes allowed the identification of key regulatory nodes. To achieve this, we implemented a machine-learning-ready pipeline using variance-stabilized expression of the most variable genes and Random Forest modeling to classify photosynthesis and redox states. ALAD suppression triggered upregulation of cytochrome P450s, lipoxygenases, annexins, and ferritin, ROS detoxification, and iron homeostasis. ALAD suppression induced a pronounced lipid peroxidation signature, as evidenced by elevated malondialdehyde accumulation and altered levels of linoleic and α-linolenic acids, accompanied by increased jasmonic acid (JA) accumulation. Together, the suppression of ALAD reveals a coordinated stress response integrating chloroplast architecture, photosynthetic efficiency, redox regulation, nutrient homeostasis, and defense pathways.