To evaluate the role of specific xanthophylls in light utilization, wild-type and xanthophyll mutant plants (npq1, npq2, lut2, lut2npq1 and lut2npq2) from Arabidopsis thaliana were grown under three different light regimes: 30 (low light, LL), 150 (medium light, ML) and 450 (high light, HL) mumol photons m(-2) s(-1). We studied the pigment content, growth rate, xanthophyll cycle activity, chlorophyll fluorescence parameters and the response to photoinhibition. All genotypes differed strongly in the growth rates and the resistance against photoinhibition. In particular, replacement of lutein (Lut) by violaxanthin (Vx) in the lut2npq1 mutant did not affect the growth at non-saturating light intensities (LL and ML), but led to a pronounced reduction of growth under HL conditions, indicating an important photoprotective role of Lut. This was further supported by a much higher sensitivity of all Lut-deficient plants to photoinhibition in comparison with the wild type. In contrast, replacement of Lut by zeaxanthin (Zx) in lut2npq2 led to a pronounced reduction of growth under all light regimes, most likely related to the permanent non-photochemical dissipation of excitation energy by Zx at Vx-binding sites and the destabilization of antenna proteins by binding of Zx to Lut-binding sites. The high susceptibility of lut2npq2 to photoinhibition in comparison with npq2 further indicated that the photoprotective function of Zx is abolished in the absence of Lut. Thus, it can be concluded from our work that neither Vx nor Zx is able to fulfil the essential photoprotective function at Lut-binding sites under in vivo conditions.

Upon the transition of dark-adapted plants to low light, the energy-dependent quenching (qE) of excitation energy is only transiently induced due to the only transient generation of the transthylakoid pH gradient. We investigated the transient qE (qE(TR)) in different Arabidopsis (Arabidopsis thaliana) mutants. In dark-adapted plants, qE(TR) was absent in the npq4 mutant (deficient in the PsbS protein) and the pgr1 mutant (restricted in lumen acidification). In comparison with wild-type plants, qE(TR) was reduced in the zeaxanthin (Zx)-deficient npq1 mutant and increased in the Zx-accumulating npq2 mutant. After preillumination of plants (to allow the synthesis of large amounts of Zx), the formation and relaxation of qE(TR) was accelerated in all plants (except for npq4) in comparison with the respective dark-adapted plants. The extent of qE(TR), however, was unchanged in npq1 and npq4, decreased in npq2, but increased in wild-type and pgr1 plants. Even in presence of high levels of Zx, qE(TR) in pgr1 mutants was still lower than that in wild-type plants. In the presence of the uncoupler nigericin, qE(TR) was completely abolished in all genotypes. Thus, the transient qE(TR) shows essentially the same characteristics as the steady-state qE; it is strictly dependent on the PsbS protein and a low lumen pH, but the extent of qE(TR) is largely modulated by Zx. These results indicate that qE(TR) does not represent a different quenching mechanism in comparison with the steady-state qE, but simply reflects the response of qE to the dynamics of the lumen pH during light activation of photosynthesis.

Upon the transition of dark-adapted plants to low light, the energy-dependent quenching (qE) of excitation energy is only transiently induced due to the only transient generation of the transthylakoid pH gradient. We investigated the transient qE (qE TR ) in different Arabidopsis (Arabidopsis thaliana) mutants. In dark-adapted plants, qE TR was absent in the npq4 mutant (deficient in the PsbS protein) and the pgr1 mutant (restricted in lumen acidification). In comparison with wild-type plants, qE TR was reduced in the zeaxanthin (Zx)-deficient npq1 mutant and increased in the Zx-accumulating npq2 mutant. After preillumination of plants (to allow the synthesis of large amounts of Zx), the formation and relaxation of qE TR was accelerated in all plants (except for npq4) in comparison with the respective dark-adapted plants. The extent of qE TR , however, was unchanged in npq1 and npq4, decreased in npq2, but increased in wild-type and pgr1 plants. Even in presence of high levels of Zx, qE TR in pgr1 mutants was still lower than that in wild-type plants. In the presence of the uncoupler nigericin, qE TR was completely abolished in all genotypes. Thus, the transient qE TR shows essentially the same characteristics as the steady-state qE; it is strictly dependent on the PsbS protein and a low lumen pH, but the extent of qE TR is largely modulated by Zx. These results indicate that qE TR does not represent a different quenching mechanism in comparison with the steady-state qE, but simply reflects the response of qE to the dynamics of the lumen pH during light activation of photosynthesis.

Arabidopsis thaliana plants grown from ethyl methane sulfonate-treated seeds were screened for so-called que mutants, which are affected in non-photochemical energy quenching. Based on video imaging of chlorophyll fluorescence an energy dissipation mutant, que1, was identified, isolated and characterized. Similar to the npq mutants, the que1 mutant showed a drastically reduced capacity for pH-dependent energy dissipation, qE, but without affecting the Delta pH-dependent conformational changes at 535 nm (DeltaA (535)), which have been supposed to be obligatorily correlated with qE and to reflect pH-regulated binding of zeaxanthin to the PsbS protein. Western blot and DNA sequence analysis revealed that neither a reduced expression of the PsbS protein nor a mutation in the PsbS gene was responsible for the missing qE in que1. Measurements of 9-aminoacridine fluorescence quenching showed that the acidification of the thylakoid lumen was also not affected in the mutant. Furthermore, que1 was able to convert violaxanthin to zeaxanthin. However, unusual characteristics of zeaxanthin formation in the mutant pointed at an altered availability of violaxanthin for de-epoxidation. This was further accompanied by a decrease of the photochemical quenching of chlorophyll fluorescence (qP), an increase of the portion of oxidized P700 and a reduction of the electron transport rate. These characteristics indicate changes in the organization of the thylakoid membrane that affect linear electron transport (but not lumen acidification) and the formation of energy dissipation in photosystem II. Preliminary genetic analysis revealed that the phenotype of que1 is related to two different mutations, mapped to the lower arms of chromosomes 1 and 4.

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