Current understanding

Mechanism of the PSII Repair Process
Photo-oxidative damage to the D1 protein of photosystem II reaction center is an inevitable process that can occur at any light intensity. For survival, plants have evolved a PSII repair mechanism to regenerate active complexes from the ones that are damaged. The repair process is in continuous operation under normal and adverse photosynthesis conditions and affords chloroplast recovery from photodamage. The term ‘photosystem II damage and repair cycle’ (Fig. 1) has been established for this naturally occurring phenomenon in all oxygenic-evolving organisms.

Fig. 1. Diagram illustrating the photosystem II damage and repair cycle.

A temporal and spatial sequence of events in the PSII repair process includes:

a) Protection and stabilization of the inactive PSII
Under the photoinhibitory conditions, when the rate of photodamage exceeds the rate of the repair mechanism, photoinactivated PSII reaction centers accumulate in the thylakoid membranes. Such inactive reaction centers that contain damaged D1 protein are impaired in photochemical reactions. At this stage, the remaining active components of the photoinhibited PSII are vulnerable to massive photobleaching as photons can still be absorbed by its auxiliary chlorophyll antenna but can no longer be utilized in the photochemistry. It has been suggested that the inactive photosystem II, reversible and permanent, can be converted into an energy trap capable of efficiently quenching excess excitation energy.

There have been speculations that a xanthophyll cycle pigment zeaxanthin also plays a role in the sustained quenching due to photoinhibition. Accumulation and retention of zeaxanthin has been found to correlate with the degree of photoinhibition in overwintering evergreen plants, pea, shade leaves of Schefflera arboricola, brown algae Dictyota dichotoma and green algae Chlamydomonas reinhardtii and Dunaliella salina. These diverse observations, which cover both higher plants and algae, raise the prospective that at least part of the zeaxanthin accumulation process might be a subsequent event from photoinhibition. Functional role of zeaxanthin in protecting and stabilizing the damaged PSII is one of the ongoing research topics in this lab.

b) Partial disassembly of the damaged PSII holocomplex

Photosystem II reaction center is believed to exist as a dimer, of which each monomer consists of more than 25 transmembrane and peripheral extrinsic protein subunits. In order for the damaged PSII to return to the active state, it needs to undergo the processes of selective degradation and replacement of the non-functional D1 protein. As a prerequisite for the D1 degradation, a core complex of the photodamaged PSII has to be partially disassembled from its sizable holocomplex and migrates to the non-appressed thylakoid membrane where it is exposed to specific proteases and the D1 biosynthesis machineries.

In higher plant PSII, several major subunits including D1, D2, PsbH, CP43 and LHCII are phosphorylated upon illumination. Although phosphorylation of the LHCII involves in balancing of light energy absorption between PSII and PSI, the reversible phosphorylation of the other PSII components has been found to play an important role in the regulation of D1 protein turnover. Phosphorylation of the PSII core proteins does not prevent monomerization of the PSII but rather functions as a protective mechanism to inhibit premature degradation of the damaged D1 before it reaches the stroma-exposed region. Subsequent dephosphorylation of the PSII proteins in the stroma lamellae allows coordinated D1 degradation and biosynthesis to take place.

In a unicellular green alga Dunaliella salina, photodamaged and disassembled PSII reaction center has been identified and isolated as a distinct 160 kDa complex on SDS-PAGE. The 160 kDa complex was found to be a cross-linked derivative of D1, D2, CP47 and a chloroplast-localized heat-shock protein 70 (HSP70B). Native conformation of the 160 kDa PSII repair intermediate was found to be a large protein complex migrating at about 320 kDa on native PAGE. The colorless appearance of this 320 kDa complex on native gel prior to staining confirmed the notion that this PSII repair intermediate has been disassembled from its peripheral chlorophyll a/b light-harvesting antenna. Nevertheless, at present the picture is not fully clear regarding the relationship between the 160/320 kDa complexes.

At this point the regulatory mechanism of the damaged and disassembled PSII reaction center in green algae still needs further investigation. Identification and characterization of the PSII repair intermediate similar to the 160 kDa in other organisms would help complete the overall picture of the complex and its formation. In addition, functions of the molecular chaperone HSP70B and possibly other unidentified proteins in facilitating the repair process also need more detailed characterization. We are interested in addressing these aspects of the PSII repair process.

c) Biodegradation of inactive D1 protein by specific proteases

During the repair process, photodamaged D1 has to be selectively degraded and replaced with a new functional copy. Analyses under in vivo and in vitro systems revealed various breakdown products of D1 proteins. A primary cleavage site after an acceptor-side photoinhibition was found to reside in a stromal loop connecting D and E helices. On the other hand, a cleavage at a lumenal loop connecting helix A and B or a lumenal loop between C and D helices could be observed following a donor-side-induced photodamage to the PSII reaction center. Nevertheless, regardless of the diverse degradation pattern of the D1 protein, it was generally believed that specific proteases tightly associated with the thylakoid membranes are responsible for the degradation of photoinactivated D1 protein.

The protease that performs the primary cleavage of the photoinhibited D1 protein has been identified by in vitro and in vivo experiments as a chloroplast-localized Deg protease family, which belongs to a family of prokaryotic trypsin-type Deg/Htr serine proteases. The results of this primary cleavage are an N-terminal fragment of 23-kDa and a C-terminus of 10 kDa, of which the reaction was found to be GTP dependent. Subsequent degradation of the 23-kDa D1 fragment requires a chloroplast homologue of the bacterial FtsH protease, ATP and a divalent metal ion, primarily Zn.

Although many evidences are pointing toward Deg and FtsH in performing the degradation of the photodamaged D1, the molecular mechanism of the degradation is not fully understood. One cannot exclude possibility that other yet identified proteins or proteases may interact with the Deg and FtsH or facilitate the degradation process. In fact, there has been suggestion that degradation and replacement of the damaged D1 protein are strictly coordinated. It is likely, therefore, that D1 degradation in vivo may involve formation of a large multicomponent degradation and biosynthesis complex.

d) Biosynthesis, reinsertion of the newly active D1 into the PSII core complex

Once the damaged D1 protein is degraded, a new functional copy is de novo synthesized and incorporated into the core complex using the chloroplast translation machineries.

e) Reassembly of fully active PSII and its incorporation in the grana partition

The newly repaired PSII core complex is reassembled with the structural and peripheral antenna complexes in the grana partition of the thylakoid membranes. The fully reassembled complex is now active and ready to function again.

Fig. 2. The schematic diagram showing the working hypothesis for the PSII damage and repair cycle in model green algae.

Photoprotective Mechanisms

Photoprotection entails various mechanisms employed by photosynthetic organisms to minimize the harmful effects of excess photon absorption. These mechanisms include physiological responses to lower the level of incident light such as movement of leaves and chloroplasts. At the molecular level, scavenging of reactive oxygen species by antioxidant molecules, non-photochemical dissipation of excess excitation energy as heat and modification of photosynthetic machineries to enhance photon usage and reduce light absorption can also help diminish the photo-oxidative damage.

Non-photochemical quenching is a mechanism by which excess energy from singlet-excited chlorophyll is quenched and safely dissipated at heat. Generally, NPQ can be observed by a decrease in Chl fluorescence emission. Three different types of NPQ can be distinguished by their relaxation kinetics following dark incubation as well as their responses to various inhibitors. Temporally, energy- or pH-dependent quenching (qE) relaxes within a few minutes, followed by state-transitions (qT) in many minutes and then by photoinhibitory quenching (qI), which relaxes on a time scale of hours.

Longer-term acclimation to irradiance stress entails the assembly of a truncated light-harvesting antenna size for the photosystems and adjustments in the stoichiometry between PSI and PSII. Such acclimation mechanisms are manifested as an overall reduced content of cellular Chl and the thylakoid membrane space. These adjustments help attenuate light absorption, regulate and better balance the relative activities of the light versus the carbon reactions. Photoacclimation processes optimize photosynthetic reactions and, thus, bring about attenuation of the rate of PSII photodamage.

Despite of these known responses to counteract the harmful effect of excessive light levels, different photosynthetic organisms exhibit different degree of tolerance to irradiance stresses. Using two green algae with different level of light sensitivities, Chlamydomonas reinhardtii and Dunaliella salina, as models, we are performing comparative investigation of their molecular response to high light stress. Our hope is to understand the factor(s) that confer tolerance to light stress.

Recent Publications

INTERNATIONAL RESEARCH PUBLICATIONS

1. Yokthongwattana K, Thaipratum R, Melis A and Svasti J Excitation quenching properties of Dunaliella salina (green alga) wild type and zea1, a mutant constitutively accumulating zeaxanthin. In preparation.
2. Yokthongwattana K, Savchenko T, Polle JEW and Melis A (2005) Isolation and characterization of a xanthophyll-rich fraction from the thylakoid membrane of Dunaliella salina (green algae). Photochem. Photobiol. Sci., 4: 1028 - 1034. (IF 2007 = 2.208)
3. Chen H-C, Yokthongwattana K, Newton AJ and Melis A (2003) SulP, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in Chlamydomonas reinhardtii. Planta, 218: 98-106. (IF 2007 = 3.058)
4. Jin E, Yokthongwattana K, Polle JEW and Melis A (2003) Role of the reversible xanthophyll cycle in the photosystem-II damage and repair cycle in Dunaliella salina (green alga). Plant Physiol., 132: 352-364. (IF 2007 = 6.367)
5. Yokthongwattana K, Chrost B, Behrman S, Casper-Lindley C and Melis A (2001) Photosystem II damage and repair cycle in the green alga Dunaliella salina: involvement of a chloroplast-localized HSP70. Plant Cell Physiol., 42: 1389-1397. (IF 2007 = 3.654)

INTERNATIONAL BOOK CHAPTERS, REVIEW ARTICLES

1. Yokthongwattana K, Jin E and Melis A (2008) Chloroplast acclimation, photodamage and repair reactions of photosystem-II in the model green alga Dunaliella salina. In: Ben-Amotz A, Polle JEW, Rao DVS (eds) The alga Dunaliella : biodiversity, physiology, genomics and biotechnology. Science Publishers, New Hampshire, USA, in press.
2. Yokthongwattana K and Melis A (2006) Photoinhibition and recovery in oxygenic photosynthesis: mechanism of a photosystem-II damage and repair cycle, In Demmig-Adams B, Adams III WW, Mattoo AK (eds) Photoprotection, photoinhibition, gene regulation and environment. Advances in Photosynthesis Series, Springer, Dordrecht, The Netherlands, pp. 175-191.

more information at
http://www.sc.mahidol.ac.th/academics/staff/AC_k/Kittisak_Y.htm