Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii: non‐photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient

Abstract

SummaryFluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthinaccumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25-35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a 'dimmer switch'. Similar, but quantitatively different, behaviour was observed in the intensity and Correspondence to: Robert Clegg. Tel: 1-217-244-8143; fax: 1-217244-7187; e-mail: rclegg@uiuc.edu fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P-level and during the slow fluorescence transient, compared to wild type and the npq1 mutant.The fluorescence lifetime-resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The aboveresults are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so-called 'State Transitions', and on non-photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two 'lifetime pools' centred on slower and faster fluorescence lifetimes.

Acclimation of Chlamydomonas reinhardtii to Different Growth Irradiances

“…Zeaxanthin in particular, beside ROS scavenging, is involved in quenching of singlet (11,69) and in some cases triplet (70) chlorophyll excited states. In C. reinhardtii accumulation of zeaxanthin has been correlated to shortening of fluorescence lifetime imaging in vivo (71). Because zeaxanthin is not involved in qE, this effect is likely due to constitutive quenching of singlet excited states of Chl reported upon binding of zeaxanthin to Lhc proteins (4,(72)(73)(74), which is the molecular basis for qI component of NPQ (11).…”

The Non-Photochemical Quenching of the Electronically Excited State of Chlorophyll a in Plants: Definitions, Timelines, Viewpoints, Open Questions

“…Gilmore et al (1996a) showed that both intensity and lifetime of Chl a fluorescence of PS II (the fluorescence lifetime is a direct measure of the quantum yield of fluorescence) were independent of antenna size differences between wild type barley and the chlorina mutant that lacked LHCIIb. [Note that change in fluorescence intensity can simply be due to change in the concentration of Chl; thus, measurement of lifetime of fluorescence is crucial in reaching firm conclusions; see, e.g., Holub et al 2000Holub et al , 2007. This suggested involvement of the innermost peripheral antenna complexes (i.e., CP24, CP26, and CP29) in qE.…”

Avaliação de furanocumarinas como inibidores da fotossíntese através de ensaios de fluorescência da clorofila a

“…Contudo, existe um grupo de metodologias que permite estudar detalhes da fotossíntese sem destruir nenhum tecido fotossintético, baseado na quantificação da emissão da fluorescência da clorofila a. 5 Essas técnicas por serem indiretas, precisas, econômicas, rápidas e não destrutivas, nos últi-mos anos têm levado a uma grande variedade de estudos in vivo do aparato fotossintético em diversos sistemas biológicos. A principal vantagem é que a medida da fluorescência oferece um grande número de informações sobre os sistemas biológicos.…”