Phloretin as a C4 photosynthesis disruptor: Metabolic, enzymatic, and physiological insights

Phloretin, a dihydrochalcone, was investigated for its effects on the physiology and C4 photosynthetic metabolism of maize (Zea mays). Seedlings were grown hydroponically and treated with 500 μM phloretin for seven days. Phloretin significantly reduced root length and fresh and dry weights (24-31 %), whereas stem growth and chlorophyll content remained unaffected. Leaf pyruvate levels declined sharply, suggesting disruption of carbon metabolism. Depletion analyses confirmed phloretin uptake from the nutrient solution, indicating systemic absorption by the plant. Enzyme kinetics using purified pyruvate orthophosphate dikinase revealed a mixed-type inhibition, with increased K_M and reduced V_max in the presence of phloretin, highlighting its direct effect on a C4-specific enzyme. Gas exchange analyses showed substantial reductions in net photosynthesis, stomatal conductance, ATP production, and carboxylation efficiency, with no change in mesophyll conductance. ATP production rates (J_ATP) and carboxylation by phosphoenolpyruvate carboxylase and Rubisco (V_p and V_c) were significantly lower in treated plants, even at saturating CO₂. A marked increase in bundle sheath leakiness (up to 5.7-fold) was observed, indicating disruption of the carbon-concentrating mechanism. Chlorophyll a fluorescence revealed unaltered PSII structural integrity (maximum quantum yield of PSII, F_v/F_m), but significant reductions in effective quantum yield of PSII (ϕPSII), electron transport rate (ETR), and photochemical quenching coefficient (qP), along with elevated non-photochemical quenching (NPQ)-suggesting downstream photochemical limitation. Finally, the pronounced root inhibition, compared to unaffected stems, suggests a phloretin-induced source-sink imbalance, likely due to restricted assimilate supply and altered allocation. Collectively, these findings support phloretin's potential as an inhibitor of C4 metabolism and a candidate scaffold for herbicide development. Further studies are needed to explore chemical optimization and application strategies.