Chloroplast protein translocation pathways and ubiquitin-dependent regulation at a glance.

Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.

Interplay of four auxiliary factors is required for the assembly of photosystem I reaction center subcomplex.

The photosystem I (PSI) complex consisting of reaction center (RC) subunits, several peripheral subunits and many co-factors, is present in the thylakoid membranes of chloroplasts and cyanobacteria. The assembly of RC subunits (PsaA/B) that bind electron transfer co-factors and antenna pigments is an intricate process, and is mediated by several auxiliary factors such as Ycf3, Y3IP1/CGL59, Ycf4 and Ycf37/PYG7/CGL71. However, their precise molecular mechanisms in RC assembly remain to be addressed. Here we purified four PSI auxiliary factors by affinity chromatography, and characterized co-purified PSI assembly intermediates. We suggest that Ycf3 assists the initial assembly of newly synthesized PsaA/B subunits into an RC subcomplex, while Y3IP1 may be involved in transferring the RC subcomplex from Ycf3 to the Ycf4 module that stabilizes it. CGL71 may form an oligomer that transiently interacts with the PSI RC subcomplex, physically protecting it under oxic conditions until association with the peripheral PSI subunits occurs. Together, our results reveal the interplay among four auxiliary factors required for the stepwise assembly of the PSI RC.

Assembly Apparatus of Light-Harvesting Complexes: Identification of Alb3.1-cpSRP-LHCP Complexes in the Green Alga Chlamydomonas reinhardtii.

The unicellular green alga, Chlamydomonas reinhardtii, contains many light-harvesting complexes (LHCs) associating chlorophylls a/b and carotenoids; the major LHCIIs (types I, II, III and IV) and minor light-harvesting complexes, CP26 and CP29, for photosystem II, as well as nine LHCIs (LHCA1-9), for photosystem I. A pale green mutant BF4 exhibited impaired accumulation of LHCs due to deficiency in the Alb3.1 gene, which encodes the insertase involved in insertion, folding and assembly of LHC proteins in the thylakoid membranes. To elucidate the molecular mechanism by which ALB3.1 assists LHC assembly, we complemented BF4 to express ALB3.1 fused with no, single or triple Human influenza hemagglutinin (HA) tag at its C-terminus (cAlb3.1, cAlb3.1-HA or cAlb3.1-3HA). The resulting complemented strains accumulated most LHC proteins comparable to wild-type (WT) levels. The affinity purification of Alb3.1-HA and Alb3.1-3HA preparations showed that ALB3.1 interacts with cpSRP43 and cpSRP54 proteins of the chloroplast signal recognition particle (cpSRP) and several LHC proteins; two major LHCII proteins (types I and III), two minor LHCII proteins (CP26 and CP29) and eight LHCI proteins (LHCA1, 2, 3, 4, 5, 6, 8 and 9). Pulse-chase labeling experiments revealed that the newly synthesized major LHCII proteins were transiently bound to the Alb3.1 complex. We propose that Alb3.1 interacts with cpSRP43 and cpSRP54 to form an assembly apparatus for most LHCs in the thylakoid membranes. Interestingly, photosystem I (PSI) proteins were also detected in the Alb3.1 preparations, suggesting that the integration of LHCIs to a PSI core complex to form a PSI-LHCI subcomplex occurs before assembled LHCIs dissociate from the Alb3.1-cpSRP complex.

The slow S to M rise of chlorophyll a fluorescence reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii.

The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between photosystem I (PS I) and photosystem II (PS II) to provide balanced photosynthesis. Chlorophyll (Chl) a fluorescence induction (the so-called OJIPSMT transient) is a signature of several photosynthetic reactions. Here, we show that the slow (seconds to minutes) S to M fluorescence rise is reduced or absent in the stt7 mutant (which is locked in state 1) in C. reinhardtii. This suggests that the SM rise in wild type C. reinhardtii may be due to state 2 (low fluorescence state; larger antenna in PS I) to state 1 (high fluorescence state; larger antenna in PS II) transition, and thus, it can be used as an efficient and quick method to monitor state transitions in algae, as has already been shown in cyanobacteria (Papageorgiou et al. 1999, 2007; Kana et al. 2012). We also discuss our results on the effects of (1) 3-(3,4-dichlorophenyl)-1,4-dimethyl urea, an inhibitor of electron transport; (2) n-propyl gallate, an inhibitor of alternative oxidase (AOX) in mitochondria and of plastid terminal oxidase in chloroplasts; (3) salicylhydroxamic acid, an inhibitor of AOX in mitochondria; and (4) carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an uncoupler of phosphorylation, which dissipates proton gradient across membranes. Based on the data presented in this paper, we conclude that the slow PSMT fluorescence transient in C. reinhardtii is due to the superimposition of, at least, two phenomena: qE dependent non-photochemical quenching of the excited state of Chl, and state transitions.

Moderate heat stress induces state transitions in Arabidopsis thaliana.

The effect of temperature on the photosynthetic machinery is crucial for the fundamental understanding of plant physiology and the bioengineering of heat-tolerant varieties. In our study, Arabidopsis thaliana was exposed to mild (40°C), short-term heat stress in the dark to evaluate the heat-triggered phosphorylation and migration of light harvesting complex (LHC) II in both wild-type (wt) and mutant lacking STN7 kinase. The 77K emission spectra revealed an increase in PSI relative to PSII emission similar to increases observed in light-induced state I to state II transitions in wt but not in stn7 mutant. Immunoblotting results indicated that the major LHCII was phosphorylated at threonine sites under heat stress in wt plants but not in the mutant. These results support the proposition that mild heat stress triggers state transitions in the dark similar to light-induced state transitions, which involve phosphorylation of LHCII by STN7 kinase. Pre-treatment of Arabidopsis leaves with inhibitor DBMIB, altered the extent of LHCII phosphorylation and PSI fluorescence emission suggests that activation of STN7 kinase may be dependent on Cyt b(6)/f under elevated temperatures in dark. Furthermore, fast Chl a transient of temperature-exposed leaves of wt showed a decrease in the F(v)/F(m) ratio due to both an increase in F(o) and a decrease in F(m). In summary, our findings indicate that a mild heat treatment (40°C) induces state transitions in the dark resulting in the migration of phosphorylated LHCII from the grana to the stroma region.

Structural and functional changes of PSI-LHCI supercomplexes of Chlamydomonas reinhardtii cells grown under high salt conditions.

The eVect of high salt concentration (100 mM NaCl) on the organization of photosystem I-light harvesting complex I supercomplexes (PSI-LHCI) of Chlamydomonas reinhardtii was studied. The electron transfer activity was reduced by 39% in isolated PSI-LHCI supercomplexes. The visible circular dichroism (CD) spectra associated with strongly coupled chlorophyll (Chl) dimers were reduced in intensity, indicating that pigment-pigment interactions were disrupted. This data is consistent with results from Xuorescence streak camera spectroscopy, which suggest that red-shifted pigments in the PSI-LHCI antenna had been lost. Denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI reaction center proteins PsaD, PsaE and PsaF were reduced due to salt stress. PsaE is almost completely absent under high salt conditions. It is known that the membrane-extrinsic subunits PsaD and E form the ferredoxin-docking site. Our results indicate that the PSI-LHCI supercomplex is damaged by reactive oxygen species at high salt concentration, with particular impact on the ferredoxin-docking site and the PSILHCI interface.

The photosystem I assembly apparatus consisting of Ycf3-Y3IP1 and Ycf4 modules.

In oxygenic photosynthesis, light energy is converted into redox energy by two photosystems (PSI and PSII). PSI forms one of the largest multiprotein complexes in thylakoid membranes consisting of a core complex, peripheral light-harvesting complexes (LHCIs) and cofactors. Although the high-resolution structure of the PSI-LHCI complex has been determined, the assembly process remains unclear due to the rapid nature of the assembly process. Here we show that two conserved chloroplast-encoded auxiliary factors, Ycf3 and Ycf4, form modules that mediate PSI assembly. The first module consists of the tetratricopeptide repeat protein Ycf3 and its interacting partner, Y3IP1, and mainly facilitates the assembly of reaction center subunits. The second module consists of oligomeric Ycf4 and facilitates the integration of peripheral PSI subunits and LHCIs into the PSI reaction center subcomplex. We reveal that these two modules are major mediators of the PSI-LHCI assembly process.

Antimycin A sensitive pathway independent from PGR5 cyclic electron transfer triggers non-photochemical reduction of PQ pool and state transitions in Arabidopsis thaliana.

We investigated the mechanism involved in triggering state transitions at 40°C in Arabidopsis thaliana. Leaves (1-6 week old) exposed to 40°C exhibited state II transition indicating its role as one of the earliest stress responsive mechanism apart from regulation of light energy distribution between photosystem (PS)II and PSI. Post illumination transients (rise in Fo') revealed that non-photochemical reduction of PQ pool at 40°C in dark is responsible for activation of STN7 kinase, consequently light harvesting complex (LHC)II phosphorylation leading to state II condition. Later, in pgr5 mutant, non-photochemical reduction of PQ pool was observed indicating the involvement of alternative electron transfer routes. In chlororespiratory mutant crr2-2, state II transition occurred signifying that the reduction of PQ pool is independent from NDH mediated cyclic electron transfer. Further, antimycin A inhibitor studies in wt and mutants revealed its inhibitory action on non-photochemical reduction of PQ pool affecting both LHCII phosphorylation and migration to PSI which leads to state I. Thus, our study showed that antimycin A sensitive pathway independent from PGR5 dependent cyclic electron transfer, is responsible for inducing non-photochemical reduction of PQ pool and state transitions at 40°C.

Heat- and light-induced detachment of the light harvesting complex from isolated photosystem I supercomplexes.

In a previous study, using photosystem I enriched stroma thylakoid membrane vesicles, we have shown that the light harvesting complexes of this photosystem are prone to heat- and light-induced, thermo-optically driven detachment from the supercomplex [43]. We have also shown that the splitting of the supercomplex occurs in a gradual and specific manner, selectively affecting the different constituents of the antenna complexes. Here we further analyse these heat- and light-induced processes in isolated Photosystem I supercomplex using circular dichroism and 77K fluorescence emission spectroscopy and immuno blotting, and obtain further details on the sequence of events of the dissociation process as well as on the thermal stability of the different components. Our absorption and circular dichroism spectroscopy and immuno blotting data show that the dissociation of LHCI from PSI-LHCI supercomplex starts above 50°C. Also, the low temperature fluorescence emission spectra depicts decrease of maximum fluorescence emission at 730nm and an increase of the intensity at 685nm, and about 10nm blue-shifts, from 730 to 720nm and from 685 to 676nm, respectively, indicating the heat (50°C) induced detachment of LHCI from PSI core complexes. The reaction centre proteins are highly stable even at high temperatures. Lhca2 is more heat stable than the other light harvesting protein complexes of PSI, whereas Lhca4 and Lhca3 are rather labile. Combined heat and light treatments significantly enhances the disorganization of PSI-LHCI supercomplexes, indicating a thermo-optic mechanism, which might have significant role under combined heat and light stress conditions.

Change in fast Chl a fluorescence transients, 2 dimensional protein profile and pigment protein interactions during state transitions in Arabidopsis thaliana.

Photosynthetic organisms during acclimation to light, differences in the amount of energy absorbed by photosystems leads to an imbalance in the energy distribution between photosystem (PS) II and PSI. Here, we describe the changes in fast chlorophyll (Chl) a fluorescence transients (OJIP) in wild type and stn7 under state I and state II light conditions. Fluorescence quenching in the OJIP transients recorded from state II exposed wt leaves is due to mobilization of LHCII to PSI. Similar kind of quenching was not observed in stn7 plants exposed to state II light. OJIP transients can be used to study the changes in Chl a fluorescence upon state transitions in A. thaliana. Immunoblotting and 2 dimensional gel electrophoresis studies have shown that phosphorylated Lhcb2 under state II condition exhibited 4 isoforms, whereas dephosphorylated Lhcb2 exhibited 3 isoforms in state I. Phosphorylation and migration of LHCII to PSI resulted in changes in the pigment protein profile of the thylakoid membranes in state II from wt. The increase in circular dichroism (CD) signals at 663 nm and 679 nm was due to association of chirally active trimeric LHCII to PSI-LHCI supercomplex leading to macro-aggregation of pigment-pigment complexes in state II pre-illuminated conditions in wt A. thaliana.

Effect of cold temperature on regulation of state transitions in Arabidopsis thaliana.

Low temperature is one of the most important abiotic factors limiting growth, development and distribution of plants. The effect of cold temperature on phosphorylation and migration of LHCII has been studied by 77K fluorescence emission spectroscopy and immuno-blotting in Arabidopsis thaliana. It has been reported that the mechanism of state transitions has been well operated at optimum growth temperatures. In this study, exposure of leaves to cold conditions (10 °C for 180 min) along with low light treatment (for 3h) did not show any increase in F726 which corresponds to fluorescence from PSI supercomplex, whereas low light at optimal temperature (26±2 °C) could enhanced F726. Therefore these results conclude that low light at cold condition did not enhance PSI absorption cross-section. We have also observed low levels of LHCII phosphorylation in cold exposed leaves in dark or low light. Though LHCII phosphorylation was detectable, the lateral movement of phosphorylated LHCII is reduced due to high granal stacking in cold treated leaves either in light or dark. Apart from these results, it is suggested that increased OJ phase and decreased JI and IP phases of Chl a fluorescence transients were due to reduced electron transport processes in cold treated samples.

Anaerobiosis induced state transition: a non photochemical reduction of PQ pool mediated by NDH in Arabidopsis thaliana.

BACKGROUND: Non photochemical reduction of PQ pool and mobilization of LHCII between PSII and PSI are found to be linked under abiotic stress conditions. The interaction of non photochemical reduction of PQ pool and state transitions associated physiological changes are critically important under anaerobic condition in higher plants. METHODOLOGY/FINDINGS: The present study focused on the effect of anaerobiosis on non-photochemical reduction of PQ pool which trigger state II transition in Arabidopsis thaliana. Upon exposure to dark-anaerobic condition the shape of the OJIP transient rise is completely altered where as in aerobic treated leaves the rise is unaltered. Rise in F(o) and F(J) was due to the loss of oxidized PQ pool as the PQ pool becomes more reduced. The increase in F(o)' was due to the non photochemical reduction of PQ pool which activated STN7 kinase and induced LHCII phosphorylation under anaerobic condition. Further, it was observed that the phosphorylated LHCII is migrated and associated with PSI supercomplex increasing its absorption cross-section. Furthermore, evidences from crr2-2 (NDH mutant) and pgr5 mutants (deficient in non NDH pathway of cyclic electron transport) have indicated that NDH is responsible for non photochemical reduction of the PQ pool. We propose that dark anaerobic condition accelerates production of reducing equivalents (such as NADPH by various metabolic pathways) which reduce PQ pool and is mediated by NDH leading to state II transition. CONCLUSIONS/SIGNIFICANCE: Anaerobic condition triggers non photochemical reduction of PQ pool mediated by NDH complex. The reduced PQ pool activates STN7 kinase leading to state II transition in A. thaliana.

Proteomic analysis of thylakoid membranes.

Chlamydomonas is a model organism to study photosynthesis. Thylakoid membranes comprise several proteins belonging to photosystems I and II. In this chapter, we show the accurate proteomic measurements in thylakoid membranes. The chlorophyll-containing membrane protein complexes were precipitated using chloroform/methanol solution. These complexes were separated using two-dimensional gel electrophoresis, and the resolved spots were exercised from the gel matrix and digested with trypsin. These peptide fragments were separated by MALDI-TOF, and the isotopic masses were blasted to a MASCOT server to obtain the protein sequence. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The method discussed here would be a useful method for the separation and identification of thylakoid membrane proteins.

Novel binding studies of human serum albumin with trans-feruloyl maslinic acid.

Human serum albumin (HSA) is a predominant protein in the blood. Most drugs can bind to HSA and be transported to target locations of the body. For this study, we have extracted 3-trans-feruloyl maslinic acid (FMA) from the medicinal plant Tetracera asiatica, its a non-fluorescent derivative have potent anti-cancer, anti-HIV, anti-diabetic, and anti-inflammatory activities. The binding constant of the compound with HSA, calculated from fluorescence data, was found as K(FMA)=1.42+/-0.01 x 10(8) M(-1), which corresponds to 10.9 kcal M(-1) of free energy. Furthermore, microTOF-Q mass spectrometry data showed binding of FMA at nanomolar concentrations of FMA to free HSA. The study detected a mass increase from 66,560 Da (free HSA) to 67,919 Da (HSA+drug). This indicated a strong binding of FMA to HSA, resulting in an increase of the protein's absorbance and fluorescence. The secondary structure of HSA+FMA (0.1 mM) complexes showed the protein secondary structure became partially unfolded upon interaction of FMA with HSA, as well as indicating that HSA-FMA complexes were formed. Docking experiments uncovered the binding mode of FMA in HSA molecule. It was found that FMA binds strongly in different places with hydrogen bonding at IB domain of Arg 114, Leu 115 and Asp 173.