Phycobilisome protein ApcG interacts with PSII and regulates energy transfer in Synechocystis.

Photosynthetic organisms harvest light using pigment-protein complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show in Synechocystis sp. PCC 6803 that the recently discovered PBS linker protein ApcG (sll1873) interacts specifically with PSII through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG causes reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with PSII is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with the thylakoid membrane, suggesting a regulatory role mediated by phosphorylation of ApcG. Low-temperature fluorescence measurements showed decreased PSI fluorescence in apcG deletion and complementation strains. The PSI fluorescence was the lowest in the phospho-mimicking complementation strain, while the pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and imply that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI.

Characterization of the preferred cation cofactors of chloroplast protein kinases in Arabidopsis thaliana.

Chloroplasts sense a variety of stimuli triggering several acclimation responses. One prominent response is the mechanism of state transitions, which enables rapid adaption to changes in illumination. Here, we investigated the link between divalent cations (calcium, magnesium, and manganese) and protein kinase activity in Arabidopsis chloroplasts. Our results show that manganese ions are the strongest activator of kinase activity in chloroplasts followed by magnesium ions, whereas calcium ions are not able to induce kinase activity. Additionally, the phosphorylation of specific protein bands is strongly reduced in chloroplasts of a cmt1 mutant, which is impaired in manganese import into chloroplasts, as compared to the wild-type. These findings provide insights for the future characterization of chloroplast protein kinase activity and potential target proteins.

Pathway Engineering, Re-targeting, and Synthetic Scaffolding Improve the Production of Squalene in Plants.

Plants are increasingly becoming an option for sustainable bioproduction of chemicals and complex molecules like terpenoids. The triterpene squalene has a variety of biotechnological uses and is the precursor to a diverse array of triterpenoids, but we currently lack a sustainable strategy to produce large quantities for industrial applications. Here, we further establish engineered plants as a platform for production of squalene through pathway re-targeting and membrane scaffolding. The squalene biosynthetic pathway, which natively resides in the cytosol and endoplasmic reticulum, was re-targeted to plastids, where screening of diverse variants of enzymes at key steps improved squalene yields. The highest yielding enzymes were used to create biosynthetic scaffolds on co-engineered, cytosolic lipid droplets, resulting in squalene yields up to 0.58 mg/gFW or 318% higher than a cytosolic pathway without scaffolding during transient expression. These scaffolds were also re-targeted to plastids where they associated with membranes throughout, including the formation of plastoglobules or plastidial lipid droplets. Plastid scaffolding ameliorated the negative effects of squalene biosynthesis and showed up to 345% higher rates of photosynthesis than without scaffolding. This study establishes a platform for engineering the production of squalene in plants, providing the opportunity to expand future work into production of higher-value triterpenoids.

The plastoglobule-localized protein AtABC1K6 is a Mn2+-dependent kinase necessary for timely transition to reproductive growth.

The Absence of bc1 Complex (ABC1) is an ancient, atypical protein kinase family that emerged prior to the archaeal-eubacterial divergence. Loss-of-function mutants in ABC1 genes are linked to respiratory defects in microbes and humans and to compromised photosynthetic performance and stress tolerance in plants. However, demonstration of protein kinase activity remains elusive, hampering their study. Here, we investigate a homolog from Arabidopsis thaliana, AtABC1K6, and demonstrate in vitro autophosphorylation activity, which we replicate with a human ABC1 ortholog. We also show that AtABC1K6 protein kinase activity requires an atypical buffer composition, including Mn2+ as a divalent cation cofactor and a low salt concentration. AtABC1K6 associates with plastoglobule lipid droplets of A. thaliana chloroplasts, along with five paralogs. We show that the protein kinase activity associated with isolated A. thaliana plastoglobules was inhibited at higher salt concentrations, but could accommodate Mg2+ as well as Mn2+, indicating salt sensitivity, but not the requirement for Mn2+, may be a general characteristic of ABC1 proteins. Finally, loss of functional AtABC1K6 impairs the developmental transition from vegetative to reproductive growth. This phenotype was complemented by the wild-type sequence of AtABC1K6, but not by a kinase-dead point mutant in the unique Ala-triad of the ATP-binding pocket, demonstrating the physiological relevance of the protein's kinase activity. We suggest that ABC1s are bona fide protein kinases with a unique regulatory mechanism. Our results open the door to detailed functional and mechanistic studies of ABC1 proteins and plastoglobules.

Insights into topology and membrane interaction characteristics of plastoglobule-localized AtFBN1a and AtLOX2.

Plant chloroplasts harbor ubiquitous lipid droplets called plastoglobules. While physically connected to the thylakoid membrane, they are characterized by a unique set of about 30 proteins specifically associated with the plastoglobule. How these proteins selectively target the plastoglobule remains unknown. Protease shaving assays with isolated Arabidopsis thaliana thylakoid and plastoglobule show that a ca. 25 kD portion of the abundant structural protein of plastoglobules, Fibrillin 1a, is protected from protease digestion. Mapping of protease cleavage sites and experimentally identified phosphorylation sites onto a homology model of Fibrillin 1a indicates that this protected sequence corresponds to the C-terminal lipocalin-like domain, implicated in specific lipid binding. In contrast, protease shaving and membrane washing assays with another plastoglobule-associated protein harboring a C-terminal PLAT domain, Lipoxygenase 2, is consistent with an exposed PLAT domain positioned parallel with, and upon, the surface of the plastoglobule. We propose a model where conserved lipid-binding domains associate with either the surface or neutral core of the lipid droplet. Our study provides insight into the topology and membrane interactions of two plastoglobule-localized proteins.

Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress.

Plants require rapid responses to adapt to environmental stresses. This includes dramatic changes in the size and number of plastoglobule lipid droplets within chloroplasts. Although the morphological changes of plastoglobules are well documented, little is known about the corresponding molecular changes. To address this gap, we have compared the quantitative proteome, oligomeric state, prenyl-lipid content and kinase activities of Arabidopsis thaliana plastoglobules under unstressed and 5-day light-stressed conditions. Our results show a specific recruitment of proteins related to leaf senescence and jasmonic acid biosynthesis under light stress, and identify nearly half of the plastoglobule proteins in high native molecular weight masses. Additionally, a specific increase in plastoglobule carotenoid abundance under the light stress was consistent with enhanced thylakoid disassembly and leaf senescence, supporting a specific role for plastoglobules in senescence and thylakoid remodeling as an intermediate storage site for photosynthetic pigments. In vitro kinase assays of isolated plastoglobules demonstrated kinase activity towards multiple target proteins, which was more pronounced in the plastoglobules of unstressed than light-stressed leaf tissue, and which was diminished in plastoglobules of the abc1k1/abc1k3 double-mutant. These results strongly suggest that plastoglobule-localized ABC1 kinases hold endogenous kinase activity, as these were the only known or putative kinases identified in the isolated plastoglobules by deep bottom-up proteomics. Collectively, our study reveals targeted changes to the protein and prenyl-lipid composition of plastoglobules under light stress that present strategies by which plastoglobules appear to facilitate stress adaptation within chloroplasts.

Lipid droplets throughout the evolutionary tree.

Intracellular lipid droplets are utilized for lipid storage and metabolism in organisms as evolutionarily diverse as animals, fungi, plants, bacteria, and archaea. These lipid droplets demonstrate great diversity in biological functions and protein and lipid compositions, yet fundamentally share common molecular and ultrastructural characteristics. Lipid droplet research has been largely fragmented across the diversity of lipid droplet classes and sub-classes. However, we suggest that there is great potential benefit to the lipid community in better integrating the lipid droplet research fields. To facilitate such integration, we survey the protein and lipid compositions, functional roles, and mechanisms of biogenesis across the breadth of lipid droplets studied throughout the natural world. We depict the big picture of lipid droplet biology, emphasizing shared characteristics and unique differences seen between different classes. In presenting the known diversity of lipid droplets side-by-side it becomes necessary to offer for the first time a consistent system of categorization and nomenclature. We propose a division into three primary classes that reflect their sub-cellular location: i) cytoplasmic lipid droplets (CYTO-LDs), that are present in the eukaryotic cytoplasm, ii) prokaryotic lipid droplets (PRO-LDs), that exist in the prokaryotic cytoplasm, and iii) plastid lipid droplets (PL-LDs), that are found in plant plastids, organelles of photosynthetic eukaryotes. Within each class there is a remarkable array of sub-classes displaying various sizes, shapes and compositions. A more integrated lipid droplet research field will provide opportunities to better build on discoveries and accelerate the pace of research in ways that have not been possible.

Plastoglobular protein 18 is involved in chloroplast function and thylakoid formation.

Plastoglobules are lipoprotein particles that are found in different types of plastids. They contain a very specific and specialized set of lipids and proteins. Plastoglobules are highly dynamic in size and shape, and are therefore thought to participate in adaptation processes during either abiotic or biotic stresses or transitions between developmental stages. They are suggested to function in thylakoid biogenesis, isoprenoid metabolism, and chlorophyll degradation. While several plastoglobular proteins contain identifiable domains, others provide no structural clues to their function. In this study, we investigate the role of plastoglobular protein 18 (PG18), which is conserved from cyanobacteria to higher plants. Analysis of a PG18 loss-of-function mutant in Arabidopsis thaliana demonstrated that PG18 plays an important role in thylakoid formation; the loss of PG18 results in impaired accumulation, assembly, and function of thylakoid membrane complexes. Interestingly, the mutant accumulated less chlorophyll and carotenoids, whereas xanthophyll cycle pigments were increased. Accumulation of photosynthetic complexes is similarly affected in both a Synechocystis and an Arabidopsis PG18 mutant. However, the ultrastructure of cyanobacterial thylakoids is not compromised by the lack of PG18, probably due to its less complex architecture.

An active Mitochondrial Complex II Present in Mature Seeds Contains an Embryo-Specific Iron-Sulfur Subunit Regulated by ABA and bZIP53 and Is Involved in Germination and Seedling Establishment.

Complex II (succinate dehydrogenase) is an essential mitochondrial enzyme involved in both the tricarboxylic acid cycle and the respiratory chain. In Arabidopsis thaliana, its iron-sulfur subunit (SDH2) is encoded by three genes, one of them (SDH2.3) being specifically expressed during seed maturation in the embryo. Here we show that seed SDH2.3 expression is regulated by abscisic acid (ABA) and we define the promoter region (-114 to +49) possessing all the cis-elements necessary and sufficient for high expression in seeds. This region includes between -114 and -32 three ABRE (ABA-responsive) elements and one RY-enhancer like element, and we demonstrate that these elements, although necessary, are not sufficient for seed expression, our results supporting a role for the region encoding the 5' untranslated region (+1 to +49). The SDH2.3 promoter is activated in leaf protoplasts by heterodimers between the basic leucine zipper transcription factors bZIP53 (group S1) and bZIP10 (group C) acting through the ABRE elements, and by the B3 domain transcription factor ABA insensitive 3 (ABI3). The in vivo role of bZIP53 is further supported by decreased SDH2.3 expression in a knockdown bzip53 mutant. By using the protein synthesis inhibitor cycloheximide and sdh2 mutants we have been able to conclusively show that complex II is already present in mature embryos before imbibition, and contains mainly SDH2.3 as iron-sulfur subunit. This complex plays a role during seed germination sensu-stricto since we have previously shown that seeds lacking SDH2.3 show retarded germination and now we demonstrate that low concentrations of thenoyltrifluoroacetone, a complex II inhibitor, also delay germination. Furthermore, complex II inhibitors completely block hypocotyl elongation in the dark and seedling establishment in the light, highlighting an essential role of complex II in the acquisition of photosynthetic competence and the transition from heterotrophy to autotrophy.