A new regulatory role for the chloroplast ATP synthase

One of the challenges of modern biology is the synthesis of vast amounts of data into a deep understanding of organism structure and function. At present, the most visible effort toward meeting this challenge is the use of computing technology to effectively reassemble organisms that have been fragmented and processed into DNA sequences, relative abundances of RNA transcripts, peptide patterns, or profiles of metabolites (1–8). An important complement to this approach is the use of nondestructive methods to observe function in intact organisms as they live. Quantitative analysis of specific functions at the molecular level is often difficult in intact organisms, owing to their complexity, but such analyses must be attempted, if only to validate models derived from the fragmentation and computation approach. In cases where the function of interest is perturbed by the extraction of chemical information or occurs too rapidly to be resolved by such extraction, in vivo study is essential.

The intimacy of plants with their physical environment, including the fact that they cannot move, has rendered them highly sensitive and responsive to physical factors.

The article in this issue of PNAS by Kanazawa and Kramer (9) is an elegant example of how sophisticated hypotheses of function can be tested in intact organisms. This study applies a combination of nonfocusing optics (10) and absorbance difference spectroscopy to study H⁺ flux through the chloroplast ATPases of plant leaves as they adjust their photosynthetic systems to different irradiances and external levels of CO₂. Plants and other photosynthetic organisms exhibit rich absorption and fluorescence differences between their dark and illuminated states (11–14). In the green region of the spectrum, the charge difference generated across the thylakoid membranes of chloroplasts by illumination causes a change in the absorption spectra of carotenoid pigments in the membranes, the so-called “electrochromic shift” (e.g., ref. 15). The charge difference results from H⁺ pumping into the thylakoid lumen by photosynthetic electron transport. Decay of the charge difference during a brief interval of darkness, observed by means of the electrochromic shift, indicates the efflux of H⁺ from the lumen via the chloroplast ATPase (16). Nonfocusing optics allow this decay to be observed in leaf tissue, which is optically very dense and highly scattering, while appropriate time resolution and referencing of the measurement allow it to be distinguished from interfering absorbance changes that proceed at different rates. The importance of observing H⁺ flux through the ATPases in a live leaf is that it allows testing of several established hypotheses for how plants dynamically regulate their photosynthetic systems to minimize photooxidative damage that can occur during periods of environmental stress.

It has often been said that the intimacy of plants with their physical environment, including the fact that they cannot move significantly to escape unfavorable conditions, has rendered them highly sensitive and responsive to physical factors. A case of this is the capacity of plants to reconcile the rate at which they absorb solar energy with the rate at which they use it for metabolism. Sunlight varies constantly in nature, and metabolic rate is determined by independent variations of temperature, water, mineral nutrients, and CO₂. On a clear day at noon, for example, the absorption of solar energy by a leaf may be high, but use of the energy by carbon and nitrogen assimilation may be nearly zero owing to low temperature. The excess solar energy absorbed under such conditions can drive the formation of reactive O₂ species that cause photooxidative damage to photosynthetic cells (17–19). A primary mechanism for avoiding photooxidative damage is adjustment of the efficiency of photosystem II (20, 21). Under weak light and optimal conditions for metabolism, the photochemical efficiency of photosystem II exceeds 90% on a quantum basis. Under strong light and unfavorable conditions, however, the quantum efficiency of photosystem II can be down-regulated to less than 10% owing to a dramatic increase in the conversion of absorbed light into heat before photochemistry occurs. Such increased thermal conversion protects against photooxidation because it reduces the energy available to form reactive O₂ species in the photosynthetic system, while the heat that is generated is insufficient to cause harm. It is reversible on a time scale of minutes to hours so that high photosynthetic efficiency recovers under favorable conditions. This reversible increase in thermal conversion in photosystem II is referred to as “nonphotochemical quenching” (NPQ), because it decreases the yield of chlorophyll fluorescence from photosystem II independently of the quenching effect of photosynthetic electron transport (22). Two processes are known to be part of the mechanism of NPQ. One is the conversion of violaxanthin to zeaxanthin in the thylakoid membranes by activation of a membrane-associated violaxanthin de-epoxidase (23–25). The other is protonation of the PsbS subunit of the photosystem II complex (26, 27). Both of these processes are caused directly by acidification of the thylakoid lumen and, under many conditions, NPQ is roughly proportional to the rate of linear electron transport, the path that evolves oxygen, reduces NADP⁺, and pumps H⁺ into the thylakoid lumen. What has been difficult to account for until now is the observation that high rates of NPQ can occur when linear electron transport is strongly limited by lack of NADP⁺, as occurs when carbon assimilation is slowed by low temperature or when the stomata of leaves close during drought and limit the entry of CO₂. NPQ is particularly important under such conditions because the potential for photooxidation is high.

Several hypotheses have been proposed to account for high rates of NPQ when linear electron transport is limited. The most prominent invoke alternate paths of electron transport, such as cyclic electron transport around photosystem I (28, 29) or a variation of linear electron transport in which O₂ accepts electrons at photosystem I in place of NADP⁺ and the resulting superoxide is safely converted to water by a photosystem I-associated antioxidant system (the “water–water cycle;” refs. 30 and 31). These alternate paths do not require NADP⁺, but do pump H⁺ into the thylakoid lumen and could maintain a protective level of NPQ when carbon assimilation is limited. Rates of these alternate pathways are a small fraction of the linear path, however, and up-regulation sufficient to maintain NPQ at the needed levels has not been proven. Kanazawa and Kramer (9) now present and test with these others an alternative hypothesis: that reduced conductance of the chloroplast ATP synthase to H⁺ allows low pH in the thylakoid lumen and consequent high rates of NPQ to be maintained when linear electron transport is slowed by unfavorable conditions. Thus, the chloroplast ATP synthase assumes an important role in regulating adjustments of the photosynthetic system to environmental conditions by varying the relationship of photosynthetic electron transport to NPQ. The mechanism by which H⁺ conductivity of the chloroplast ATP synthase is altered and how the mechanism perceives the need for NPQ when linear electron transport is limited are now pressing topics of interest. Questions also arise about how changes in H⁺ conductivity of the chloroplast ATP synthase intermesh with chloroplast metabolism and whether similar processes occur in mitochondria.

Study of the integrated function of organisms, or significant subsystems of organisms, in the light of genomic information has been termed systems biology (e.g., ref. 32). In many respects, modern photosynthesis research approaches systems biology. Gene sequences, detailed structural information, and most of the protein-protein interactions of the photosynthetic system are known (33–37). A vast array of mutants have been made in a diversity of species and their phenotypes analyzed under many conditions. Despite this high level of progress, many questions about functions of the photosynthetic system and their adaptive value remain unanswered or even unasked. For example, it is unclear whether electron transport through the photosystem I complex follows one or two paths in vivo and whether such bifurcation has an adaptive significance to the organism (38). Observation of such functions in living organisms is a necessary part of understanding them. In many cases, spectroscopic methods are the best tools for this purpose. Absorption, fluorescence, and magnetic resonance spectroscopies have been applied extensively to study isolated photosystems and thylakoid membranes, but have been used much less with intact samples because of their optical and chemical complexity. Spectroscopic methods refined for application to more intact samples, as illustrated in Kanazawa and Kramer (9), will contribute much toward achieving systems biology in photosynthetic organisms and perhaps others as well (14).