An ABHD17-like hydrolase screening system to identify de-S-acylation enzymes of protein substrates in plant cells.

Protein S-acylation is an important post-translational modification in eukaryotes, regulating the subcellular localization, trafficking, stability, and activity of substrate proteins. The dynamic regulation of this reversible modification is mediated inversely by protein S-acyltransferases and de-S-acylation enzymes, but the de-S-acylation mechanism remains unclear in plant cells. Here, we characterized a group of putative protein de-S-acylation enzymes in Arabidopsis thaliana, including 11 members of Alpha/Beta Hydrolase Domain-containing Protein 17-like acyl protein thioesterases (ABAPTs). A robust system was then established for the screening of de-S-acylation enzymes of protein substrates in plant cells, based on the effects of substrate localization and confirmed via the protein S-acylation levels. Using this system, the ABAPTs, which specifically reduced the S-acylation levels and disrupted the plasma membrane localization of five immunity-related proteins, were identified respectively in Arabidopsis. Further results indicated that the de-S-acylation of RPM1-Interacting Protein 4, which was mediated by ABAPT8, resulted in an increase of cell death in Arabidopsis and Nicotiana benthamiana, supporting the physiological role of the ABAPTs in plants. Collectively, our current work provides a powerful and reliable system to identify the pairs of plant protein substrates and de-S-acylation enzymes for further studies on the dynamic regulation of plant protein S-acylation.

A mitochondria-targeted coenzyme Q peptoid induces superoxide dismutase and alleviates salinity stress in plant cells.

Salinity is a serious challenge to global agriculture and threatens human food security. Plant cells can respond to salt stress either by activation of adaptive responses, or by programmed cell death. The mechanisms deciding the respective response are far from understood, but seem to depend on the degree, to which mitochondria can maintain oxidative homeostasis. Using plant PeptoQ, a Trojan Peptoid, as vehicle, it is possible to transport a coenzyme Q10 (CoQ10) derivative into plant mitochondria. We show that salinity stress in tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow-2) can be mitigated by pretreatment with plant PeptoQ with respect to numerous aspects including proliferation, expansion, redox homeostasis, and programmed cell death. We tested the salinity response for transcripts from nine salt-stress related-genes representing different adaptive responses. While most did not show any significant response, the salt response of the transcription factor NtNAC, probably involved in mitochondrial retrograde signaling, was significantly modulated by the plant PeptoQ. Most strikingly, transcripts for the mitochondrial, Mn-dependent Superoxide Dismutase were rapidly and drastically upregulated in presence of the peptoid, and this response was disappearing in presence of salt. The same pattern, albeit at lower amplitude, was seen for the sodium exporter SOS1. The findings are discussed by a model, where plant PeptoQ modulates retrograde signalling to the nucleus leading to a strong expression of mitochondrial SOD, what renders mitochondria more resilient to perturbations of oxidative balance, such that cells escape salt induced cell death and remain viable.

Alternative oxidase (AOX) over-expression improves cell expansion and elongation in cotton seedling exposed to cool temperatures.

KEY MESSAGE: Evidence that supports a relation between AOX expression and improvement in plant height, internode length, and total leaf area under cool temperature is shown. Cell expansion and elongation appear to be enhanced when AOX expression was increased. Cotton growth is sensitive to cool temperature during germination and early seedling development. Delayed emergence, seedling damage, and increased risk to disease are common. Late seasonal cool weather is a major factor limiting the consistent production of high-quality cotton lint in West Texas. Alternative oxidase functions in the inner membrane of the mitochondria via an alternative respiration pathway and serves as a multifunctional system for amelioration of abiotic and biotic stresses. Cotton seedling emergence and growth exposed to cool temperatures was examined in plants with enhanced AOX expression. Thirteen T1 seed lines showed 3 to 1 segregation for the T-DNA containing the tobacco AOX1 gene. Two over-expressing, single-copy, homozygous AOX lines (94-20T and 66-6T) and Null line (94-3N) were selected for examination. The transcript levels were ≈ 2 to 6 fold higher in the AOX lines compared to those of the Null line and wild-type in stem, leaf, root and boll tissues. The research examined the hypothesis that transgenic cotton with enhanced AOX expression will have enhanced growth traits under suboptimal cool temperatures. Improved plant height, internode length, plant height and internode length from second node, and total leaf area under cool temperatures were observed in AOX over-expression lines. This may be attributed to improved cell expansion and elongation characteristics in the AOX line.

Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.

Phylogenetic analysis of proteins involved in the stringent response in plant cells.

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ppx families indicated that plant gppA/ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol.

Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

MAIN CONCLUSION: Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H+-ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H+-ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H+-ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H+-ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

TaMCA1, a regulator of cell death, is important for the interaction between wheat and Puccinia striiformis.

Metacaspase orthologs are conserved in fungi, protozoa and plants, however, their roles in plant disease resistance are largely unknown. In this study, we identified a Triticum aestivum metacaspase gene, TaMCA1, with three copies located on chromosomes 1A, 1B and 1D. The TaMCA1 protein contained typical structural features of type I metacaspases domains, including an N-terminal pro-domain. Transient expression analyses indicated that TaMCA1 was localized in cytosol and mitochondria. TaMCA1 exhibited no caspase-1 activity in vitro, but was able to inhibit cell death in tobacco and wheat leaves induced by the mouse Bax gene. In addition, the expression level of TaMCA1 was up-regulated following challenge with the Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaMCA1 via virus-induced gene silencing (VIGS) enhanced plant disease resistance to Pst, and the accumulation of hydrogen peroxide (H2O2). Further study showed that TaMCA1 decreased yeast cell resistance similar to the function of yeast metacaspase, and there was no interaction between TaMCA1 and TaLSD1. Based on these combined results, we speculate that TaMCA1, a regulator of cell death, is important during the compatible interaction of wheat and Pst.

Mechanisms and effective control of physiological browning phenomena in plant cell cultures.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells.

ATP synthase.

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

Agrobacterium tumefaciens mediated transient expression of plant cell wall-degrading enzymes in detached sunflower leaves.

For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S-driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA-Based Overexpression Vector) systems. For 6-day leaf incubations, codon-optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two-phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6-fold. Production also increased up to 4.2-fold when the leaf incubation temperature was elevated above the typical temperature, 20C, to 30C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co-expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall-degrading enzymes in sunflower.

[Phytaspases: aspartate-specific proteases involved in plant cell death].

Structure and properties of the recently discovered aspartate-specific cell death-related plant proteases named phytaspases are reviewed and compared to those of animal apoptotic proteases, caspases. Caspases (cysteine-dependent proteases) and phytaspases (serine-dependent proteases) are structurally very different, yet they share cleavage specificity and a role in programmed cell death. We demonstrate here that the distinctions in structural organization of animal and plant death proteases define differences in the strategies to regulate functioning of these proteolytic enzymes in the two kingdoms.

Carbohydrate-active enzymes in pythium and their role in plant cell wall and storage polysaccharide degradation.

Carbohydrate-active enzymes (CAZymes) are involved in the metabolism of glycoconjugates, oligosaccharides, and polysaccharides and, in the case of plant pathogens, in the degradation of the host cell wall and storage compounds. We performed an in silico analysis of CAZymes predicted from the genomes of seven Pythium species (Py. aphanidermatum, Py. arrhenomanes, Py. irregulare, Py. iwayamai, Py. ultimum var. ultimum, Py. ultimum var. sporangiiferum and Py. vexans) using the "CAZymes Analysis Toolkit" and "Database for Automated Carbohydrate-active Enzyme Annotation" and compared them to previously published oomycete genomes. Growth of Pythium spp. was assessed in a minimal medium containing selected carbon sources that are usually present in plants. The in silico analyses, coupled with our in vitro growth assays, suggest that most of the predicted CAZymes are involved in the metabolism of the oomycete cell wall with starch and sucrose serving as the main carbohydrate sources for growth of these plant pathogens. The genomes of Pythium spp. also encode pectinases and cellulases that facilitate degradation of the plant cell wall and are important in hyphal penetration; however, the species examined in this study lack the requisite genes for the complete saccharification of these carbohydrates for use as a carbon source. Genes encoding for xylan, xyloglucan, (galacto)(gluco)mannan and cutin degradation were absent or infrequent in Pythium spp.. Comparative analyses of predicted CAZymes in oomycetes indicated distinct evolutionary histories. Furthermore, CAZyme gene families among Pythium spp. were not uniformly distributed in the genomes, suggesting independent gene loss events, reflective of the polyphyletic relationships among some of the species.

Measuring PLD activity in vivo.

Phospholipase D (PLD) hydrolyzes structural phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) into phosphatidic acid (PA) and free choline/ethanolamine. In plants, this activity can be stimulated by a wide variety of biotic and abiotic stresses (Li et al., Biochim Biophys Acta 1791:927-935, 2009; Testerink and Munnik, J Exp Bot 62(7):2349-2361, 2011). This chapter describes a protocol for the measurement of PLD activity in vivo. The protocol takes advantage of a unique property of PLD, i.e., its ability to substitute a primary alcohol, such as 1-butanol, for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of phosphatidylbutanol (PBut), which is a specific and unique reporter for PLD activity. The assay is highly sensitive for detecting PLD activity in vivo, following stimulation of intact plant cells, seedlings, and tissues, being a valuable method for studying the regulation of plant PLD activity in vivo.

Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta.

N-Glycan processing is one of the most important cellular protein modifications in plants and as such is essential for plant development and defense mechanisms. The accuracy of Golgi-located processing steps is governed by the strict intra-Golgi localization of sequentially acting glycosidases and glycosyltransferases. Their differential distribution goes hand in hand with the compartmentalization of the Golgi stack into cis-, medial-, and trans-cisternae, which separate early from late processing steps. The mechanisms that direct differential enzyme concentration are still unknown, but the formation of multienzyme complexes is considered a feasible Golgi protein localization strategy. In this study, we used two-photon excitation-Förster resonance energy transfer-fluorescence lifetime imaging microscopy to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Following the coexpression of fluorescent protein-tagged amino-terminal Golgi-targeting sequences (cytoplasmic-transmembrane-stem [CTS] region) of enzyme pairs in leaves of tobacco (Nicotiana spp.), we observed that all tested cis- and medial-Golgi enzymes, namely Arabidopsis (Arabidopsis thaliana) Golgi α-mannosidase I, Nicotiana tabacum ß1,2-N-acetylglucosaminyltransferase I, Arabidopsis Golgi α-mannosidase II (GMII), and Arabidopsis ß1,2-xylosyltransferase, form homodimers and heterodimers, whereas among the late-acting enzymes Arabidopsis ß1,3-galactosyltransferase1 (GALT1), Arabidopsis α1,4-fucosyltransferase, and Rattus norvegicus α2,6-sialyltransferase (a nonplant Golgi marker), only GALT1 and medial-Golgi GMII were found to form a heterodimer. Furthermore, the efficiency of energy transfer indicating the formation of interactions decreased considerably in a cis-to-trans fashion. The comparative fluorescence lifetime imaging of several full-length cis- and medial-Golgi enzymes and their respective catalytic domain-deleted CTS clones further suggested that the formation of protein-protein interactions can occur through their amino-terminal CTS region.

Simulation of variation potential in higher plant cells.

Variation potential (VP), a propagating electrical signal unique to plants, induces a number of changes in many physiological processes. However, the mechanisms of its generation and propagation are still under discussion and require experimental and theoretical analysis, including VP simulations. The mathematical model for VP formation in plants has been worked out and is based on our previous description of electrophysiological processes in higher plant cells, including plasma membrane ion transport systems (K(+), Cl(-) and Ca(2+) channels, H(+) and Ca(2+)-ATPase, 2H(+)/Cl(-) symporter and H(+)/K(+) antiporter) and their regulation, ion concentration changes in cells and extracellular spaces and buffers in cytoplasm and apoplast. In addition, the VP model takes into account wound substance diffusion, which is described by a one-dimensional diffusion equation, and ligand-gated Ca(2+) channels, which are activated by this substance. The VP model simulates the experimental dependence of amplitude, velocity and shape of VP on the distance from the wounding site and describes the influence of metabolic inhibitors, divalent cation chelators and anion channel blockers on the generation of this electrical reaction, as shown in experiments. Thus, our model favorably simulates VP in plants and theoretically supports the role of wound substance diffusion and Ca(2+) influx in VP development.

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death.

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H2O2. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.