Auxin co-receptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root solely by modulation of nuclear auxin pathway.

The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

DIOXYGENASE FOR AUXIN OXIDATION 1 catalyzes the oxidation of IAA amino acid conjugates.

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

Distinct metabolism of N-glucosides of isopentenyladenine and trans-zeatin determines cytokinin metabolic spectrum in Arabidopsis.

The diversity of cytokinin (CK) metabolites suggests their interconversions are the predominant regulatory mechanism of CK action. Nevertheless, little is known about their directionality and kinetics in planta. CK metabolite levels were measured in 2-wk-old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling. Apart from elevated levels of trans-zeatin (tZ) metabolites upon application of N6 -(Δ2 -isopentenyl)adenine (iP), we observed no conversions between the individual CK-types - iP, tZ, dihydrozeatin (DHZ) and cis-zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ-type CKs was observed after application of tZ, suggesting low baseline activity of zeatin reductase. Among N-glucosides, those of iP were not converted back to iP while tZ N-glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum. We present the first large-scale study of short-term CK metabolism kinetics and show that tZ N7- and N9-glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N-glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates.

PIN-driven auxin transport emerged early in streptophyte evolution.

PIN-FORMED (PIN) transporters mediate directional, intercellular movement of the phytohormone auxin in land plants. To elucidate the evolutionary origins of this developmentally crucial mechanism, we analysed the single PIN homologue of a simple green alga Klebsormidium flaccidum. KfPIN functions as a plasma membrane-localized auxin exporter in land plants and heterologous models. While its role in algae remains unclear, PIN-driven auxin export is probably an ancient and conserved trait within streptophytes.

Transcription of specific auxin efflux and influx carriers drives auxin homeostasis in tobacco cells.

Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.

Plant Cell Lines in Cell Morphogenesis Research: From Phenotyping to -Omics.

Here we provide an overview of procedures for long-term cultivation, phenotyping, genotyping, and genetic transformation of cell cultures of tobacco cell lines BY-2 and VBI-0, and of A. thaliana, ecotype Landsberg erecta (LE) cell line. Notably, we present an improved protocol for BY-2 transformation and cloning and extend the available plant cell lines methodology toward high-throughput technologies like fluorescent-based cell sorting and transcriptomics.

Homologous recombination changes the context of Cytochrome b transcription in the mitochondrial genome of Silene vulgaris KRA.

BACKGROUND: Silene vulgaris (bladder campion) is a gynodioecious species existing as two genders - male-sterile females and hermaphrodites. Cytoplasmic male sterility (CMS) is generally encoded by mitochondrial genes, which interact with nuclear fertility restorer genes. Mitochondrial genomes of this species vary in DNA sequence, gene order and gene content. Multiple CMS genes are expected to exist in S. vulgaris, but little is known about their molecular identity. RESULTS: We assembled the complete mitochondrial genome from the haplotype KRA of S. vulgaris. It consists of five chromosomes, two of which recombine with each other. Two small non-recombining chromosomes exist in linear, supercoiled and relaxed circle forms. We compared the mitochondrial transcriptomes from females and hermaphrodites and confirmed the differentially expressed chimeric gene bobt as the strongest CMS candidate gene in S. vulgaris KRA. The chimeric gene bobt is co-transcribed with the Cytochrome b (cob) gene in some genomic configurations. The co-transcription of a CMS factor with an essential gene may constrain transcription inhibition as a mechanism for fertility restoration because of the need to maintain appropriate production of the necessary protein. Homologous recombination places the gene cob outside the control of bobt, which allows for the suppression of the CMS gene by the fertility restorer genes. We found the loss of three editing sites in the KRA mitochondrial genome and identified four sites with highly distinct editing rates between KRA and another S. vulgaris haplotypes (KOV). Three of these highly differentially edited sites were located in the transport membrane protein B (mttB) gene. They resulted in differences in MttB protein sequences between haplotypes. CONCLUSIONS: Frequent homologous recombination events that are widespread in plant mitochondrial genomes may change chromosomal configurations and also the control of gene transcription including CMS gene expression. Posttranscriptional processes, e.g. RNA editing shall be evaluated in evolutionary and co-evolutionary studies of mitochondrial genes, because they may change protein composition despite the sequence identity of the respective genes. The investigation of natural populations of wild species such as S. vulgaris are necessary to reveal important aspects of CMS missed in domesticated crops, the traditional focus of the CMS studies.

Dynamics of the Pollen Sequestrome Defined by Subcellular Coupled Omics.

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

Background and Aim: The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods: In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results: ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions: The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.

Characterization of pollen-expressed bZIP protein interactions and the role of ATbZIP18 in the male gametophyte.

KEY MESSAGE : bZIP TF network in pollen. Transcriptional control of gene expression represents an important mechanism guiding organisms through developmental processes and providing plasticity towards environmental stimuli. Because of their sessile nature, plants require effective gene regulation for rapid response to variation in environmental and developmental conditions. Transcription factors (TFs) provide such control ensuring correct gene expression in spatial and temporal manner. Our work reports the interaction network of six bZIP TFs expressed in Arabidopsis thaliana pollen and highlights the potential functional role for AtbZIP18 in pollen. AtbZIP18 was shown to interact with three other pollen-expressed bZIP TFs-AtbZIP34, AtbZIP52, and AtbZIP61 in yeast two-hybrid assays. AtbZIP18 transcripts are highly expressed in pollen, and at the subcellular level, an AtbZIP18-GFP fusion protein was located in the nucleus and cytoplasm/ER. To address the role of AtbZIP18 in the male gametophyte, we performed phenotypic analysis of a T-DNA knockout allele, which showed slightly reduced transmission through the male gametophyte. Some of the phenotype defects in atbzip18 pollen, although observed at low penetrance, were similar to those seen at higher frequency in the T-DNA knockout of the interacting partner, AtbZIP34. To gain deeper insight into the regulatory role of AtbZIP18, we analysed atbzip18/- pollen microarray data. Our results point towards a potential repressive role for AtbZIP18 and its functional redundancy with AtbZIP34 in pollen.

Thorium impact on tobacco root transcriptome.

Thorium is natural actinide metal with potential use in nuclear energetics. Contamination by thorium, originated from mining activities or spills, represents environmental risk due to its radioactivity and chemical toxicity. A promising approach for cleaning of contaminated areas is phytoremediation, which need to be based, however, on detail understanding of the thorium effects on plants. In this study we investigated transcriptomic response of tobacco roots exposed to 200µM thorium for one week. Thorium application resulted in up-regulation of 152 and down-regulation of 100 genes (p-value <0.01, fold change ≥2). The stimulated genes were involved in components of jasmonic acid and salicylic acid signaling pathways and various abiotic (e.g. oxidative stress) and biotic stress (e.g. pathogens, wounding) responsive genes. Further, up-regulation of phosphate starvation genes and down-regulation of genes involved in phytic acid biosynthesis indicated that thorium disturbed phosphate uptake or signaling. Also expression of iron responsive genes was influenced. Negative regulation of several aquaporins indicated disturbance of water homeostasis. Genes potentially involved in thorium transport could be zinc-induced facilitator ZIF2, plant cadmium resistance PCR2, and ABC transporter ABCG40. This study provides the first insight at the processes in plants exposed to thorium.

NtGNL1a ARF-GEF acts in endocytosis in tobacco cells.

BACKGROUND: Processes of anterograde and retrograde membrane trafficking play an important role in cellular homeostasis and dynamic rearrangements of the plasma membrane (PM) in all eukaryotes. These processes depend on the activity of adenosine ribosylation factors (ARFs), a family of GTP-binding proteins and their guanine exchange factors (GEFs). However, knowledge on the function and specificity of individual ARF-GEFs for individual steps of membrane trafficking pathways is still limited in plants. RESULTS: In this work, treatments with various trafficking inhibitors showed that the endocytosis of FM 4-64 is largely dynamin-dependent and relies on proteins containing endocytic tyrosine-based internalization motif and intact cytoskeleton. Interestingly, brefeldin A (BFA), reported previously as an inhibitor of anterograde membrane trafficking in plants, appeared to be the most potent inhibitor of endocytosis in tobacco. In concert with this finding, we demonstrate that the point mutation in the Sec7 domain of the GNOM-LIKE protein1a (NtGNL1a) confers intracellular trafficking pathway-specific BFA resistance. The internalization of FM 4-64 and trafficking of PIN-FORMED1 (PIN1) auxin efflux carrier in BY-2 tobacco cells were studied to reveal the function of the ARF-GEF NtGNL1a in these. CONCLUSIONS: Altogether, our observations uncovered the role of NtGNL1a in endocytosis, including endocytosis of PM proteins (as PIN1 auxin efflux carrier). Moreover these data emphasize the need of careful evaluation of mode of action of non-native inhibitors in various species. In addition, they demonstrate the potential of tobacco BY-2 cells for selective mapping of ARF-GEF-regulated endomembrane trafficking pathways.

Tobacco susceptibility to Potato virus Y(NTN) infection is affected by grafting and endogenous cytokinin content.

Faster or stronger response to pathogen occurs if plants undergo prior priming. Cytokinins seem to be also involved in plant priming and in response to pathogens. Susceptibility to Potato virus Y(NTN) (PVY(NTN)) was studied in transgenic cytokinin overproducing (Pssu-ipt) tobacco and compared with nontransgenic plants. Since cytokinin overproduction inhibits development of plant roots and grafting overcomes this limitation, both types were grown as rooted and/or grafted plants to check also the effect of grafting. Control rooted tobacco (C), the most susceptible to PVY(NTN), showed always symptoms during the infection together with the rising virus content and a systemic response, such as accumulation of H2O2, salicylic acid (SA) and other phenolic acids, and stress-induced enzyme activities. In transgenic and grafted plants, the response to PVY(NTN) was dependent on protective mechanisms activated prior to the inoculation. In Pssu-ipt tobacco, cytokinin active forms and SA contents exceeded manifold their content in C. Grafting promoted the accumulation of phenolics, but SA, and stimulated peroxidase activities. Thus, the pre-infection barrier established in both transgenic and grafted plants helped to suppress partly the virus multiplication and resulted in milder symptom development. However, only the synergic effect of both grafting and the high cytokinins led to PVY(NTN) tolerance in transgenic grafts. Possible mechanisms were discussed.

Transcription of atp1 is influenced by both genomic configuration and nuclear background in the highly rearranged mitochondrial genomes of Silene vulgaris.

An extraordinary variation in mitochondrial DNA sequence exists in angiosperm Silene vulgaris. The atp1 gene is flanked by very variable regions, as deduced from four completely sequenced mitochondrial genomes of this species. This diversity contributed to a highly variable transcript profile of this gene observed across S. vulgaris populations. We examined the atp1 transcript in the KOV mitochondrial genome and found three 5' ends, created most likely by the combination of transcription initiation and RNA processing. Most atp1 transcripts terminated about 70 bp upstream of the translation stop codon, which was present in only 10 % of them. Controlled crosses between a KOV mother and a geographically distant pollen donor (Krasnoyarsk, Russia) showed that nuclear background also affected atp1 transcription. The distant pollen donor introduced the factor(s) preventing the formation of a long 2,100 nt-transcript, because this long atp1 transcript reappeared in the progeny from self-crosses. The highly rearranged mitochondrial genomes with a variation in gene flanking regions make S. vulgaris an excellent model for the study of mitochondrial gene expression in plants.

Intraspecific variation in mitochondrial genome sequence, structure, and gene content in Silene vulgaris, an angiosperm with pervasive cytoplasmic male sterility.

In angiosperms, mitochondrial-encoded genes can cause cytoplasmic male sterility (CMS), resulting in the coexistence of female and hermaphroditic individuals (gynodioecy). We compared four complete mitochondrial genomes from the gynodioecious species Silene vulgaris and found unprecedented amounts of intraspecific diversity for plant mitochondrial DNA (mtDNA). Remarkably, only about half of overall sequence content is shared between any pair of genomes. The four mtDNAs range in size from 361 to 429 kb and differ in gene complement, with rpl5 and rps13 being intact in some genomes but absent or pseudogenized in others. The genomes exhibit essentially no conservation of synteny and are highly repetitive, with evidence of reciprocal recombination occurring even across short repeats (< 250 bp). Some mitochondrial genes exhibit atypically high degrees of nucleotide polymorphism, while others are invariant. The genomes also contain a variable number of small autonomously mapping chromosomes, which have only recently been identified in angiosperm mtDNA. Southern blot analysis of one of these chromosomes indicated a complex in vivo structure consisting of both monomeric circles and multimeric forms. We conclude that S. vulgaris harbors an unusually large degree of variation in mtDNA sequence and structure and discuss the extent to which this variation might be related to CMS.

Mosaic origins of a complex chimeric mitochondrial gene in Silene vulgaris.

Chimeric genes are significant sources of evolutionary innovation that are normally created when portions of two or more protein coding regions fuse to form a new open reading frame. In plant mitochondria astonishingly high numbers of different novel chimeric genes have been reported, where they are generated through processes of rearrangement and recombination. Nonetheless, because most studies do not find or report nucleotide variation within the same chimeric gene, evolution after the origination of these chimeric genes remains unstudied. Here we identify two alleles of a complex chimera in Silene vulgaris that are divergent in nucleotide sequence, genomic position relative to other mitochondrial genes, and expression patterns. Structural patterns suggest a history partially influenced by gene conversion between the chimeric gene and functional copies of subunit 1 of the mitochondrial ATP synthase gene (atp1). We identified small repeat structures within the chimeras that are likely recombination sites allowing generation of the chimera. These results establish the potential for chimeric gene divergence in different plant mitochondrial lineages within the same species. This result contrasts with the absence of diversity within mitochondrial chimeras found in crop species.

Characterization of phosphoenolpyruvate carboxylase from mature maize seeds: properties of phosphorylated and dephosphorylated forms.

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 µmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to L-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition.

Transcription profiles of mitochondrial genes correlate with mitochondrial DNA haplotypes in a natural population of Silene vulgaris.

BACKGROUND: Although rapid changes in copy number and gene order are common within plant mitochondrial genomes, associated patterns of gene transcription are underinvestigated. Previous studies have shown that the gynodioecious plant species Silene vulgaris exhibits high mitochondrial diversity and occasional paternal inheritance of mitochondrial markers. Here we address whether variation in DNA molecular markers is correlated with variation in transcription of mitochondrial genes in S. vulgaris collected from natural populations. RESULTS: We analyzed RFLP variation in two mitochondrial genes, cox1 and atp1, in offspring of ten plants from a natural population of S. vulgaris in Central Europe. We also investigated transcription profiles of the atp1 and cox1 genes. Most DNA haplotypes and transcription profiles were maternally inherited; for these, transcription profiles were associated with specific mitochondrial DNA haplotypes. One individual exhibited a pattern consistent with paternal inheritance of mitochondrial DNA; this individual exhibited a transcription profile suggestive of paternal but inconsistent with maternal inheritance. We found no associations between gender and transcript profiles. CONCLUSIONS: Specific transcription profiles of mitochondrial genes were associated with specific mitochondrial DNA haplotypes in a natural population of a gynodioecious species S. vulgaris.Our findings suggest the potential for a causal association between rearrangements in the plant mt genome and transcription product variation.

Regulation of phosphoenolpyruvate carboxylase in PVY(NTN)-infected tobacco plants.

The effect of viral infection on the regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in Nicotiana tabacum L. leaves was studied. PEPC activity was 3 times higher in infected plant leaves compared to healthy plants. Activity of plant PEPC can be regulated, e.g., by de novo synthesis or reversible phosphorylation. The reason for the increase of PEPC activity as a consequence of PVY(NTN) infection was studied. The amount of PEPC determined by Western blot analysis or by relative estimation of PEPC mRNA by real-time PCR did not differ in control and PVY(NTN)-infected plants. Changes in posttranslational modification of PEPC by phosphorylation were evaluated by comparing activity of the native and the dephosphorylated enzyme. The infected plants were characterized by a higher decrease of the enzyme activity after its dephosphorylation, which indicated a higher phosphorylation level. Immunochemical detection of phosphoproteins by Western blot analysis showed a more intensive band corresponding to PEPC from the infected material. This strengthens the hypothesis of an infection-related phosphorylation, which could be part of the plant's response to pathogen attack. The physiological implications of the increase in PEPC activity during PVY(NTN) infection are discussed.