Marcel Kuntz

Fibril assembly and carotenoid overaccumulation in chromoplasts: a model for supramolecular lipoprotein structures.

Chromoplast development in ripening bell pepper fruits is characterized by a massive synthesis of carotenoid pigments, resulting in their distinctive red color. We have shown that 95% of these pigments accumulate in chromoplasts in specific lipoprotein fibrils. In addition to carotenoids, purified fibrils contain galactolipids, phospholipids, and a single, 32-kD protein, designated fibrillin, which has antigenically related counterparts in other species. Fibrils were reconstituted in vitro when purified fibrillin was combined with carotenoids and polar lipids in the same stoichiometric ratio found in fibrils in vivo. Antibodies directed against fibrillin were used to isolate a fibrillin cDNA clone and, in immunological studies, to follow its accumulation during the chloroplast-to-chromoplast transition under different conditions. A model for fibril architecture is proposed wherein carotenoids accumulate in the center of the fibrils and are surrounded by a layer of polar lipids, which in turn are surrounded by an outer layer of fibrillin. Topological analysis of purified fibrils verified this structure. Collectively, these results suggest that the process of fibril self-assembly in chromoplasts is an example of a general phenomenon shared among cells that target excess membrane lipids into deposit structures to avoid their destabilizing or toxic effects. In addition, we have shown that abscisic acid stimulates this phenomenon in chromoplasts, whereas gibberellic acid and auxin delay it.

Evaluation of Genetically Engineered Crops Using Transcriptomic, Proteomic, and Metabolomic Profiling Techniques

A major principle and guiding tool for the food safety assessment of genetically engineered (GE) crops is the concept of “substantial equivalence” according to principles outlined in the Organization for Economic Cooperation and Development (OECD) consensus documents ([OECD, 2006][1]) and

Biochemistry and Molecular Biology of Chromoplast Development

Plant cells contain a unique class of organelles, designated the plastids, which distinguish them from animal cells. According to the largely accepted endosymbiotic theory of evolution, plastids are descendants of prokaryotes. This process requires several adaptative changes which involve the maintenance and the expression of part of the plastid genome, as well as the integration of the plastid activity to the cellular metabolism. This is illustrated by the diversity of plastids encountered in plant cells. For instance, in tissues undergoing color changes, i.e., flowers and fruits, the chromoplasts produce and accumulate excess carotenoids. In this paper we attempt to review the basic aspects of chromoplast development.

A dual strategy to cope with high light in Chlamydomonas reinhardtii

To protect photosynthetic organisms from photodamage, excess energy in photosystem II must be dissipated. In C. reinhardtii, two alternative mechanisms (energy thermal dissipation [qE] and kinase-mediated migration of light harvesting proteins [qT]) synergistically modulate photoprotection via the reversible migration of LHCSR3, a key qE effector in this alga, between photosystems II and I during qT. Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.

Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating ξ‐carotene desaturation

We have cloned a cDNA from the plant Capsicum annuum which encodes a novel enzyme mediating the dehydrogenation of ξ‐carotene and neurosporene to lycopene when expressed in E. coli cells accumulating ξ‐carotene or neurosporene. This enzyme is unable to dehydrogenate either phytoene or lycopene. The deduced amino acid sequence suggests that this cDNA encodes a polypeptide whose mature size is ca. 59 kDa and which is synthesized as a precursor with a NH2‐terminal extension resembling transit peptides for plastid targeting. Sequence comparison reveals 33–35% similarity with previously cloned plant or cyanobacterial phytoene desaturases. In contrast, only limited sequence similarity is found with a ξ‐carotene desaturase from the cyanobacterium Anabaena.

The Biosynthetic Capacities of the Plastids and Integration Between Cytoplasmic and Chloroplast Processes

Plastids are semiautonomous organelles derived from cyanobacterial ancestors. Following endosymbiosis, plastids have evolved to optimize their functions, thereby limiting metabolic redundancy with other cell compartments. Contemporary plastids have also recruited proteins produced by the nuclear genome of the host cell. In addition, many genes acquired from the cyanobacterial ancestor evolved to code for proteins that are targeted to cell compartments other than the plastid. Consequently, metabolic pathways are now a patchwork of enzymes of diverse origins, located in various cell compartments. Because of this, a wide range of metabolites and ions traffic between the plastids and other cell compartments. In this review, we provide a comprehensive analysis of the well-known, and of the as yet uncharacterized, chloroplast/cytosol exchange processes, which can be deduced from what is currently known about compartmentation of plant-cell metabolism.

Dual Role of the Plastid Terminal Oxidase in Tomato

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.

Plastid terminal oxidase (PTOX) has the potential to act as a safety valve for excess excitation energy in the alpine plant species Ranunculus glacialis L.

Ranunculus glacialis leaves were tested for their plastid terminal oxidase (PTOX) content and electron flow to photorespiration and to alternative acceptors. In shade-leaves, the PTOX and NAD(P)H dehydrogenase (NDH) content were markedly lower than in sun-leaves. Carbon assimilation/light and Ci response curves were not different in sun- and shade-leaves, but photosynthetic capacity was the highest in sun-leaves. Based on calculation of the apparent specificity factor of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), the magnitude of alternative electron flow unrelated to carboxylation and oxygenation of Rubisco correlated to the PTOX content in sun-, shade- and growth chamber-leaves. Similarly, fluorescence induction kinetics indicated more complete and more rapid reoxidation of the plastoquinone (PQ) pool in sun- than in shade-leaves. Blocking electron flow to assimilation, photorespiration and the Mehler reaction with appropriate inhibitors showed that sun-leaves were able to maintain higher electron flow and PQ oxidation. The results suggest that PTOX can act as a safety valve in R. glacialis leaves under conditions where incident photon flux density (PFD) exceeds the growth PFD and under conditions where the plastoquinone pool is highly reduced. Such conditions can occur frequently in alpine climates due to rapid light and temperature changes.

Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant.

The physiological role of the plastid terminal oxidase (PTOX) involved in plastoquinol oxidation in chloroplasts has been investigated in vivo in tomato leaves. Enzyme activity was assessed by non-invasive methods based on the analysis of the kinetics of chlorophyll fluorescence changes. In the dark, the maximum PTOX rate was smaller than 1 electron per second per PSII. This value was further decreased upon light acclimation, and became almost negligible upon inhibition of the photosynthetic performances by reducing the CO(2) availability. In contrast, prolonged exposure to high light resulted in an increase of the overall PTOX activity, which was paralleled by an increased protein accumulation. Under all the conditions tested the enzyme activity always remained about two orders of magnitude lower than that of electron flux through the linear photosynthetic electron pathway. Therefore, PTOX cannot have a role of a safety valve for photogenerated electrons, while it could be involved in acclimation to high light. Moreover, by playing a major role in the control of the stromal redox poise, PTOX is also capable of modulating the balance between linear and cyclic electron flow around PSI during the deactivation phase of carbon assimilation that follows a light to dark transition.