RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO2 ) and ribulose-1,5-bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD-like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.

LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells.

To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.

Functional organization of photosystem II antenna complexes: CP29 under the spotlight.

In the first step of the photosynthetic process, light is absorbed by the pigments associated with the antenna proteins, known as light-harvesting complexes (Lhcs), which in vivo are functionally organized as hetero-oligomers. The architecture of the pigments, chlorophylls, and carotenoids bound to each LHC is responsible for the efficient excitation energy transfer resulting in photochemistry. So far, the only LHC studied in depth was LHCII, the most abundant membrane protein of plants, while less information was available for the other antennae. In particular, despite the availability of the structure of CP29 obtained at near atomic resolution in 2011 (Pan et al., 2011), the mismatch in pigment content and spectroscopic properties between CP29 in solution and in the crystal has hampered the possibility to use the structure to interpret the experimental data. In this work, we purified CP29 and its larger assembly (CP29-LHCII-CP24) from the membrane in very mild conditions using a His-tag, and we have studied their pigment binding and spectroscopic properties. In addition, we have performed mutation analysis in vivo to obtain mutants of CP29 lacking individual chlorophylls. The peculiar properties of this antenna support its role in directing the energy flow from the external antennae to the reaction center.

Excitation dynamics and structural implication of the stress-related complex LHCSR3 from the green alga Chlamydomonas reinhardtii.

LHCSR3 is a member of the Light-Harvesting Complexes (LHC) family, which is mainly composed of pigment-protein complexes responsible for collecting photons during the first steps of photosynthesis. Unlike related LHCs, LHCSR3 is expressed in stress conditions and has been shown to be essential for the fast component of photoprotection, non-photochemical quenching (NPQ), in the green alga Chlamydomonas reinhardtii. In plants, which do not possess LHCSR homologs, NPQ is triggered by the PSBS protein. Both PSBS and LHCSR3 possess the ability to sense pH changes but, unlike PSBS, LHCSR3 binds multiple pigments. In this work we have analyzed the properties of the pigments bound to LHCSR3 and their excited state dynamics. The data show efficient excitation energy transfer between pigments with rates similar to those observed for the other LHCs. Application of an exciton model based on a template of LHCII, the most abundant LHC, satisfactorily explains the collected steady state and time-resolved spectroscopic data, indicating that LHCSR3 has a LHC-like molecular architecture, although it probably binds less pigments. The model suggests that most of the chlorophylls have similar energy and interactions as in LHCII. The most striking difference is the localization of the lowest energy state, which is not on the Chlorophyll a (Chl a) 610-611-612 triplet as in all the LHCB antennas, but on Chl a613, which is located close to the lumen and to the pH-sensing region of the protein.

Regulation of light harvesting in the green alga Chlamydomonas reinhardtii: the C-terminus of LHCSR is the knob of a dimmer switch.

Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.

Revisiting the Role of Xanthophylls in Nonphotochemical Quenching.

Photoprotective nonphotochemical quenching (NPQ) of absorbed solar energy is vital for survival of photosynthetic organisms, and NPQ modifications significantly improve plant productivity. However, the exact NPQ quenching mechanism is obscured by discrepancies between reported mechanisms, involving xanthophyll-chlorophyll (Xan-Chl) and Chl-Chl interactions. We present evidence of an experimental artifact that may explain the discrepancies: strong laser pulses lead to the formation of a novel electronic species in the major plant light-harvesting complex (LHCII). This species evolves from a high excited state of Chl a and is absent with weak laser pulses. It resembles an excitonically coupled heterodimer of Chl a and lutein (or other Xans at site L1) and acts as a de-excitation channel. Laser powers, and consequently amounts of artifact, vary strongly between NPQ studies, thereby explaining contradicting spectral signatures attributed to NPQ. Our results offer pathways toward unveiling NPQ mechanisms and highlight the necessity of careful attention to laser-induced artifacts.

In vitro reconstitution of light-harvesting complexes of plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 1987(1), who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

Human polymorphic variants of the NEIL1 DNA glycosylase.

In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864-1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type beta,delta-elimination reaction but primarily yielded the beta-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and gamma-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome.

Progress in maize gene discovery: a project update.

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon ( RescueMu). Current achievements of the MGDP (NSF 98-72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor. In addition, ZmDB contains links to other plant and genetics websites.