Water stress and senescence increase calcium oxalate crystals and decrease their solubility in amaranth leaves

Abstract

Calcium oxalate (CaOx) crystals are widespread in plants, yet their physiological roles remain debated. Although calcium sequestration is the most widely accepted function, some studies propose that CaOx crystals can serve as a carbon source, implying calcium release. These studies report decreases in CaOx crystals under water stress and other CO2-limiting conditions, suggesting that crystals are oxidized to CO2 to sustain the Calvin cycle. To test this hypothesis, we subjected 39-day-old Amaranthus cruentus plants to 10 days of water stress, followed by rewatering and a 16-day recovery period. Crystal abundance was analyzed by polarization microscopy, first in fixed leaves and then in optically cleared leaves. In control plants, CaOx crystals were present from the leaf-primordium stage and increased throughout leaf expansion, remaining abundant until late senescence. Water stress accelerated this pattern, and rewatering had no detectable effect. Optical clearing was intended to improve crystal visualization but unexpectedly dissolved crystals in some leaves. Serendipitously, we found that crystal solubility decreased with leaf age in control plants. Water stress accelerated this loss of solubility, whereas rewatering restored solubility to levels characteristic of younger leaves. Natural senescence increased CaOx crystal abundance and rendered them increasingly recalcitrant; water stress accelerated both processes. These findings challenge the carbon-source hypothesis.