RESUMEN
Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants' survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state. Interestingly, we found that chlorophyll (Chl) fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, indicating that they serve as a site for qH-energy dissipation and providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein, and lipid contents of antenna with active or inactive qH but did not detect any evident differences. Finally, we investigated whether specific subunits of the major antenna complexes were required for qH but found that qH was insensitive to trimer composition. Because we previously observed that qH can occur in the absence of specific xanthophylls, and no evident changes in pigments, proteins, or lipids were detected, we tentatively propose that the energy-dissipative state reported here may stem from Chl-Chl excitonic interaction.
Asunto(s)
Clorofila , Complejos de Proteína Captadores de Luz , Complejo de Proteína del Fotosistema II , Plantas , Clorofila/química , Luz , Complejos de Proteína Captadores de Luz/química , Fotosíntesis , Complejo de Proteína del Fotosistema II/química , Plantas/química , Tilacoides/química , Xantófilas/químicaRESUMEN
The photosystem II (PSII) protein PsbS and the enzyme violaxanthin deepoxidase (VDE) are known to influence the dynamics of energy-dependent quenching (qE), the component of nonphotochemical quenching (NPQ) that allows plants to respond to fast fluctuations in light intensity. Although the absence of PsbS and VDE has been shown to change the amount of quenching, there have not been any measurements that can detect whether the presence of these proteins alters the type of quenching that occurs. The chlorophyll fluorescence lifetime probes the excited-state chlorophyll relaxation dynamics and can be used to determine the amount of quenching as well as whether two different genotypes with the same amount of NPQ have similar dynamics of excited-state chlorophyll relaxation. We measured the fluorescence lifetimes on whole leaves of Arabidopsis thaliana throughout the induction and relaxation of NPQ for wild type and the qE mutants, npq4, which lacks PsbS; npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin; and npq1 npq4, which lacks both VDE and PsbS. These measurements show that although PsbS changes the amount of quenching and the rate at which quenching turns on, it does not affect the relaxation dynamics of excited chlorophyll during quenching. In addition, the data suggest that PsbS responds not only to ΔpH but also to the Δψ across the thylakoid membrane. In contrast, the presence of VDE, which is necessary for the accumulation of zeaxanthin, affects the excited-state chlorophyll relaxation dynamics.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Complejo de Proteína del Fotosistema II/metabolismo , Zeaxantinas/metabolismo , Arabidopsis/enzimología , Clorofila/química , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Oxidorreductasas/metabolismoRESUMEN
We describe a technique to measure the fluorescence decay profiles of intact leaves during adaptation to high light and subsequent relaxation to dark conditions. We show how to ensure that photosystem II reaction centers are closed and compare data for wild type Arabidopsis thaliana with conventional pulse-amplitude modulated (PAM) fluorescence measurements. Unlike PAM measurements, the lifetime measurements are not sensitive to photobleaching or chloroplast shielding, and the form of the fluorescence decay provides additional information to test quantitative models of excitation dynamics in intact leaves.
Asunto(s)
Biología Molecular/métodos , Complejo de Proteína del Fotosistema II/química , Hojas de la Planta/química , Adaptación Fisiológica , Arabidopsis/química , Arabidopsis/metabolismo , Clorofila/química , Cloroplastos/química , Cloroplastos/metabolismo , Diseño de Equipo , Fluorescencia , Luz , Biología Molecular/instrumentación , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismo , Spinacia oleracea/químicaRESUMEN
The light-harvesting complexes of plants have evolved the ability to switch between efficient light harvesting and quenching forms to optimize photosynthesis in response to the environment. Several distinct mechanisms, collectively termed "nonphotochemical quenching" (NPQ), provide flexibility in this response. Here we report the isolation and characterization of a mutant, suppressor of quenching 1 (soq1), that has high NPQ even in the absence of photosystem II subunit S (PsbS), a protein that is necessary for the rapidly reversible component of NPQ. The formation of NPQ in soq1 was light intensity-dependent, and it exhibited slow relaxation kinetics and other characteristics that distinguish it from known NPQ components. Treatment with chemical inhibitors or an uncoupler, as well as crosses to mutants known to affect other NPQ components, showed that the NPQ in soq1 does not require a transthylakoid pH gradient, zeaxanthin formation, or the phosphorylation of light-harvesting complexes, and it appears to be unrelated to the photosystem II damage-and-repair cycle. Measurements of pigments and chlorophyll fluorescence lifetimes indicated that the additional NPQ in soq1 is the result of a decrease in chlorophyll excited-state lifetime and not pigment bleaching. The SOQ1 gene was isolated by map-based cloning, and it encodes a previously uncharacterized thylakoid membrane protein with thioredoxin-like and ß-propeller domains located in the lumen and a haloacid-dehalogenase domain exposed to the chloroplast stroma. We propose that the role of SOQ1 is to prevent formation of a slowly reversible form of antenna quenching, thereby maintaining the efficiency of light harvesting.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Complejos de Proteína Captadores de Luz/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteínas de las Membranas de los Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Cruzamientos Genéticos , Fluorescencia , Immunoblotting , Complejos de Proteína Captadores de Luz/genética , Modelos Biológicos , Tiorredoxinas/aislamiento & purificación , Proteínas de las Membranas de los Tilacoides/genéticaRESUMEN
Energy-dependent quenching (qE) in photosystem II (PSII) is a pH-dependent response that enables plants to regulate light harvesting in response to rapid fluctuations in light intensity. In this review, we aim to provide a physical picture for understanding the interplay between the triggering of qE by a pH gradient across the thylakoid membrane and subsequent changes in PSII. We discuss how these changes alter the energy transfer network of chlorophyll in the grana membrane and allow it to switch between an unquenched and quenched state. Within this conceptual framework, we describe the biochemical and spectroscopic measurements and models that have been used to understand the mechanism of qE in plants with a focus on measurements of samples that perform qE in response to light. In addition, we address the outstanding questions and challenges in the field. One of the current challenges in gaining a full understanding of qE is the difficulty in simultaneously measuring both the photophysical mechanism of quenching and the physiological state of the thylakoid membrane. We suggest that new experimental and modeling efforts that can monitor the many processes that occur on multiple timescales and length scales will be important for elucidating the quantitative details of the mechanism of qE.