Arabidopsis Thaliana Cell Suspension Cultures Research Articles

Modulation of mitochondrial activity by S-nitrosoglutathione reductase in Arabidopsis thaliana transgenic cell lines

The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOROE) or antisense (GSNORAS) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOROE had the lowest SNO and NO levels and GSNORAS the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOROE cells grown under nutritional stress. Moreover, GSNOROE could not increase alternative oxidase (AOX) activity under nutritional stress. GSNORAS showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.

Early transcriptional defense responses in Arabidopsis cell suspension culture under high-light conditions.

The early transcriptional defense responses and reactive oxygen species (ROS) production in Arabidopsis (Arabidopsis thaliana) cell suspension culture (ACSC), containing functional chloroplasts, were examined at high light (HL). The transcriptional analysis revealed that most of the ROS markers identified among the 449 transcripts with significant differential expression were transcripts specifically up-regulated by singlet oxygen ((1)O(2)). On the contrary, minimal correlation was established with transcripts specifically up-regulated by superoxide radical or hydrogen peroxide. The transcriptional analysis was supported by fluorescence microscopy experiments. The incubation of ACSC with the (1)O(2) sensor green reagent and 2',7'-dichlorofluorescein diacetate showed that the 30-min-HL-treated cultures emitted fluorescence that corresponded with the production of (1)O(2) but not of hydrogen peroxide. Furthermore, the in vivo photodamage of the D1 protein of photosystem II indicated that the photogeneration of (1)O(2) took place within the photosystem II reaction center. Functional enrichment analyses identified transcripts that are key components of the ROS signaling transduction pathway in plants as well as others encoding transcription factors that regulate both ROS scavenging and water deficit stress. A meta-analysis examining the transcriptional profiles of mutants and hormone treatments in Arabidopsis showed a high correlation between ACSC at HL and the fluorescent mutant family of Arabidopsis, a producer of (1)O(2) in plastids. Intriguingly, a high correlation was also observed with ABA deficient1 and more axillary growth4, two mutants with defects in the biosynthesis pathways of two key (apo)carotenoid-derived plant hormones (i.e. abscisic acid and strigolactones, respectively). ACSC has proven to be a valuable system for studying early transcriptional responses to HL stress.

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state (13)C-labeling experiments using [1-(13)C]-, [2-(13)C]-, and [U-(13)C(6)]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.

Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures

Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 °C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

Nitric oxide is involved in cadmium-induced programmed cell death in Arabidopsis suspension cultures.

Exposure to cadmium (Cd(2+)) can result in cell death, but the molecular mechanisms of Cd(2+) cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 microm CdCl(2) and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl(2) treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N(G)-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd(2+)-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd(2+) cytotoxicity in plants.

Fine-tuning of early events in the jasmonate response

Jasmonates (JAs) control many aspects of plant defense and development, for instance by inhibiting growth and eliciting secondary metabolism. The mechanisms by which JAs regulate these processes are currently under intensive investigation. Examination of transcriptional changes upon methyl jasmonate (MeJA) perception in a fast-growing Arabidopsis thaliana cell suspension culture revealed a quick and direct dual effect of JAs on the plant\\'s cellular processes. Simultaneously, JA-elicited Arabidopsis cells activated phenylpropanoid metabolism and repressed cell cycle progression. Early JA response genes were predominantly implicated in transcriptional regulation and JA biosynthesis. In two parallel screens, we identified both early responsive transcriptional activators (ORA47 and MYC2) and transcriptional repressors (STZ/ZAT10 and AZF2) that putatively regulate the expression of the JA biosynthesis gene LOX3. In this addendum, we provide additional data demonstrating that MYC2, STZ/ZAT10, and AZF2 might also control the expression of JAZ1/TIFY10a that encodes a key repressor in the JA signaling cascade.Addendum to: Pauwels L, Morreel K, De Witte E, Lammertyn F, Van Montagu M, Boerjan W, Inzé D, Goossens A. Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc Natl Acad Sci USA 2008; 105:1380-5.

Complex I Dysfunction Redirects Cellular and Mitochondrial Metabolism in Arabidopsis

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants. However, the plant enzyme has structural and functional features that set it apart from its counterparts in other organisms, raising questions about the physiological significance of this complex in plants. We have developed an experimental model in which rotenone, a classic complex I inhibitor, has been applied to Arabidopsis (Arabidopsis thaliana) cell suspension cultures in order to dissect early metabolic adjustments involved in cell acclimation to mitochondrial dysfunction. Rotenone induced a transitory decrease in cellular respiration (0-4 h after treatment). Cell respiration then progressively recovered and reached a steady state at 10 to 12 h after treatment. Complex I inhibition by rotenone did not induce obvious oxidative stress or cell death but affected longer term cell growth. Integrated analyses of gene expression, the mitochondrial proteome, and changes in primary metabolism indicated that rotenone treatment caused changes in mitochondrial function via alterations in specific components. A physical disengagement of glycolytic activities associated with the mitochondrial outer membrane was observed, and the tricarboxylic acid cycle was altered. Amino acid and organic acid pools were also modified by rotenone treatment, with a marked early decrease of 2-oxoglutarate, aspartate, and glutamine pools. These data demonstrate that, in Arabidopsis cells, complex I inhibition by rotenone induces significant remodeling of metabolic pathways involving the mitochondria and other compartments and point to early metabolic changes in response to mitochondrial dysfunction.

Efficient and High-Throughput Vector Construction and Agrobacterium-Mediated Transformation of Arabidopsis thaliana Suspension-Cultured Cells for Functional Genomics

We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells

In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ring-shaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death.

Production of reactive oxygen species in Arabidopsis thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum: implications for basal resistance

The present understanding of ROS generation in the defence response of Arabidopsis thaliana is reviewed. Evidence suggests that the apoplastic oxidative burst generated during basal resistance is peroxidase-dependent. The ROS generated during this basal resistance may serve to activate NADPH oxidase during the R-gene-mediated hypersensitive response. The processes involved in the production of reactive oxygen species in A. thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum are investigated in the present work. This system appears analogous to the production of ROS during the basal resistance response in French bean, which is peroxidase-dependent. A panel of modulators effective in other pathogen elicitor and plant cell systems has been used to investigate the Arabidopsis signalling pathways and the plant cell responses involved. Thus as in other systems, an early calcium influx into the cytosolic compartment, a rapid efflux of K(+) and Cl(-), and extracellular alkalinization of elicited cell cultures has been found. However the alkalinization is not sufficient to stimulate the apoplastic oxidative burst by itself, unlike in French bean, although vectorial ion fluxes are needed. A secretory component which is sensitive to monensin and N-ethylmaleimide and insensitive to brefeldin A may also be necessary for the release and provision of substrates for peroxidase-dependent generation of H(2)O(2).

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.

The D-Type Cyclin CYCD3;1 Is Limiting for the G1-to-S-Phase Transition inArabidopsis

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.

Starvation‐induced expression of autophagy‐related genes in Arabidopsis

Autophagy is a catabolic process for degradation of cytoplasmic components in the vacuolar apparatus. A genome-wide survey recently showed evolutionary conservation among autophagy genes in yeast, mammals and plants. To elucidate the molecular and subcellular machinery responsible for the sequestration and subsequent digestion of intracellular material in plants, we utilized a combination of morphological and molecular methods (confocal laser-scanning microscopy, transmission electron microscopy and real-time PCR respectively). Autophagy in Arabidopsis thaliana suspension-cultured cells was induced by carbon starvation, which triggered an immediate arrest of cell growth together with a rapid degradation of cellular proteins. We followed the onset of these responses and, in this report, provide a clear functional classification for the highly polymorphic autophagosomes by which the cell sequesters and degrades a portion of its own cytoplasm. Quantification of autophagy-related structures shows that cells respond to the stress signal by a rapid and massive, but transient burst of autophagic activity, which adapts to the stress signal. We also monitored the real-time expressions of AtATG3, AtATG4a, AtATG4b, AtATG7 and AtATG8a-AtATG8i genes, which are orthologues of yeast genes involved in the Atg8 ubiquitination-like conjugation pathway and are linked to autophagosome formation. We show that these autophagy-related genes are transiently up-regulated in a co-ordinated manner at the onset of starvation. Sucrose starvation induces autophagy and up-regulates orthologues of the yeast Atg8 conjugation pathway genes in Arabidopsis cultured cells. The AtATG3, AtATG4a, AtATG4b, AtATG7 and AtATG8a-AtATG8i genes are expressed in successive waves that parallel the biochemical and cytological remodelling that takes place. These genes thus serve as early markers for autophagy in plants.

Identification ofArabidopsis salt and osmotic stress responsive proteins using two-dimensional difference gel electrophoresis and mass spectrometry

Arabidopsis thaliana cell suspension cultures have been used to investigate the effects of salinity and hyperosmotic stress on plant cellular proteins. We show that 200 mM NaCl and 400 mM sorbitol treatments induce extracellular medium acidification in Arabidopsis cell cultures, a typical response of plant cells to salt and hyperosmotic stress. Using (35)S-labelled amino acids, we demonstrated that NaCl causes a transient suppression of de novo protein synthesis, from which the cells recover within 4 h. Changes in the abundance of cellular proteins 6 h post NaCl and sorbitol treatments were analysed by 2-DE. Of a total of 2,949 protein spots detected on the gels, 266 showed significant changes in abundance across five independent experiments. Using MALDI-TOF MS, we identified 75 salt and sorbitol responsive spots. These fall into 10 functional categories that include H(+) transporting ATPases, signal transduction related proteins, transcription/translation related proteins, detoxifying enzymes, amino acid and purine biosynthesis related proteins, proteolytic enzymes, heat-shock proteins, carbohydrate metabolism-associated proteins and proteins with no known biological functions.

Apoplastic Calmodulin Receptor-like Binding Proteins in Suspension-cultured Cells of Arabidopsis thaliana

Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-3H]thymidine into nuclear DNA. Radioligand binding analysis with 35S-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the Kd approximately 9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with Kd values of approximately 47.5 and 830 nM. Chemical crosslinking of 35S-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.

Thaxtomin A induces programmed cell death in Arabidopsis thaliana suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.

Proteomic Identification of S-Nitrosylated Proteins in Arabidopsis

Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.

Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress).

NADH kinase (NADHK; ATP:NADH 2'-phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg2+ and NAD+, with respective K(m) values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD+, the specificity constant (V(max)/K(m)) for NADH was 100-fold greater than for NAD+. The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PP(i) or poly-P(i) could not substitute as phospho donors. PP(i) acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD+. This study suggests that, in Arabidopsis, NADP+/NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool.

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors.

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.

Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization.

A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH 5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.