In oxygenic photosynthetic eukaryotes, the hydroxylated carotenoid zeaxanthin is produced from preexisting violaxanthin upon exposure to excess light conditions. Zeaxanthin binding to components of the photosystem II (PSII) antenna system has been investigated thoroughly and shown to help in the dissipation of excess chlorophyll-excited states and scavenging of oxygen radicals. However, the functional consequences of the accumulation of the light-harvesting complex I (LHCI) proteins in the photosystem I (PSI) antenna have remained unclarified so far. In this work we investigated the effect of zeaxanthin binding on photoprotection of PSI–LHCI by comparing preparations isolated from wild-type
Arabidopsis thaliana (i.e., with violaxanthin) and those isolated from the
A.
thaliana nonphotochemical quenching 2 mutant, in which violaxanthin is replaced by zeaxanthin. Time-resolved fluorescence measurements showed that zeaxanthin binding leads to a previously unrecognized quenching effect on PSI–LHCI fluorescence. The efficiency of energy transfer from the LHCI moiety of the complex to the PSI reaction center was down-regulated, and an enhanced PSI resistance to photoinhibition was observed both in vitro and in vivo. Thus, zeaxanthin was shown to be effective in inducing dissipative states in PSI, similar to its well-known effect on PSII. We propose that, upon acclimation to high light, PSI–LHCI changes its light-harvesting efficiency by a zeaxanthin-dependent quenching of the absorbed excitation energy, whereas in PSII the stoichiometry of LHC antenna proteins per reaction center is reduced directly.In eukaryotic photosynthetic organisms, photosystem I (PSI) and photosystem II (PSII) comprise a core complex hosting cofactors involved in electron transport and an outer antenna system made of light-harvesting complexes (LHCs): Lhcas for PSI and Lhcbs for PSII. The core complexes bind chlorophyll a (Chl a) and β-carotene, whereas the outer antenna system, in addition to Chl a, binds chlorophyll b (Chl b) and xanthophylls. Despite their overall similarity, PSI and PSII differ in the rate at which they trap excitation energy at the reaction center (RC), with PSI being faster than PSII (
1–
9). They also differ in their structure (
10–
12). PSI is monomeric and carries its antenna moiety on only one side as a half-moon–shaped structure whose size is not modulated by growth conditions (
13,
14). PSII, on the other hand, is found mainly as a dimeric core surrounded by an inner layer of antenna proteins (Lhcb4–6) and an outer layer of heterotrimeric LHCII complexes (Lhcb 1–3) whose stoichiometry varies depending on the growth conditions (
7,
12,
13,
15). Acclimation to high irradiance leads to a lower number of trimers per PSII RC accompanied by loss of the monomeric Lhcb6. These slow acclimative responses regulate the excitation pressure on the PSII RC, preventing saturation of the electron transport chain (
16) and the oxidative stress in high light (HL), leading to photoinhibition. The response to rapid changes in light level is managed by turning on some photoprotective mechanisms, such as the nonphotochemical quenching (NPQ) of the excess energy absorbed by PSII (
16), which is activated by the acidification of the thylakoid lumen and protonation of the trigger protein PsbS or LhcSR. Low luminal pH also activates violaxanthin de-epoxidase (VDE), catalyzing the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin (
17,
18), a scavenger of reactive oxygen species (ROS) produced by excess light (
9,
13). Zeaxanthin also enhances NPQ, as observed in vivo by a decrease of PSII fluorescence (
19). The short-term effects of exposure to HL on PSI have been disregarded thus far. Because of its rapid photochemistry, PSI shows low fluorescence emission, implying a low
1Chl* concentration and a low probability that chlorophyll triplet states will be formed by intersystem crossing. This characteristic suggests that the formation of oxygen singlet excited states (
1O*
2) is reduced and that NPQ phenomena in photoprotection are less relevant in PSI (
20,
21). Nevertheless, several reports have shown that, especially in the cold (
22–
29), PSI can exhibit photo-inhibition, with its Lhca proteins being the primary target (
24,
30). Upon synthesis in HL, zeaxanthin binding could be traced to two different types of binding site. One, designated “V1,” is located in the periphery of LHCII trimers (
31–
33). The second, designated “L2,” has an inner location in the dimeric Lhca1–4 and the monomeric Lhcb4–6 members of the LHC family (
34–
37). Experimental determination of the efficiency of the violaxanthin-to-zeaxanthin exchange yielded a maximal score in the Lhca3 and Lhca4 subunits (
24,
25). Interestingly, Lhca1/4 and Lhca2/3 are bound to the PSI core as dimers that can be isolated in fractions identified as “LHCI-730” and “LHCI-680,” respectively, both accumulating zeaxanthin to a de-epoxidation index of ∼0.2 (
20,
38). Lhca3 and Lhca4 carry low-absorption-energy chlorophyll forms known as “red forms” (
39,
40) that are responsible for the red-shifted PSI emission peak at 730–740 nm at 77 K. The molecular basis for red forms is an excitonic interaction of two chromophores: chlorophylls 603 and 609 located a few angstroms from the xanthophyll in site L2, which can be either violaxanthin or zeaxanthin depending on light conditions (
41,
42). It is unclear whether the binding of zeaxanthin to the PSI–LHCI complex has specific physiological function(s) or is simply a result of its common origin with Lhcb proteins.The goal of this study was to understand whether zeaxanthin plays a role in PSI–LHCI photoprotection. To investigate the role of zeaxanthin bound to Lhca proteins, we analyzed the changes in antenna size and Chl a fluorescence dynamics in PSI supercomplexes binding either violaxanthin or zeaxanthin. We found a zeaxanthin-dependent regulation of PSI antenna size and an enhanced resistance to excess light upon zeaxanthin binding. These results show that dynamic changes in the efficiency of light use and in photoprotection capacity are not exclusive to PSII, as previously thought; instead, eukaryotic photosynthetic organisms modulate the function of both photosystems in a coordinated manner.
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