In plant cells, plastids form elongated extensions called stromules, the regulation and purposes of which remain unclear. Here, we quantitatively explore how different stromule structures serve to enhance the ability of a plastid to interact with other organelles: increasing the effective space for interaction and biomolecular exchange between organelles. Interestingly, electron microscopy and confocal imaging showed that the cytoplasm in Arabidopsis thaliana and Nicotiana benthamiana epidermal cells is extremely thin (around 100 nm in regions without organelles), meaning that inter-organelle interactions effectively take place in 2D. We combine these imaging modalities with mathematical modeling and new in planta experiments to demonstrate how different stromule varieties (single or multiple, linear or branching) could be employed to optimize different aspects of inter-organelle interaction capacity in this 2D space. We found that stromule formation and branching provide a proportionally higher benefit to interaction capacity in 2D than in 3D. Additionally, this benefit depends on optimal plastid spacing. We hypothesize that cells can promote the formation of different stromule architectures in the quasi-2D cytoplasm to optimize their interaction interface to meet specific requirements. These results provide new insight into the mechanisms underlying the transition from low to high stromule numbers, the consequences for interaction with smaller organelles, how plastid access and plastid to nucleus signaling are balanced and the impact of plastid density on organelle interaction.

The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Plant cell walls are sophisticated carbohydrate-rich structures representing the immediate contact surface with the extracellular environment, often serving as the first barrier against biotic and abiotic stresses. Notably, a variety of perturbations in plant cell walls result in upregulated jasmonate (JA) production, a phytohormone with essential roles in defense and growth responses. Hence, cell wall-derived signals can initiate intracellular JA-mediated responses and the elucidation of the underlying signaling pathways could provide novel insights into cell wall maintenance and remodeling, as well as advance our understanding on how is JA biosynthesis initiated. This Mini Review will describe current knowledge about cell wall-derived damage signals and their effects on JA biosynthesis, as well as provide future perspectives.

Main conclusionSolanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation.Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

Recognition of pathogen-associated molecular patterns (PAMPs) induces multiple defense mechanisms to limit pathogen growth. Here, we show that the Arabidopsis thaliana tandem zinc finger protein 9 (TZF9) is phosphorylated by PAMP-responsive mitogen-activated protein kinases (MAPKs) and is required to trigger a full PAMP-triggered immune response. Analysis of a tzf9 mutant revealed attenuation in specific PAMP-triggered reactions such as reactive oxygen species accumulation, MAPK activation and, partially, the expression of several PAMP-responsive genes. In accordance with these weaker PAMP-triggered responses, tzf9 mutant plants exhibit enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000. Visualization of TZF9 localization by fusion to green fluorescent protein revealed cytoplasmic foci that co-localize with marker proteins of processing bodies (P-bodies). This localization pattern is affected by inhibitor treatments that limit mRNA availability (such as cycloheximide or actinomycin D) or block nuclear export (leptomycin B). Coupled with its ability to bind the ribohomopolymers poly(rU) and poly(rG), these results suggest involvement of TZF9 in post-transcriptional regulation, such as mRNA processing or storage pathways, to regulate plant innate immunity.

Arabidopsis caffeoyl coenzyme A dependent O-methyltransferase 1 (CCoAOMT1) and caffeic acid O-methyltransferase 1 (COMT1) display a similar substrate profile although with distinct substrate preferences and are considered the key methyltransferases (OMTs) in the biosynthesis of lignin monomers, coniferyl and sinapoylalcohol. Whereas CCoAOMT1 displays a strong preference for caffeoyl coenzyme A, COMT1 preferentially methylates 5-hydroxyferuloyl CoA derivatives and also performs methylation of flavonols with vicinal aromatic dihydroxy groups, such as quercetin. Based on different knockout lines, phenolic profiling, and immunohistochemistry, we present evidence that both enzymes fulfil distinct, yet different tasks in Arabidopsis anthers. CCoAOMT1 besides its role in vascular tissues can be localized to the tapetum of young stamens, contributing to the biosynthesis of spermidine phenylpropanoid conjugates. COMT1, although present in the same organ, is not localized in the tapetum, but in two directly adjacent cells layers, the endothecium and the epidermal layer of stamens. In vivo localization and phenolic profiling of comt1 plants provide evidence that COMT1 neither contributes to the accumulation of spermidine phenylpropanoid conjugates nor to the flavonol glycoside pattern of pollen grains.

Water-soluble chlorophyll protein (WSCP) has been found in many Brassicaceae, most often in leaves. In many cases, its expression is stress-induced, therefore, it is thought to be involved in some stress response. In this work, recombinant WSCP from Arabidopsis thaliana (AtWSCP) is found to form chlorophyll-protein complexes in vitro that share many properties with recombinant or native WSCP from Brassica oleracea, BoWSCP, including an unusual heat resistance up to 100°C in aqueous solution. A polyclonal antibody raised against the recombinant apoprotein is used to identify plant tissues expressing AtWSCP. The only plant organs containing significant amounts of AtWSCP are the gynoecium in open flowers and the septum of developing siliques, specifically the transmission tract. In fully grown but still green siliques, the protein has almost disappeared. Possible implications for AtWSCP functions are discussed.

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C–C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

Brassicaceous plants are characterized by a pronounced metabolic flux toward sinapate, produced by the shikimate/phenylpropanoid pathway, which is converted into a broad spectrum of O-ester conjugates. The abundant sinapate esters in Brassica napus and Arabidopsis thaliana reflect a well-known metabolic network, including UDP-glucose:sinapate glucosyltransferase (SGT), sinapoylglucose:choline sinapoyltransferase (SCT), sinapoylglucose:l-malate sinapoyltransferase (SMT) and sinapoylcholine (sinapine) esterase (SCE). 1-O-Sinapoylglucose, produced by SGT during seed development, is converted to sinapine by SCT and hydrolyzed by SCE in germinating seeds. The released sinapate feeds via sinapoylglucose into the biosynthesis of sinapoylmalate in the seedlings catalyzed by SMT. Sinapoylmalate is involved in protecting the leaves against the deleterious effects of UV-B radiation. Sinapine might function as storage vehicle for ready supply of choline for phosphatidylcholine biosynthesis in young seedlings. The antinutritive character of sinapine and related sinapate esters hamper the use of the valuable seed protein of the oilseed crop B. napus for animal feed and human nutrition. Due to limited variation in seed sinapine content within the assortment of B. napus cultivars, low sinapine lines cannot be generated by conventional breeding giving rise to genetic engineering of sinapate ester metabolism as a promising means. In this article we review the progress made throughout the last decade in identification of genes involved in sinapate ester metabolism and characterization of the encoded enzymes. Based on gene structures and enzyme recruitment, evolution of sinapate ester metabolism is discussed. Strategies of targeted metabolic engineering, designed to generate low-sinapate ester lines of B. napus, are evaluated.

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.