Abstract: Phosphorus (P) deficiency induces a dark-green leaf phenotype in many species due to anthocyanin accumulation. However, the association of accumulated anthocyanin with the dark-green leaf phenotype in modern non-pigmented rice cultivars has been questioned due to mutations in the anthocyanin activator genes responsible for pigmentation in rice leaves. Here, we investigated the molecular regulation of the dark-green phenotype observed in phosphorus-deficient rice leaves (Oryza sativa L. cv. KDML105). Comparative transcriptomic analysis revealed that a total of 5,036 genes were differentially expressed under P deficiency, with enriched pathways related to lipid metabolism, chlorophyll degradation, and photosynthesis regulation. Physiological measurements indicated substantially increased chlorophyll content under P deficiency despite the downregulation of chlorophyll biosynthesis genes. The upregulation of transcription factors that repress chlorophyll degradation and galactolipid enhancement in the thylakoid membrane were associated with chloroplast stability and chlorophyll retention. Additionally, changes in leaf anatomical structure under P stress likely contributed to the phenotype by concentrating chlorophyll per unit leaf area. These transcriptomic predictions were validated by independent qRT-PCR, and transmission electron microscopy together with hyperspectral reflectance indices confirmed that the retained chlorophyll resides in structurally intact chloroplasts and underlies the greener leaf optical signature. Our findings provide a comprehensive framework for understanding the P-deficiency-induced dark-green phenotype in rice leaves, offering insights into potential targets for the explanation of P homeostasis adaptation in modern rice cultivars.

Key words: phosphorus deficiency, dark-green phenotype, rice, transcriptomics