A novel O-methyltransferase Cp4MP-OMT catalyses the final step in the biosynthesis of the volatile 1,4-dimethoxybenzene in pumpkin (Cucurbita pepo) flowers.

BACKGROUND: Floral scents play a crucial role in attracting insect pollinators. Among the compounds attractive to pollinators is 1,4-dimethoxybenzene (1,4-DMB). It is a significant contributor to the scent profile of plants from various genera, including economically important Cucurbita species. Despite its importance, the biosynthetic pathway for the formation of 1,4-DMB was not elucidated so far. RESULTS: In this study we showed the catalysis of 1,4-DMB in the presence of 4-methoxyphenol (4-MP) by protein extract from Styrian oil pumpkin (Cucurbita pepo) flowers. Based on this finding, we identified a novel O-methyltransferase gene, Cp4MP-OMT, whose expression is highly upregulated in the volatile-producing tissue of pumpkin flowers when compared to vegetative tissues. OMT activity was verified by purified recombinant Cp4MP-OMT, illustrating its ability to catalyse the methylation of 4-MP to 1,4-DMB in the presence of cofactor SAM (S-(5'-adenosyl)-L-methionine). CONCLUSIONS: Cp4MP-OMT is a novel O-methyltransferase from C. pepo, responsible for the final step in the biosynthesis of the floral scent compound 1,4-DMB. Considering the significance of 1,4-DMB in attracting insects for pollination and in the further course fruit formation, enhanced understanding of its biosynthetic pathways holds great promise for both ecological insights and advancements in plant breeding initiatives.

The molecular identity of the characean OH- transporter: a candidate related to the SLC4 family of animal pH regulators.

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH-, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH-/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH-/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Brefeldin A inhibits clathrin-dependent endocytosis and ion transport in Chara internodal cells.

BACKGROUND: The Characeae are multicellular green algae, which are closely related to higher plants. Their internodal cells are a convenient model to study membrane transport and organelle interactions. RESULTS: In this study, we report on the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on internodal cells of Chara australis. BFA induced the commonly observed agglomeration of Golgi bodies and trans Golgi network into 'brefeldin compartments' at concentrations between 6 and 500 µM and within 30-120 min treatment. In contrast to most other cells, however, BFA inhibited endocytosis and significantly decreased the number of clathrin-coated pits and clathrin-coated vesicles at the plasma membrane. BFA did not inhibit secretion of organelles at wounds induced by puncturing or local light damage but prevented the formation of cellulosic wound walls probably because of insufficient membrane recycling. We also found that BFA inhibited the formation of alkaline and acid regions along the cell surface ('pH banding pattern') which facilitates carbon uptake required for photosynthesis; we hypothesise that this is due to insufficient recycling of ion transporters. During long-term treatments over several days, BFA delayed the formation of complex 3D plasma membranes (charasomes). Interestingly, BFA had no detectable effect on clathrin-dependent charasome degradation. Protein sequence analysis suggests that the peculiar effects of BFA in Chara internodal cells are due to a mutation in the guanine-nucleotide exchange factor GNOM required for recruitment of membrane coats via activation of ADP-ribosylation factor proteins. CONCLUSIONS AND SIGNIFICANCE: This work provides an overview on the effects of BFA on different processes in C. australis. It revealed similarities but also distinct differences in vesicle trafficking between higher plant and algal cells. It shows that characean internodal cells are a promising model to study interactions between seemingly distant metabolic pathways.

Dissecting the subcellular membrane proteome reveals enrichment of H+ (co-)transporters and vesicle trafficking proteins in acidic zones of Chara internodal cells.

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H+ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H+ ATPase sequence suggests a different regulation compared to higher plant PM H+ ATPases. A higher abundance of H+ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes.

Surface pH changes suggest a role for H+/OH- channels in salinity response of Chara australis.

To understand salt stress, the full impact of salinity on plant cell physiology has to be resolved. Electrical measurements suggest that salinity inhibits the proton pump and opens putative H+/OH- channels all over the cell surface of salt sensitive Chara australis (Beilby and Al Khazaaly 2009; Al Khazaaly and Beilby 2012). The channels open transiently at first, causing a characteristic noise in membrane potential difference (PD), and after longer exposure remain open with a typical current-voltage (I/V) profile, both abolished by the addition of 1 mM ZnCl2, the main known blocker of animal H+ channels. The cells were imaged with confocal microscopy, using fluorescein isothiocyanate (FITC) coupled to dextran 70 to illuminate the pH changes outside the cell wall in artificial fresh water (AFW) and in saline medium. In the early saline exposure, we observed alkaline patches (bright fluorescent spots) appearing transiently in random spatial distribution. After longer exposure, some of the spots became fixed in space. Saline also abolished or diminished the pH banding pattern observed in the untreated control cells. ZnCl2 suppressed the alkaline spot formation in saline and the pH banding pattern in AFW. The osmotic component of the saline stress did not produce transient bright spots or affect banding. The displacement of H+ from the cell wall charges, the H+/OH- channel conductance/density, and self-organization are discussed. No homologies to animal H+ channels were found. Salinity activation of the H+/OH- channels might contribute to saline response in roots of land plants and leaves of aquatic angiosperms.

Clathrin in Chara australis: Molecular Analysis and Involvement in Charasome Degradation and Constitutive Endocytosis.

Charasomes are convoluted plasma membrane domains in characean green algae. They are known to form in response to light via secretion of trans-Golgi network (TGN) vesicles and local inhibition of endocytosis. Charasomes are involved in the acidification of their aqueous environment, thereby facilitating photosynthesis-dependent carbon uptake. Charasome formation is reversible to allow cells to adapt to different light conditions. Here, we show that darkness-induced degradation of charasomes involves the formation of coated pits and coated vesicles. The darkness-induced degradation of charasomes can be inhibited by 1-2 µM ikarugamycin (IKA), which is considered to be a specific inhibitor of clathrin-dependent endocytosis. At a much higher concentration (100 µM), IKA also significantly reduces the internalization of styryl dyes, indicating uptake via clathrin-coated vesicles (CV). We are the first to present evidence, based on fine structure investigation, that IKA does not interfere with the formation of clathrin coat, but inhibits the detachment and/or further processing of coated vesicles. Both charasome degradation and constitutive endocytosis are also significantly inhibited by sterol complexing agents (methyl-ß-cyclodextrin and filipin). The absence of an additive effect, when applied together with IKA, suggests that charasome degradation and constitutive endocytosis (measured via styryl dye uptake) is not inhibited due to membrane retrieval via lipid rafts, but due to clathrin coat formation requirement of a specific set of sterols. Analysis of Chara australis clathrin proteins revealed two heavy chains and several light chains with sequence peculiarities, suggesting functional and/or species specific differences. The data obtained indicate that clathrin plays a central role not only in constitutive endocytosis but also in the degradation of charasomes, thereby representing a valuable system for studying targeted exo- and endocytosis.

Is Wortmannin-Induced Reorganization of the trans-Golgi Network the Key to Explain Charasome Formation?

Wortmannin, a fungal metabolite and an inhibitor of phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases, is widely used for the investigation and dissection of vacuolar trafficking routes and for the identification of proteins located at multivesicular bodies (MVBs). In this study, we applied wortmannin on internodal cells of the characean green alga Chara australis. Wortmannin was used at concentrations of 25 and 50 µM which, unlike in other cells, arrested neither constitutive, nor wounding-induced endocytosis via coated vesicles. Wortmannin caused the formation of "mixed compartments" consisting of MVBs and membranous tubules which were probably derived from the trans-Golgi network (TGN) and within these compartments MVBs fused into larger organelles. Most interestingly, wortmannin also caused pronounced changes in the morphology of the TGNs. After transient hypertrophy, the TGNs lost their coat and formed compact, three-dimensional meshworks of anastomosing tubules containing a central core. These meshworks had a size of up to 4 µm and a striking resemblance to charasomes, which are convoluted plasma membrane domains, and which serve to increase the area available for transporters. Our findings indicate that similar mechanisms are responsible for the formation of charasomes and the wortmannin-induced reorganization of the TGN. We hypothesize that both organelles grow because of a disturbance of clathrin-dependent membrane retrieval due to inhibition of PI3 and/or PI4 kinases. This leads to local inhibition of clathrin-mediated endocytosis during charasome formation in untreated cells and to inhibition of vesicle release from the TGN in wortmannin-treated cells, respectively. The morphological resemblance between charasomes and wortmannin-modified TGN compartments suggests that homologous proteins are involved in membrane curvature and organelle architecture.

Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae.

RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE-containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.

Vesicular trafficking in characean green algae and the possible involvement of a VAMP72-family protein.

The RAB5 GTPase ARA6 (AtARA6) of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses.

Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae.

RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4 kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.

The effect of translationally controlled tumour protein (TCTP) on programmed cell death in plants.

BACKGROUND: Translationally controlled tumour protein (TCTP), a well known protein of the animal kingdom, was shown to be a Ca(2+)-binding protein with important functions in many different cellular processes (e.g. protection against stress and apoptosis, cell growth, cell cycle progression, and microtubule organization). However, only little is known about TCTP in plants. Transcript and protein levels of plant TCTPs were shown to be altered by various stress conditions (e.g. cold, salt, draught, aluminium, and pathogen infection), and Arabidopsis thaliana TCTP (AtTCTP) was described as an important regulator of growth. The aim of this study was to further characterize plant TCTP relating to one of its major functions in animals: the protection against cell death. RESULTS: We used two different activators of programmed cell death (PCD) in plants: the mammalian pro-apoptotic protein BAX and tunicamycin, an inhibitor of glycosylation and trigger of unfolded protein response (UPR). Over-expression of AtTCTP significantly decreased cell death in tobacco leaf discs in both studies. A (45)Ca overlay assay showed AtTCTP to be a Ca(2+)-binding protein and localization experiments revealed cytosolic distribution of AtTCTP-GFP in Arabidopsis seedlings. CONCLUSIONS: Our study showed cytoprotective effects of plant TCTP for the first time. Furthermore, we showed the ability of AtTCTP to bind to Ca(2+) and its cytosolic distribution within the cell. If these results are combined, two putative modes of action can be assumed: 1) AtTCTP acts as Ca(2+) sequester, preventing PCD by reducing cytosolic Ca(2+) levels as described for animals. 2) AtTCTP could directly or indirectly interact with other cytosolic or membrane-bound proteins of the cell death machinery, thereby inhibiting cell death progression. As no homologous proteins of the anti-apoptotic machinery of animals were found in plants, and functional homologues still remain to be elucidated, future work will provide more insight.

Investigations on N-rich protein (NRP) of Arabidopsis thaliana under different stress conditions.

Stress response and adaptation are important physiological mechanisms in plants. As plants are not able to avoid stressful environments by moving away, as animals, they have developed diverse mechanisms to respond to stressful situations. One of the genes involved in these mechanisms is NRP (Asparagine-rich protein or N-rich protein). In this study, NRP expression, protein localization and nrp knockout plants were investigated for further understanding of NRP function. NaCl-induced salt stress, oxidative stress (ozone exposure) and mechanical perturbation (touch treatment) were used to induce abiotic stress. NRP expression was up-regulated in the early phase of stress response to all three elicitors. Stressed nrp knockout seedlings revealed a more pronounced growth inhibition compared to wildtype (salt and osmotic stress). Seedlings showed NRP-GFP expression in the apical meristem, leaf veins, central cylinder, root hair zone and root tip. Analyses of NRP-GFP localization in root cells and protoplasts revealed cytosolic distribution under non-stress conditions and translocation of NRP-GFP to mitochondria due to stress response. Summarizing, our findings point to a contribution of NRP in signal transduction of the initial phase of general stress response in Arabidopsis thaliana.

Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum.

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min(-1). Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.