Non‐invasive, whole‐plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants

Abstract

These authors contributed equally to this work. ‡ Shared senior authorship. SUMMARYLeaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high-sensitivity, non-invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image-based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less-mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1-5 phot2-1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual-imaging platform also allowed us to develop a straightforward approach to correct non-photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy-dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the overall photosynthetic performance of higher plants.

NPQ(T): a chlorophyll fluorescence parameter for rapid estimation and imaging of non‐photochemical quenching of excitons in photosystem‐II‐associated antenna complexes

“…) that extended dark periods can substantially perturb photosynthesis, so we used the minimum dark period that is sufficient for the vast majority of the rapid relaxation of F m ′ to occur (Dutta et al . ; Cruz et al . ), although this period can be arbitrarily extended to assess different NPQ relaxation components, for example, the q T component that relaxes over the tens of minutes timescale.…”

“…Hypothetically, measurement NPQ (T) should be insensitive to photorelocation, because it occurs over minutes (Dutta et al . ), and F m ′ and F 0 ′ are collected within a period of seconds. In other words, whereas the NPQ signal should be substantially affected by chloroplast movements, the NPQ (T) parameter should not.…”

“…Reflectance measurements were obtained as described previously (Dutta et al . ), with a second charge‐coupled device (CCD) camera (KP‐FD145GV monochrome; Hitachi Kokusai Electric Inc., Tokyo, Japan; http://www.hitachi-kokusai.co.jp) with lens covered by a 650BP100 bandpass filter (Omega Optical Inc., Brattleboro, VT, USA; http://www.omegafilters.com) to capture reflected red light (625 nm) from the pulsed LEDs normally used to excite chlorophyll a fluorescence.…”

MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network

“…In its simplest application, PAM fluorometry measures the fluorescence yield changes induced by saturating pulses of actinic light, where F 0 and F s represent the fluorescence emission in dark- and light-adapted samples, and F m and $F_{\mathrm{normalm}}^{\mathrm{normal\prime }}$ describe the maximum fluorescence yield during a saturating light pulse applied to dark-adapted and light-exposed leaves (figure 4 b ). Measuring the saturation-pulse-induced maximal fluorescence yield after a few minutes' dark period ($F_{\mathrm{normalm}}^{\mathrm{normal\prime }\mathrm{normal\prime }}$) allows estimates of the contributions of rapidly reversible ‘energy-dependent’ quenching ( q E ) and long-lived quenching ( q I ) ascribed to photoinhibition, as well as contributions from state transitions [16] and chloroplast movements [33,34]. Applying far-red illumination during a short dark period to oxidize electron carriers allows us to estimate $F_0^{\mathrm{\prime }}$ , the minimal fluorescence yield with oxidized Q A .…”

A few enlarged chloroplasts are less efficient in photosynthesis than a large population of small chloroplasts in Arabidopsis thaliana

“…Chloroplast size and the number of chloroplasts per cell are regulated by both genetic and environmental factors. Rapid advances have been made in research on the regulatory mechanisms of chloroplast division in recent years due, in part, to the isolation of accumulation and replication of chloroplast ( arc ) mutants in A. thaliana 4–9 . arc mutants exhibit alterations in chloroplast size and number of mesophyll cells.…”