Felix Kessler

FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana

Tetrapyrroles such as chlorophylls and bacteriochlorophylls play a fundamental role in the energy absorption and transduction activities of photosynthetic organisms. Because of these molecules, however, photosynthetic organisms are also prone to photooxidative damage. They had to evolve highly efficient strategies to control tetrapyrrole biosynthesis and to prevent the accumulation of free intermediates that potentially are extremely destructive when illuminated. In higher plants, the metabolic flow of tetrapyrrole biosynthesis is regulated at the step of δ-aminolevulinic acid synthesis. This regulation previously has been attributed to feedback control of Glu tRNA reductase, the first enzyme committed to tetrapyrrole biosynthesis, by heme. With the recent discovery of chlorophyll intermediates acting as signals that control both nuclear gene activities and tetrapyrrole biosynthesis, it seems likely that heme is not the only regulator of this pathway. A genetic approach was used to identify additional factors involved in the control of tetrapyrrole biosynthesis. In Arabidopsis thaliana, we have found a negative regulator of tetrapyrrole biosynthesis, FLU, which operates independently of heme and seems to selectively affect only the Mg2+ branch of tetrapyrrole biosynthesis. The identity of this protein was established by map-based cloning and sequencing the FLU gene. FLU is a nuclear-encoded plastid protein that, after import and processing, becomes tightly associated with plastid membranes. It is unrelated to any of the enzymes known to be involved in tetrapyrrole biosynthesis. Its predicted features suggest that FLU mediates its regulatory effect through interaction with enzymes involved in chlorophyll synthesis.

The major protein import receptor of plastids is essential for chloroplast biogenesis.

Light triggers the developmental programme in plants that leads to the production of photosynthetically active chloroplasts from non-photosynthetic proplastids. During this chloroplast biogenesis, the photosynthetic apparatus is rapidly assembled, mostly from nuclear-encoded imported proteins, which are synthesized in the cytosol as precursors with cleavable amino-terminal targeting sequences called transit sequences. Protein translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes recognize these transit sequences, leading to the precursors being imported. The Toc complex in the pea consists of three major components, Toc75, Toc34 and Toc159 (formerly termed Toc86). Toc159, which is an integral membrane GTPase, functions as a transit-sequence receptor. Here we show that Arabidopsis thaliana Toc159 (atToc159) is essential for the biogenesis of chloroplasts. In an Arabidopsis mutant (ppi2) that lacks atToc159, photosynthetic proteins that are normally abundant are transcriptionally repressed, and are found in much smaller amounts in the plastids, although ppi2 does not affect either the expression or the import of less abundant non-photosynthetic plastid proteins. These findings indicate that atToc159 is required for the quantitative import of photosynthetic proteins. Two proteins that are related to atToc159 (atToc120 and atToc132) probably help to maintain basal protein import in ppi2, and so constitute components of alternative, atToc159-independent import pathways.

Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes.

Plastoglobules are lipoprotein particles inside chloroplasts. Their numbers have been shown to increase during the upregulation of plastid lipid metabolism in response to oxidative stress and during senescence. In this study, we used state-of-the-art high-pressure freezing/freeze-substitution methods combined with electron tomography as well as freeze-etch electron microscopy to characterize the structure and spatial relationship of plastoglobules to thylakoid membranes in developing, mature, and senescing chloroplasts. We demonstrate that plastoglobules are attached to thylakoids through a half-lipid bilayer that surrounds the globule contents and is continuous with the stroma-side leaflet of the thylakoid membrane. During oxidative stress and senescence, plastoglobules form linkage groups that are attached to each other and remain continuous with the thylakoid membrane by extensions of the half-lipid bilayer. Using three-dimensional tomography combined with immunolabeling techniques, we show that the plastoglobules contain the enzyme tocopherol cyclase (VTE1) and that this enzyme extends across the surface monolayer into the interior of the plastoglobules. These findings demonstrate that plastoglobules function as both lipid biosynthesis and storage subcompartments of thylakoid membranes. The permanent structural coupling between plastoglobules and thylakoid membranes suggests that the lipid molecules contained in the plastoglobule cores (carotenoids, plastoquinone, and tocopherol [vitamin E]) are in a dynamic equilibrium with those located in the thylakoid membranes.

Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles.

Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana. We demonstrate specific plastoglobule association of members of the plastid lipid-associated proteins/fibrillin family as well as known metabolic enzymes, including the tocopherol cyclase (VTE1), a key enzyme of tocopherol (vitamin E) synthesis. Moreover, comparative analysis of chloroplast membrane fractions shows that plastoglobules are a site of vitamin E accumulation in chloroplasts. Thus, in addition to their lipid storage function, we propose that plastoglobules are metabolically active, taking part in tocopherol synthesis and likely other pathways.

The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.

STAY-GREEN and Chlorophyll Catabolic Enzymes Interact at Light-Harvesting Complex II for Chlorophyll Detoxification during Leaf Senescence in Arabidopsis

This work shows that the chloroplast-localized components of the chlorophyll catabolic pathway dynamically interact with each other, possibly forming a multiprotein complex specifically localizing to light-harvesting complex II. This interaction likely channels chlorophyll breakdown intermediates and thereby prevents potential chlorophyll-derived phototoxicity during leaf senescence. During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel’s green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.

Arabidopsis Tic110 Is Essential for the Assembly and Function of the Protein Import Machinery of Plastids

The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.

The Function and Diversity of Plastid Protein Import Pathways: A Multilane GTPase Highway into Plastids

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus‐encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non‐photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc–Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.