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Publications

Yuxi NiuShizue MatsubaraLadislav NedbalDušan Lazár ✉️

Plant, Cell & Environment | https://doi.org/10.1111/pce.14879

Published: June 2024

Plants have evolved multiple regulatory mechanisms to cope with natural light fluctuations. The interplay between these mechanisms leads presumably to the resilience of plants in diverse light patterns. We investigated the energy-dependent nonphotochemical quenching (qE) and cyclic electron transports (CET) in light that oscillated with a 60-s period with three different amplitudes. The photosystem I (PSI) and photosystem II (PSII) function-related quantum yields and redox changes of plastocyanin and ferredoxin were measured in Arabidopsis thaliana wild types and mutants with partial defects in qE or CET. The decrease in quantum yield of qE due to the lack of either PsbS- or violaxanthin de-epoxidase was compensated by an increase in the quantum yield of the constitutive nonphotochemical quenching. The mutant lacking NAD(P)H dehydrogenase (NDH)-like-dependent CET had a transient significant PSI acceptor side limitation during the light rising phase under high amplitude of light oscillations. The mutant lacking PGR5/PGRL1-CET restricted electron flows and failed to induce effective photosynthesis control, regardless of oscillation amplitudes. This suggests that PGR5/PGRL1-CET is important for the regulation of PSI function in various amplitudes of light oscillation, while NDH-like-CET acts’ as a safety valve under fluctuating light with high amplitude. The results also bespeak interplays among multiple photosynthetic regulatory mechanisms.

Nature Methods | https://doi.org/10.1038/s41592-023-02063-y

Published: November 2023

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.

In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light.

 

Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport.

 

The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics.

 

Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.

ChemPhysChem | doi.org/10.1002/cphc.202200295
Published: 17 August 2022

We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine-wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. Several non-redundant resonant observables are simultaneously retrieved from a single experiment with phase-sensitive detection. HIGHLIGHT is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far. Moreover, labels out of targeted locations can be discarded in an inhomogeneous spatial profile of illumination. HIGHLIGHT opens roads for simplified optical setups at reduced cost and easier maintenance.

Preparatory research

Titration without separation, e.g. quantification of a target species in living cells, is a challenge of analytical chemistry. We perform the selective detection of a target using the kinetics involved in a photochemical process and develop a correlation method that we illustrate by the titration of a fluorescent photoswitcher and the target of a photoswitching sensor. Correlating an input time series and a well-chosen weighting function associated with a variable characteristic time yields a spectrum of characteristic times. The upper integration limit of the correlation output can be chosen to match the argument of an extremum of the spectrum with a characteristic time of the input time series in order to quantify the target. A similar procedure is followed to optimize the signal-to-noise ratio. Selectivity and signal-to-noise ratio associated with 15 weighting functions are theoretically predicted. The results are applied to the titration of the reversibly photoswitchable fluorescent protein Dronpa-2 and the titration of calcium using a reversibly photoswitchable fluorescent sensor. The performance of the correlation method is favorably compared to the one of other dynamic contrast protocols.

Plants growing in nature often experience fluctuating irradiance. However, in the laboratory, the dynamics of photosynthesis are usually explored by instantaneously exposing dark-adapted plants to constant light and examining the dark-to-light transition, which is a poor approximation of natural phenomena. With the aim creating a better approximation, we exposed leaves of pea (Pisum sativum) to oscillating light and measured changes in the functioning of PSI and PSII, and of the proton motive force at the thylakoid membrane. We found that the dynamics depended on the oscillation period, revealing information about the underlying regulatory networks. As demonstrated for a selected oscillation period of 60 s, the regulation tries to keep the reaction centers of PSI and PSII open. We present an evaluation of the data obtained, and discuss the involvement of particular processes in the regulation of photosynthesis. The forced oscillations provided an information-rich fingerprint of complex regulatory networks. We expect future progress in understanding these networks from experiments involving chemical interventions and plant mutants, and by using mathematical modeling and systems identification and control tools.

Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.

Plant Physioly | 10.1093/plphys/kiab317

Foundations of photosynthesis research have been established mainly by studying the response of plants to changing light, typically to sudden exposure to a constant light intensity after dark acclimation or light flashes. This approach remains valid and powerful, but can be limited by requiring dark acclimation before time-domain measurements and often assumes that rate constants determining the photosynthetic response do not change between dark and light acclimation. We show that these limits can be overcome by measuring plant responses to sinusoidally modulated light of varying frequency. By its nature, such frequency-domain characterization is performed in light-acclimated plants with no need for prior dark acclimation. Amplitudes, phase shifts, and upper harmonic modulation extracted from the data for a wide range of frequencies can target different kinetic domains and regulatory feedbacks. The occurrence of upper harmonic modulation reflects nonlinear phenomena, including photosynthetic regulation. To support these claims, we measured chlorophyll fluorescence emission of the green alga Chlorella sorokiniana in light that was sinusoidally modulated in the frequency range 1000-0.001 Hz. Based on these experimental data and numerical as well as analytical mathematical models, we propose that frequency-domain measurements can become a versatile tool in plant sensing.