Chlorophyll fluorescence at 680 and 730 nm and leaf photosynthesis

Chlorophyll fluorescence constitutes a simple, rapid, and non-invasive means to assess light utilization in Photosystem II (PS II). This study examines aspects relating to the accuracy and applicability of fluorescence for measurement of PS II photochemical quantum yield in intact leaves. A known source of error is fluorescence emission at 730 nm that arises from Photosystem I (PS I). We measured this PS I offset using a dual channel detection system that allows measurement of fluorescence yield in the red (660 nm < F < 710 nm) or far red (F > 710 nm) region of the fluorescence emission spectrum. The magnitude of the PS I offset was equivalent to 30% and 48% of the dark level fluorescence F(0) in the far red region for Helianthus annuus and Sorghum bicolor, respectively. The PS I offset was therefore subtracted from fluorescence yields measured in the far red spectral window prior to calculation of PS II quantum yield. Resulting values of PS II quantum yield were consistently higher than corresponding values based on emission in the red region. The basis for this discrepancy lies in the finite optical thickness of the leaf that leads to selective reabsorption by chlorophyll of red fluorescence emission originating in deeper cell layers. Consequently, red fluorescence measurements preferentially sense emission from chloroplasts in the uppermost layer of the leaf where levels of photoprotective nonphotochemical quenching are higher due to increased photon density. It is suggested that far red fluorescence, corrected for the PS I offset, provides the most reliable quantitative basis for calculation of PS II quantum yield because of reduced sensitivity of these measurements to gradients in leaf transmittance and quenching capacity.