Phytocystatin 6 is a context-dependent, tight-binding inhibitor of Arabidopsis thaliana legumain isoform ß.

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform ß (AtLEGß) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGß and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGß. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGß suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGß regulation and its broader physiological significance.

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.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

Inter- and intraspecific phytochemical variation correlate with epiphytic flower and leaf bacterial communities.

Microbes associated with flowers and leaves affect plant health and fitness and modify the chemical phenotypes of plants with consequences for interactions of plants with their environment. However, the drivers of bacterial communities colonizing above-ground parts of grassland plants in the field remain largely unknown. We therefore examined the relationships between phytochemistry and the epiphytic bacterial community composition of flowers and leaves of Ranunculus acris and Trifolium pratense. On 252 plant individuals, we characterized primary and specialized metabolites, that is, surface sugars, volatile organic compounds (VOCs), and metabolic fingerprints, as well as epiphytic flower and leaf bacterial communities. The genomic potential of bacterial colonizers concerning metabolic capacities was assessed using bacterial reference genomes. Phytochemical composition displayed pronounced variation within and between plant species and organs, which explained part of the variation in bacterial community composition. Correlation network analysis suggests strain-specific correlations with metabolites. Analysis of bacterial reference genomes revealed taxon-specific metabolic capabilities that corresponded with genes involved in glycolysis and adaptation to osmotic stress. Our results show relationships between phytochemistry and the flower and leaf bacterial microbiomes suggesting that plants provide chemical niches for distinct bacterial communities. In turn, bacteria may induce alterations in the plants' chemical phenotype. Thus, our study may stimulate further research on the mechanisms of trait-based community assembly in epiphytic bacteria.

Galactose induces formation of cell wall stubs and cell death in Arabidopsis roots.

MAIN CONCLUSION: Arabidopsis seedlings growing on low concentration of galactose stop regular root growth. Incomplete cell division with cell wall stubs, binuclear and giant cells and lignified root tips are observed. Galactose is a sugar abundant in root cell walls of Arabidopsis. Nevertheless, we found that the germination of Arabidopsis seedlings on galactose containing media causes a strong modification of the root development, as shown by analysing the root with microscopy methods ranging from the bright field over confocal to transmission electron microscopy. At concentrations of about 1 mM, the growth of the primary root stops after a few days though stem cell markers like WOX5 are still expressed. The root tip swells and forms a slightly opaque, partially lignified structure in parts of the cortex and the central cylinder. The formation of the cell plate after mitosis is impaired, often leading to cell wall stubs and binuclear cells. Some cells in the cortex and the central cylinder degenerate, while some rhizodermal and cortical cells increase massively in size. The galactose toxicity phenotype in Arabidopsis depends on the activity of galactokinase and is completely diminished in galactokinase knock-out lines. From the comparison of the galactose toxicity phenotype with those of cytokinesis mutants and plants treated with appropriate inhibitors we speculate that the toxicity syndrome of galactose is caused by interference with intracellular vesicle transport or cell wall biogenesis.

Nectaries in ferns: their taxonomic distribution, structure, function, and sugar composition.

PREMISE: Extrafloral nectaries have mainly been studied in angiosperms, but have also been reported in 39 fern species. Here we provide a global review of nectaries in ferns and examined their structure, function, and nectar sugar composition in two genera. METHODS: We searched in the literature and living plant collections of botanical gardens for indications of fern nectaries, observed nectar-feeding animals, studied the morphoanatomy in the two genera Aglaomorpha and Campyloneurum, and analyzed the total sugar concentrations and ratios of 16 species. Diurnal nectar release was observed with time-lapse photography. RESULTS: We found evidence for nectaries in 101 species of ferns from 11 genera and 6 families. Most of the nectary-bearing species were tree ferns (Cyatheaceae) and epiphytic ferns of the family Polypodiaceae. Nectaries consisted of cytoplasm-rich parenchyma with large nuclei and an epidermis with or without stomata, were attached to amphiphloic vascular bundles, and released nectar on the lower leaf surface mainly on expanding leaves during the night. Sugar concentrations varied between species (3.8-15.3%) but not between genera, and were sucrose-dominant (3 spp.), sucrose-rich (7), or hexose-rich (3). In the greenhouse, introduced ants, scale insects, and snails fed on the nectar. CONCLUSIONS: The wide taxonomic distribution, variable morphology, locations, and sugar compositions point to multiple evolutionary origins of fern nectaries. Nectar release in young leaves might attract mutualistic ants to protect leaves against herbivores only during this most vulnerable developmental stage. Even ex-situ, fern nectar is a valuable food source because it attracted several opportunistic animal species.

Photodynamic Inactivation of plant pathogens part II: fungi.

The constantly increasing demand for agricultural produce from organic and conventional farming calls for new, sustainable, and biocompatible solutions for crop protection. The overuse of fungicides leading to contamination of both produce and environment and the emergence of plant pathogenic fungi that are resistant to conventional treatments warrant the need for new methods to combat fungal infections in the field. We here deliver the follow-up study to our research on the Photodynamic Inactivation (PDI) of plant pathogenic bacteria (Glueck et al. in Photochem Photobiol Sci 18(7):1700-1708, 2019) by expanding the scope to fungal pathogens. Both fungal species employed in this study-Alternaria solani and Botrytis cinerea-cause substantial crop and economic losses. Sodium magnesium chlorophyllin (Chl, approved as food additive E140) in combination with Na2EDTA and the chlorin e6 derivative B17-0024 holding cationic moieties serve as eco-friendly photoactive compounds. Effectiveness of the antifungal PDI was measured by inhibition of growth of mycelial spheres (average diameter 2-3 mm) after incubation with the photosensitizer for 100 min and subsequent illumination using a LED array (395 nm, 106.6 J cm-2). One hundred micromolar Chl combined with 5 mM Na2EDTA was able to successfully photokill 94.1% of A. solani and 91.7% of B. cinerea samples. PDI based on B17-0024 can completely inactivate A. solani at 10 times lower concentration (10 µM); however, for B. cinerea, the concentration required for complete eradication was similar to that of Chl with Na2EDTA (100 µM). Using a plant compatibility assay based on Fragaria vesca, we further demonstrate that both photosensitizers neither affect host plant development nor cause significant leaf damage. The plants were sprayed with 300 µL of treatment solution used for PDI (one or three treatments on consecutive days) and plant growth was monitored for 21 days. Only minor leaf damage was observed in samples exposed to the chelators Na2EDTA and polyaspartic acid, but overall plant development was unaffected. In conclusion, our results suggest that sodium magnesium chlorophyllin in combination with EDTA and B17-0024 could serve as effective and safe photofungicides.

Ascorbate oxidation activates systemic defence against root-knot nematode Meloidogyne graminicola in rice.

Ascorbic acid (AA) is the major antioxidant buffer produced in the shoot tissue of plants. Previous studies on root-knot nematode (RKN; Meloidogyne graminicola)-infected rice (Oryza sativa) plants showed differential expression of AA-recycling genes, although their functional role was unknown. Our results confirmed increased dehydroascorbate (DHA) levels in nematode-induced root galls, while AA mutants were significantly more susceptible to nematode infection. External applications of ascorbate oxidase (AO), DHA, or reduced AA, revealed systemic effects of ascorbate oxidation on rice defence versus RKN, associated with a primed accumulation of H2O2 upon nematode infection. To confirm and further investigate these systemic effects, a transcriptome analysis was done on roots of foliar AO-treated plants, revealing activation of the ethylene (ET) response and jasmonic acid (JA) biosynthesis pathways in roots, which was confirmed by hormone measurements. Activation of these pathways by methyl-JA, or ethephon treatment can complement the susceptibility phenotype of the rice Vitamin C (vtc1) mutant. Experiments on the jasmonate signalling (jar1) mutant or using chemical JA/ET inhibitors confirm that the effects of ascorbate oxidation are dependent on both the JA and ET pathways. Collectively, our data reveal a novel pathway in which ascorbate oxidation induces systemic defence against RKNs.

Bimodal Pollination Systems in Andean Melastomataceae Involving Birds, Bats, and Rodents.

Floral adaptation to a single most effective functional pollinator group leads to specialized pollination syndromes. However, adaptations allowing for pollination by two functional groups (bimodal pollination systems) remain a rarely investigated conundrum. We tested whether floral scent and nectar traits of species visited by two functional pollinator groups indicate specialization on either of the two pollinator groups or adaptations of both (bimodal systems). We studied pollination biology in four species of Meriania (Melastomataceae) in the Ecuadorian Andes. Pollinator observations and exclusion experiments showed that each species was effectively pollinated by two functional groups (hummingbirds/bats, hummingbirds/rodents, flowerpiercers/rodents), nectar composition followed known bird preferences, and scent profiles gave mixed support for specialization on bats and rodents. Our results suggest that nectar-rewarding Meriania species have evolved stable bimodal pollination strategies with parallel adaptations to two functional pollinator groups. The discovery of rodent pollination is particularly important given its rarity outside of South Africa.

Transient alkalinization of the leaf apoplast stiffens the cell wall during onset of chloride salinity in corn leaves.

During chloride salinity, the pH of the leaf apoplast (pHapo) transiently alkalizes. There is an ongoing debate about the physiological relevance of these stress-induced pHapo changes. Using proteomic analyses of expanding leaves of corn (Zea mays L.), we show that this transition in pHapo conveys functionality by (i) adjusting protein abundances and (ii) affecting the rheological properties of the cell wall. pHapo was monitored in planta via microscopy-based ratio imaging, and the leaf-proteomic response to the transient leaf apoplastic alkalinization was analyzed via ultra-high performance liquid chromatography-MS. This analysis identified 1459 proteins, of which 44 exhibited increased abundance specifically through the chloride-induced transient rise in pHapo These elevated protein abundances did not directly arise from high tissue concentrations of Cl- or Na+ but were due to changes in the pHapo Most of these proteins functioned in growth-relevant processes and in the synthesis of cell wall-building components such as arabinose. Measurements with a linear-variable differential transducer revealed that the transient alkalinization rigidified (i.e. stiffened) the cell wall during the onset of chloride salinity. A decrease in t-coumaric and t-ferulic acids indicates that the wall stiffening arises from cross-linkage to cell wall polymers. We conclude that the pH of the apoplast represents a dynamic factor that is mechanistically coupled to cellular responses to chloride stress. By hardening the wall, the increased pH abrogates wall loosening required for cell expansion and growth. We conclude that the transient alkalinization of the leaf apoplast is related to salinity-induced growth reduction.

Arabidopsis MAP-Kinase 3 Phosphorylates UDP-Glucose Dehydrogenase: a Key Enzyme Providing UDP-Sugar for Cell Wall Biosynthesis.

The enzyme UDP-glucose dehydrogenase (UGD) competes with sucrose-phosphate synthase for the common photosynthesis product UDP-glucose. Sucrose-phosphate synthase is part of a pathway for the export of sucrose from source leaves to neighboring cells or the phloem. UGD is a central enzyme in a pathway for many nucleotide sugars used in local cell wall biosynthesis. Here, we identify a highly conserved phosphorylation site in UGD which is readily phosphorylated by MAP-kinase 3 in Arabidopsis. Phosphorylation occurs at a surface-exposed extra loop in all plant UGDs that is absent in UGDs from bacteria or animals. Phosphorylated sucrose-phosphate synthase is shifted to an inactive form which we did not measure for phosphorylated UGD. Plant UGDs have an extra loop which is phosphorylated by AtMPK3. Phosphorylation is not causing a reduction of UGD activity as found for the competitor enzymes and thus sets a preference for maintaining UDP-sugars at a constant level to prioritize cell wall biosynthesis.

The role of arabinokinase in arabinose toxicity in plants.

Plant cell wall polymers are synthesized by glycosyltransferases using nucleotide sugars as substrates. Most UDP-sugars are synthesized from UDP-glucose via de novo pathways but salvage pathways work in parallel to recycle sugars, which have been released during cell wall polymer and glycoprotein turnover. Here we report on the cloning and biochemical analysis of two arabinokinases in Arabidopsis. Arabinokinase is a 100 kDa protein located in the cytosol with a putative N-terminal glycosyltransferase domain and a C-terminal sugar-1-kinase domain. This unique structure is highly conserved in the plant kingdom. Arabinokinase has a high affinity for l-arabinose, which is the only sugar substrate of this GHMP (galactose; homoserine; mevalonate; phosphomevalonate) kinase. Plants that were knocked-out for arabinokinase and the previously described ara1-1 mutant were characterized. The ARA1-1 mutant form of the enzyme carries a point mutation in an α-helix. The mutation is close to the substrate binding site and changes the Km value for arabinose from 80 µm in the wild type to 17 000 µm in ARA1-1. The previous arabinose toxicity explanation is challenged by knockout plants in arabinokinase that accumulate higher levels of arabinose but do not show signs of arabinose toxicity. Analysis of marker genes from sugar signalling pathways (SnRK1 and Tor) suggest that ara1-1 misinterprets its carbon energy status. Although glucose is present in ara1-1 similar to wild type levels, it constitutively changes gene expression as typically found in wild type plants only under starvation conditions. Furthermore, ara1-1 shows increased expression of marker genes for programmed cell death as found in other lesion mimic mutants.

UDP-sugar pyrophosphorylase is essential for arabinose and xylose recycling, and is required during vegetative and reproductive growth in Arabidopsis.

Numerous nucleotide sugars are needed in plants to synthesize cell wall polymers and glycoproteins. The de novo synthesis of nucleotide sugars is of major importance. During growth, however, some polymers are broken down to monosaccharides. Reactivation of these sugars into nucleotide sugars occurs in two steps: first, by a substrate-specific sugar-1-kinase and, second, by UDP-sugar-pyrophosphorylase (USP), which has broad substrate specificity. A knock-out of the USP gene results in non-fertile pollen. By using various genetic complementation approaches we obtained a strong (>95%) knock-down line in USP that allowed us to investigate the physiological role of the enzyme during the life cycle. Mutant plants show an arabinose reduction in the cell wall, and accumulate mainly two sugars, arabinose and xylose, in the cytoplasm. The arabinogalactanproteins in usp mutants show no significant reduction in size. USP is also part of the myo-inositol oxygenation pathway to UDP-glucuronic acid; however, free glucuronic acid does not accumulate in cells, suggesting alternative conversion pathways of this monosaccharide. The knock-down plants are mostly sterile because of the improper formation of anthers and pollen sacks.

A mutation in the Arabidopsis thaliana cell wall biosynthesis gene pectin methylesterase 3 as well as its aberrant expression cause hypersensitivity specifically to Zn.

Defects in metal homeostasis factors are often accompanied by the loss of metal tolerance. Therefore, we screened for mutants with compromised growth in the presence of excess Zn(2+) in order to identify factors involved in Zn biology in plants. Here we report the isolation of six ozs (overly Zn sensitive) ethyl methanesulfonate Arabidopsis thaliana mutants with contrasting patterns of metal sensitivity, and the molecular characterization of two mutants hypersensitive specifically to Zn(2+) . Mutant ozs1 represents a non-functional allele of the vacuolar Zn transporter AtMTP1, providing additional genetic evidence for its major role in Zn(2+) tolerance in seedlings. Mutant ozs2 carries a semi-dominant mutation in the gene encoding pectin methylesterase 3 (AtPME3), an enzyme catalyzing demethylesterification of pectin. The mutation results in impaired proteolytic processing of AtPME3. Ectopic expression of AtPME3 causes strong Zn(2+) hypersensitivity that is tightly correlated with transcript abundance. Together these observations suggest detrimental effects on Golgi-localized processes. The ozs2 but not the ozs1 phenotype can be suppressed by extra Ca(2+) , indicating changes in apoplastic cation-binding capacity. However, we did not detect any changes in bulk metal-binding capacity, overall pectin methylesterification status or cell wall ultrastructure in ozs2, leading us to hypothesize that the ozs2 mutation causes hypersensitivity towards the specific interference of Zn ions with cell wall-controlled growth processes.

Myo-inositol oxygenase is important for the removal of excess myo-inositol from syncytia induced by Heterodera schachtii in Arabidopsis roots.

The enzyme myo-inositol oxygenase is the key enzyme of a pathway leading from myo-inositol to UDP-glucuronic acid. In Arabidopsis, myo-inositol oxygenase is encoded by four genes. All genes are strongly expressed in syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots. Here, we studied the effect of a quadruple myo-inositol oxygenase mutant on nematode development. We performed metabolite profiling of syncytia induced in roots of the myo-inositol oxygenase quadruple mutant. The role of galactinol in syncytia was studied using Arabidopsis lines with elevated galactinol levels and by supplying galactinol to wild-type seedlings. The quadruple myo-inositol oxygenase mutant showed a significant reduction in susceptibility to H. schachtii, and syncytia had elevated myo-inositol and galactinol levels and an elevated expression level of the antimicrobial thionin gene Thi2.1. This reduction in susceptibility could also be achieved by exogenous application of galactinol to wild-type seedlings. The primary function of myo-inositol oxygenase for syncytium development is probably not the production of UDP-glucuronic acid as a precursor for cell wall polysaccharides, but the reduction of myo-inositol levels and thereby a reduction in the galactinol level to avoid the induction of defence-related genes.

Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation.

An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80 ± 5 % and 90 ± 5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (Hypercarb(TM)) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 µL, the limits of detection for the UDP-sugars were in the 70 nmol L(-1) range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 µg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant 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.

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.

Characterization of GDP-mannose dehydrogenase from the brown alga Ectocarpus siliculosus providing the precursor for the alginate polymer.

Alginate is a major cell wall polymer of brown algae. The precursor for the polymer is GDP-mannuronic acid, which is believed to be derived from a four-electron oxidation of GDP-mannose through the enzyme GDP-mannose dehydrogenase (GMD). So far no eukaryotic GMD has been biochemically characterized. We have identified a candidate gene in the Ectocarpus siliculosus genome and expressed it as a recombinant protein in Escherichia coli. The GMD from Ectocarpus differs strongly from related enzymes in bacteria and is as distant to the bacterial proteins as it is to the group of UDP-glucose dehydrogenases. It lacks the C-terminal ∼120 amino acid domain present in bacterial GMDs, which is believed to be involved in catalysis. The GMD from brown algae is highly active at alkaline pH and contains a catalytic Cys residue, sensitive to heavy metals. The product GDP-mannuronic acid was analyzed by HPLC and mass spectroscopy. The K(m) for GDP-mannose was 95 µM, and 86 µM for NAD(+). No substrate other than GDP-mannose was oxidized by the enzyme. In gel filtration experiments the enzyme behaved as a dimer. The Ectocarpus GMD is stimulated by salts even at low molar concentrations as a possible adaptation to marine life. It is rapidly inactivated at temperatures above 30 °C.

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides.

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.