In vivo two-photon FLIM resolves photosynthetic properties of maize bundle sheath cells

Maize ( Zea mays L.) performs highly efficient C ₄ photosynthesis by dividing photosynthetic metabolism between mesophyll and bundle sheath cells. In vivo physiological measurements are indispensable for C ₄ photosynthesis research as any isolated cells or sectioned leaf often show interrupted and abnormal photosynthetic activities. Yet, direct in vivo observation regarding bundle sheath cells in the delicate anatomy of the C ₄ leaf is still challenging. In the current work, we used two-photon fluorescence-lifetime imaging microscopy (two-photon-FLIM) to access the photosynthetic properties of bundle sheath cells on intact maize leaves. The results provide spectroscopic evidence for the diminished total PSII activity in bundle sheath cells at its physiological level and show that the single PSIIs could undergo charge separation as causal. We also report an acetic acid-induced chlorophyll fluorescence quenching on intact maize leaves, which might be a physiological state related to the nonphotochemical quenching mechanism.