Alanine aminotransferase (AlaAT) has been studied in a variety of organisms due to the involvement of this enzyme in mammalian processes such as non-alcoholic hepatocellular damage, and in plant processes such as C4 photosynthesis, post-hypoxic stress response and nitrogen use efficiency. To date, very few studies have made direct comparisons of AlaAT enzymes and fewer still have made direct comparisons of this enzyme across a broad spectrum of organisms. In this study we present a direct kinetic comparison of glutamate:pyruvate aminotransferase (GPAT) activity for seven AlaATs and two glutamate:glyoxylate aminotransferases (GGAT), measuring the KM values for the enzymes analyzed. We also demonstrate that recombinant expression of AlaAT enzymes in Eschericia coli results in differences in bacterial growth inhibition, supporting previous reports of AlaAT possessing bactericidal properties, attributed to lipopolysaccharide endotoxin recognition and binding. A probable lipopolysaccharide binding region within the AlaAT enzymes, homologous to a region of a lipopolysaccharide binding protein (LBP) in humans, was also identified in this study. The AlaAT enzyme differences identified here indicate that AlaAT homologues have differentiated significantly and the roles these homologues play in vivo may also have diverged significantly. Specifically, the differing kinetics of AlaAT enzymes and how this may alter the nitrogen use efficiency in plants is discussed.
The plant Golgi apparatus and trans-Golgi network are major endomembrane trafficking hubs within the plant cell and are involved in a diverse and vital series of functions to maintain plant growth and development. Recently, a series of disparate technical approaches have been used to isolate and characterize components of these complex organelles by mass spectrometry in the model plant Arabidopsis thaliana. Collectively, these studies have increased the number of Golgi and vesicular localized proteins identified by mass spectrometry to nearly 500 proteins. We have sought to provide a brief overview of these technical approaches and bring the datasets together to examine how they can reveal insights into the secretory pathway.
Golgi; trans-Golgi network; proteomics; LOPIT; free-flow electrophoresis; Arabidopsis; SYP61
We outline a high throughput procedure that improves outlier detection in cell wall screens using FT-NIR spectroscopy of plant leaves. The improvement relies on generating a calibration set from a subset of a mutant population by taking advantage of the Mahalanobis distance outlier scheme to construct a monosaccharide range predictive model using PLS regression. This model was then used to identify specific monosaccharide outliers from the mutant population.
near infrared spectroscopy; cell wall; hemicellulose; multivariate analysis; mutant screen; pls modeling
The PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows ‘on-the-fly’ prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences.
The fungus Stagonospora nodorum is a causal agent of leaf and glume blotch disease of wheat. It has been previously shown that inactivation of heterotrimeric G protein signaling in Stagonospora nodorum caused development defects and reduced pathogenicity [P. S. Solomon et al., Mol. Plant-Microbe Interact. 17:456-466, 2004]. In this study, we sought to identify targets of the signaling pathway that may have contributed to phenotypic defects of the signaling mutants. A comparative analysis of Stagonospora nodorum wild-type and Gα-defective mutant (gna1) intracellular proteomes was performed via two-dimensional polyacrylamide gel electrophoresis. Several proteins showed significantly altered abundances when comparing the two strains. One such protein, the short-chain dehydrogenase Sch1, was 18-fold less abundant in the gna1 strain, implying that it is positively regulated by Gα signaling. Gene expression and transcriptional enhanced green fluorescent protein fusion analyses of Sch1 indicates strong expression during asexual development. Mutant strains of Stagonospora nodorum lacking Sch1 demonstrated poor growth on minimal media and exhibited a significant reduction in asexual sporulation on all growth media examined. Detailed histological experiments on sch1 pycnidia revealed that the gene is required for the differentiation of the subparietal layers of asexual pycnidia resulting in a significant reduction in both pycnidiospore size and numbers.
The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27 782 predicted phosphoserine sites distributed across 17 035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link http://phosphat.mpimp-golm.mpg.de
Knowledge of protein localisation contributes towards our understanding of protein function and of biological inter-relationships. A variety of experimental methods are currently being used to produce localisation data that need to be made accessible in an integrated manner. Chimeric fluorescent fusion proteins have been used to define subcellular localisations with at least 1100 related experiments completed in Arabidopsis. More recently, many studies have employed mass spectrometry to undertake proteomic surveys of subcellular components in Arabidopsis yielding localisation information for ∼2600 proteins. Further protein localisation information may be obtained from other literature references to analysis of locations (AmiGO: ∼900 proteins), location information from Swiss-Prot annotations (∼2000 proteins); and location inferred from gene descriptions (∼2700 proteins). Additionally, an increasing volume of available software provides location prediction information for proteins based on amino acid sequence. We have undertaken to bring these various data sources together to build SUBA, a SUBcellular location database for Arabidopsis proteins. The localisation data in SUBA encompasses 10 distinct subcellular locations, >6743 non-redundant proteins and represents the proteins encoded in the transcripts responsible for 51% of Arabidopsis expressed sequence tags. The SUBA database provides a powerful means by which to assess protein subcellular localisation in Arabidopsis ().
The Arabidopsis Mitochondrial Protein Database is an Internet-accessible relational database containing information on the predicted and experimentally confirmed protein complement of mitochondria from the model plant Arabidopsis thaliana (http://www.ampdb.bcs.uwa.edu.au/). The database was formed using the total non-redundant nuclear and organelle encoded sets of protein sequences and allows relational searching of published proteomic analyses of Arabidopsis mitochondrial samples, a set of predictions from six independent subcellular-targeting prediction programs, and orthology predictions based on pairwise comparison of the Arabidopsis protein set with known yeast and human mitochondrial proteins and with the proteome of Rickettsia. A variety of precomputed physical–biochemical parameters are also searchable as well as a more detailed breakdown of mass spectral data produced from our proteomic analysis of Arabidopsis mitochondria. It contains hyperlinks to other Arabidopsis genomic resources (MIPS, TIGR and TAIR), which provide rapid access to changing gene models as well as hyperlinks to T-DNA insertion resources, Massively Parallel Signature Sequencing (MPSS) and Genome Tiling Array data and a variety of other Arabidopsis online resources. It also incorporates basic analysis tools built into the query structure such as a BLAST facility and tools for protein sequence alignments for convenient analysis of queried results.
Experimental analyses of the proteins found in the mitochondria of yeast, humans and Arabidopsis have confirmed some expectations but given some surprises and some insights into the evolutionary origins of mitochondrial proteins.
Experimental analyses of the proteins found in the mitochondria of yeast, humans and Arabidopsis have confirmed some expectations but given some surprises and some insights into the evolutionary origins of mitochondrial proteins.
Two-dimensional gel electrophoresis (2-DE)-based proteomics approach was applied to extensively explore the molecular basis of plant development and environmental adaptation. These proteomics analyses revealed thousands of differentially expressed proteins (DEPs) closely related to different biological processes. However, little attention has been paid to how peptide mass fingerprinting (PMF) data generated by the approach can be directly utilized for the determination of protein phosphorylation. Here, we used the software tool FindMod to predict the peptides that might carry the phosphorylation modification by examining their PMF data for mass differences between the empirical and theoretical peptides and then identified phosphorylation sites using MALDI TOF/TOF according to predicted peptide data from these DEP spots in the 2-D gels. As a result, a total of 48 phosphorylation sites of 40 DEPs were successfully identified among 235 known DEPs previously revealed in the 2-D gels of elongating cotton fiber cells. The 40 phosphorylated DEPs, including important enzymes such as enolase, transketolase and UDP-L-rhamnose synthase, are presumed to participate in the functional regulation of numerous metabolic pathways, suggesting the reverse phosphorylation of these proteins might play important roles in elongating cotton fibers. The results also indicated that some different isoforms of the identical DEP revealed in our 2-DE-based proteomics analysis could be annotated by phosphorylation events. Taken together, as the first report of large-scale identification of phosphorylation sites in elongating cotton fiber cells, our study provides not only an excellent example of directly identifying phosphorylation sites from known DEPs on 2-D gels but also provides a valuable resource for future functional studies of phosphorylated proteins in this field.
Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ7-sterol-C5-desaturase (STE1/DWARF7), Δ24-sterol-Δ24-reductase (DIMINUTO/DWARF1) and Δ5,7-sterol-Δ7-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into Arabidopsis thaliana mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ5,7-sterol-Δ7-reductase and Δ24-sterol-Δ24-reductase are in addition localized to the plasma membrane, whereas Δ7-sterol-C5-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants.
Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes.
In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies.
This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.
Plant glycoside hydrolase family 9 (GH9) comprises typical endo-β-1,4-glucanase (EGases, EC126.96.36.199). Although GH9A (KORRIGAN) family genes have been reported to be involved in cellulose biosynthesis in plants, much remains unknown about other GH9 subclasses. In this study, we observed a global gene co-expression profiling and conducted a correlation analysis between OsGH9 and OsCESA among 66 tissues covering most periods of life cycles in 2 rice varieties. Our results showed that OsGH9A3 and B5 possessed an extremely high co-expression with OsCESA1, 3, and 8 typical for cellulose biosynthesis in rice. Using two distinct rice non-GH9 mutants and wild type, we performed integrative analysis of gene expression level by qRT-PCR, cellulase activities in situ and in vitro, and lignocellulose crystallinity index (CrI) in four internodes of stem tissues. For the first time, OsGH9B1, 3, and 16 were characterized with the potential role in lignocellulose crystallinity alteration in rice, whereas OsGH9A3 and B5 were suggested for cellulose biosynthesis. In addition, phylogenetic analysis and gene co-expression comparison revealed GH9 function similarity in Arabidopsis and rice. Hence, the data can provide insights into GH9 function in plants and offer the potential strategy for genetic manipulation of plant cell wall using the five aforementioned novel OsGH9 genes.
Eucalypt species are a group of flowering trees widely used in pulp production for paper manufacture. For several decades, the wood pulp industry has focused research and development efforts on improving yields, growth rates and pulp quality through breeding and the genetic improvement of key tree species. Recently, this focus has shifted from the production of high quality pulps to the investigation of the use of eucalypts as feedstocks for biofuel production. Here the structure and chemical composition of the heartwood and sapwood of Eucalyptus dunnii, E. globulus, E. pillularis, E. urophylla, an E. urophylla-E. grandis cross, Corymbia citriodora ssp. variegata, and Acacia mangium were compared using nuclear magnetic resonance spectroscopy (NMR), X-ray diffraction (XRD) and biochemical composition analysis. Some trends relating to these compositions were also identified by Fourier transform near infrared (FT-NIR) spectroscopy. These results will serve as a foundation for a more comprehensive database of wood properties that will help develop criteria for the selection of tree species for use as biorefinery feedstocks.
Bsister genes have been identified as the closest relatives of class B floral homeotic genes. Previous studies have shown that Bsister genes from eudicots are involved in cell differentiation during ovule and seed development. However, the complete function of Bsister genes in eudicots is masked by redundancy with other genes and little is known about the function of Bsister genes in monocots, and about the evolution of Bsister gene functions. Here we characterize OsMADS29, one of three MADS-box Bsister genes in rice. Our analyses show that OsMADS29 is expressed in female reproductive organs including the ovule, ovule vasculature, and the whole seed except for the outer layer cells of the pericarp. Knock-down of OsMADS29 by double-stranded RNA-mediated interference (RNAi) results in shriveled and/or aborted seeds. Histological analyses of the abnormal seeds at 7 days after pollination (DAP) indicate that the symplastic continuity, including the ovular vascular trace and the nucellar projection, which is the nutrient source for the filial tissue at early development stages, is affected. Moreover, degeneration of all the maternal tissues in the transgenic seeds, including the pericarp, ovular vascular trace, integuments, nucellar epidermis and nucellar projection, is blocked as compared to control plants. Our results suggest that OsMADS29 has important functions in seed development of rice by regulating cell degeneration of maternal tissues. Our findings provide important insights into the ancestral function of Bsister genes.
MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants.
Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.
Arabidopsis AtGH9C1 is an endo-β-1,4-glucanase possessing a carbohydrate-binding domain (CBM49). Analysis of AtGH9C1 expression by promoter-reporter GUS, RT-PCR, public transcriptome databases and GFP protein tagging demonstrated a high and selective expression of AtGH9C1 in root hairs and in the endosperm. Expression in root hair cells started prior to bulge formation and continued during hair elongation. AtGH9C1 expression increased with treatments that increase density (ACC) or length (sucrose) of root hairs. Expression in the endosperm extended sequentially to the micropylar, peripheral and chalazal compartments. A mutant with reduced AtGH9C1 expression had a delay in germination and a marked reduction in root hair presence. Complementation of the mutant partially improved both germination and root hair density. Experiments with ectopically expressed AtGH9C1-GFP with and without the CBM49, demonstrated that both forms of the protein are secreted and that CBM49 targets the protein to specific regions of the cell wall, but what makes these regions special is still unknown. The amino acid alignment of angiosperm GH9 genes with C-terminal extensions illustrate that AtGH9C1 belongs to a different clade than its tomato homolog, S1GH9C1. The latter has a CBM49 that was shown to bind crystalline cellulose. We suggest that AtGH9C1 is associated with the weakening of the cell wall during formation and growth of the root hair as well as with the sequential anterior-posterior breakdown of the endosperm cell wall that provides space for the growing embryo. Thus, is likely that the CBM49 of AtGH9C1 recognizes a form of cellulose or glucan polymer that is prevalent in the wall of these specialized tissues and that is different than the one recognized by S1GH9C1.
Saccharomyces cerevisiae CBS8066 encapsulated in semi-permeable alginate or alginate-chitosan liquid core capsules have been shown to have an enhanced tolerance towards complex dilute-acid lignocellulose hydrolysates and the lignocellulose-derived inhibitor furfural, as well as towards high temperatures. The underlying molecular reasons for these effects have however not been elucidated. In this study we have investigated the response of the encapsulation on the proteome level in the yeast cells, in comparison with cells grown freely in suspension under otherwise similar conditions. The proteomic analysis was performed on whole cell protein extracts using nLC-MS/MS with TMT® labelling and 2-D DIGE. 842 and 52 proteins were identified using each method, respectively. The abundances of 213 proteins were significantly different between encapsulated and suspended cells, with good correlation between the fold change ratios obtained by the two methods for proteins identified in both. Encapsulation of the yeast caused an up-regulation of glucose-repressed proteins and of both general and starvation-specific stress responses, such as the trehalose biosynthesis pathway, and down-regulation of proteins linked to growth and protein synthesis. The encapsulation leads to a lack of nutrients for cells close to the core of the capsule due to mass transfer limitations. The triggering of the stress response may be beneficial for the cells in certain conditions, for example leading to the increased tolerance towards high temperatures and certain inhibitors.
Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.
Plants grow and reproduce in the radioactive Chernobyl area, however there has been no comprehensive characterization of these activities. Herein we report that life in this radioactive environment has led to alteration of the developing soybean seed proteome in a specific way that resulted in the production of fertile seeds with low levels of oil and β-conglycinin seed storage proteins. Soybean seeds were harvested at four, five, and six weeks after flowering, and at maturity from plants grown in either non-radioactive or radioactive plots in the Chernobyl area. The abundance of 211 proteins was determined. The results confirmed previous data indicating that alterations in the proteome include adaptation to heavy metal stress and mobilization of seed storage proteins. The results also suggest that there have been adjustments to carbon metabolism in the cytoplasm and plastids, increased activity of the tricarboxylic acid cycle, and decreased condensation of malonyl-acyl carrier protein during fatty acid biosynthesis.
Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.
Steady state cultivation and multidimensional data analysis (metabolic fluxes, absolute proteome, and transcriptome) are used to identify parameters that control the increase in biomass yield of Lactococcus lactis from 0.10 to 0.12 C-mol C-mol−1 with an increase in specific growth rate by 5 times from 0.1 to 0.5 h−1. Reorganization of amino acid consumption was expressed by the inactivation of the arginine deiminase pathway at a specific growth rate of 0.35 h−1 followed by reduced over-consumption of pyruvate directed amino acids (asparagine, serine, threonine, alanine and cysteine) until almost all consumed amino acids were used only for protein synthesis at maximal specific growth rate. This balanced growth was characterized by a high glycolytic flux carrying up to 87% of the carbon flow and only amino acids that relate to nucleotide synthesis (glutamine, serine and asparagine) were consumed in higher amounts than required for cellular protein synthesis. Changes in the proteome were minor (mainly increase in the translation apparatus). Instead, the apparent catalytic activities of enzymes and ribosomes increased by 3.5 times (0.1 vs 0.5 h−1). The apparent catalytic activities of glycolytic enzymes and ribosomal proteins were seen to follow this regulation pattern while those of enzymes involved in nucleotide metabolism increased more than the specific growth rate (over 5.5 times). Nucleotide synthesis formed the most abundant biomonomer synthetic pathway in the cells with an expenditure of 6% from the total ATP required for biosynthesis. Due to the increase in apparent catalytic activity, ribosome translation was more efficient at higher growth rates as evidenced by a decrease of protein to mRNA ratios. All these effects resulted in a 30% decrease of calculated ATP spilling (0.1 vs 0.5 h−1). Our results show that bioprocesses can be made more efficient (using a balanced metabolism) by varying the growth conditions.
Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.