Giorgio M. Giacometti

Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo‐oxidation

Efficient photosynthesis is of fundamental importance for plant survival and fitness. However, in oxygenic photosynthesis, the complex apparatus responsible for the conversion of light into chemical energy is susceptible to photodamage. Oxygenic photosynthetic organisms have therefore evolved several protective mechanisms to deal with light energy. Rapidly inducible non‐photochemical quenching (NPQ) is a short‐term response by which plants and eukaryotic algae dissipate excitation energy as heat. This review focuses on recent advances in the elucidation of the molecular mechanisms underlying this protective quenching pathway in higher plants.

CHLOROPHYLL BINDING PROTEINS WITH ANTENNA FUNCTION IN HIGHER PLANTS and GREEN ALGAE

The absorption of light by antenna pigments creates mobile singlet state excitons which may migrate within the complex array of chlorophylls to either Photosystem I (PSI)* or PSII reaction centers. When an exciton reaches the reaction center, its energy is converted into charge separation, the exciton disappears, and a chain of redox reactions is triggered by which radiant energy is transduced. Excitons reaching the reaction centers are said to be trapped if charge separation occurs. Alternatively, they may return to the antenna pigments. During migration of excitons in the antenna matrix, the probability that they may decay as fluorescence quanta or by means of other non-radiative decay processes is finite. Owing to the high efficiency by which light is converted in the photosynthetic apparatus, energy losses are small and very little energy (approx. 2%) is emitted as fluorescence. As the purpose of photosynthesis is to convert excited state energy into chemical free energy with maximum efficiency, it might seem that any light energy emitted as fluorescence from the photosynthetic apparatus is “wasted”. However, at the present state of knowledge it cannot be excluded that fluorescence emission, at least in part, is the result of molecular control of the amount of energy reaching the reaction centers in order to avoid damage due to overexcitation and photoinhibition. In any case, the small fraction of energy emitted as fluorescence is of great importance in the investigation of excitation energy distribution and exciton migration among chlorophyll-protein complexes. The proportion of energy which is not utilized in photosynthesis and is emitted as fluorescence varies, depending mark-

Adjusted Light and Dark Cycles Can Optimize Photosynthetic Efficiency in Algae Growing in Photobioreactors

Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently.

Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization

Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.

Acclimation of Nannochloropsis gaditana to different illumination regimes: Effects on lipids accumulation

Algae are interesting potential sources of biodiesel, although research is still needed to develop efficient large scale productions. One major factor affecting productivity is light use efficiency. The effect of different light regimes on the seawater alga Nannochloropsis gaditana was accessed monitoring growth rate and photosynthetic performances. N. gaditana showed the capacity of acclimating to different light intensities, optimizing its photosynthetic apparatus to illumination. Thanks to this response, N. gaditana maintained similar growth rates under a wide range of irradiances, suggesting that this organism is a valuable candidate for outdoor productions in variable conditions. In the conditions tested here, without external CO(2) supply, light intensity alone was not found to be a major signal affecting lipids accumulation showing the absence of a direct regulatory link between the light stress and lipids accumulation. Strong illumination can nevertheless indirectly influences lipid accumulation if combined with other stresses or in the presence of excess CO(2).

Light- and pH-dependent structural changes in the PsbS subunit of photosystem II

In higher plants, the PsbS subunit of photosystem II (PSII) plays a crucial role in pH- and xanthophyll-dependent nonphotochemical quenching of excess absorbed light energy, thus contributing to the defense mechanism against photoinhibition. We determined the amino acid sequence of Zea mays PsbS and produced an antibody that recognizes with high specificity a region of the protein located in the stroma-exposed loop between the second and third putative helices. By means of this antiserum, the thylakoid membranes of various higher plant species revealed the presence of a 42-kDa protein band, indicating the formation of a dimer of the 21-kDa PsbS protein. Crosslinking experiments and immunoblotting with other antisera seem to exclude the formation of a heterodimer with other PSII protein components. The PsbS monomer/dimer ratio in isolated thylakoid membranes was found to vary with luminal pH in a reversible manner, the monomer being the prevalent form at acidic and the dimer at alkaline pH. In intact chloroplasts and whole plants, dimer-to-monomer conversion is reversibly induced by light, known to cause luminal acidification. Sucrose-gradient centrifugation revealed a prevalent association of the PsbS monomer and dimer with light-harvesting complex and PSII core complexes, respectively. The finding of the existence of a light-induced change in the quaternary structure of the PsbS subunit may contribute to understanding the mechanism of PsbS action during nonphotochemical quenching.

Degradation of Photosystem II reaction center D1-protein induced by UVB radiation in isolated thylakoids. Identification and characterization of C- and N-terminal breakdown products

Abstract The effects of UVB radiation on the stability of Photosystem II reaction center D1-protein in isolated thylakoids are investigated. A new C-terminal degradation product has been identified and characterized, produced by cleavage in the second transmembrane segment. Immunological evidence for the presence of N-terminal fragments corresponding to the remaining part of D1-protein is also provided. The appearance of these fragments is not affected by lowering the temperature from 22°C to 4°C, changing the pH from 6 to 8, adding soybean trypsin inhibitor or excluding oxygen from the thylakoid suspension.

Chromosome Scale Genome Assembly and Transcriptome Profiling of Nannochloropsis gaditana in Nitrogen Depletion

Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae. Here, we report a high-quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of the chromosomes. We identified 10646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing, and cell wall synthesis. We also analyzed the modification of the transcriptional profile in nitrogen deficiency-a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time, we observed a very significant down-regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway. Finally, we released an information resource of the genomic data of N. gaditana, available online at www.nannochloropsis.org.

New evidence suggests that the initial photoinduced cleavage of the D1-protein may not occur near the PEST sequence

When isolated reaction centres of photosystem 2 from pea or wheat are exposed to photoinhibitory illumination in the presence of an electron acceptor, breakdown products of the D1‐protein are observed having molecular masses ranging from about 24 to 10 kDa. By using antibodies raised to the C‐terminal or N‐terminal portions of D1 it was shown that the major breakdown fragment of 24 kDa was derived from the C‐terminus. This conclusion was supported by phosphorylation studies and from the digestion pattern obtained by lysine specific endoprotease‐induced proteolysis. The complementary N‐terminal breakdown fragment was found to have an apparent molecular mass of 10 kDa. The implications of these data are discussed in terms of the possible relationship between the 24 kDa C‐terminal fragment and the 23.5 kDa breakdown fragment detected in vivo by Greenberg et al. [1987, EMBO J. 6, 2865–2869] and it is suggested, based on limited proteolysis using papain, that the latter may not be derived from the N‐terminus as previously thought but also originates from the C‐terminus.

Crystal structure of the PsbQ protein of photosystem II from higher plants

The smallest extrinsic polypeptide of the water‐oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn2+ and its structure was determined by X‐ray diffraction at 1.95‐Å resolution using the multi‐wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four‐helix bundle, whereas the amino‐terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl− and Ca2+ and make them available to PSII.