Metabolic changes during wheat microspore embryogenesis induction using the highly responsive cultivar Svilena.

Androgenetically-derived haploids can be obtained by inducing embryogenesis in microspores. Thus, full homozygosity is achieved in a single generation, oppositely to conventional plant breeding programs. Here, the metabolite profile of embryogenic microspores of Triticum aestivum was acquired and integrated with transcriptomic existing data from the same samples in an effort to identify the key metabolic processes occurring during the early stages of microspore embryogenesis. Primary metabolites and transcription profiles were identified at three time points: prior to and immediately following a low temperature pre-treatment given to uninuclear microspores, and after the first nuclear division. This is the first time an integrative -omics analysis is reported in microspore embryogenesis in T. aestivum. The key findings were that the energy produced during the pre-treatment was obtained from the tricarboxylic acid (TCA) cycle and from starch degradation, while starch storage resumed after the first nuclear division. Intermediates of the TCA cycle were highly demanded from a very active amino acid metabolism. The transcription profiles of genes encoding enzymes involved in amino acid synthesis differed from the metabolite profiles. The abundance of glutamine synthetase was correlated with that of glutamine. Cytosolic glutamine synthetase isoform 1 was found predominantly after the nuclear division. Overall, energy production was shown to represent a major component of the de-differentiation process induced by the pre-treatment, supporting a highly active amino acid metabolism.

Combined nitrogen and drought stress leads to overlapping and unique proteomic responses in potato.

MAIN CONCLUSION: Nitrogen deficient and drought-tolerant or sensitive potatoes differ in proteomic responses under combined (NWD) and individual stresses. The sensitive genotype 'Kiebitz' exhibits a higher abundance of proteases under NWD. Abiotic stresses such as N deficiency and drought affect the yield of Solanum tuberosum L. tremendously. Therefore, it is of importance to improve potato genotypes in terms of stress tolerance. In this study, we identified differentially abundant proteins (DAPs) in four starch potato genotypes under N deficiency (ND), drought stress (WD), or combined stress (NWD) in two rain-out shelter experiments. The gel-free LC-MS analysis generated a set of 1177 identified and quantified proteins. The incidence of common DAPs in tolerant and sensitive genotypes under NWD indicates general responses to this stress combination. Most of these proteins were part of the amino acid metabolism (13.9%). Three isoforms of S-adenosyl methionine synthase (SAMS) were found to be lower abundant in all genotypes. As SAMS were found upon application of single stresses as well, these proteins appear to be part of the general stress response in potato. Interestingly, the sensitive genotype 'Kiebitz' showed a higher abundance of three proteases (subtilase, carboxypeptidase, subtilase family protein) and a lower abundance of a protease inhibitor (stigma expressed protein) under NWD stress compared to control plants. The comparably tolerant genotype 'Tomba', however, displayed lower abundances of proteases. This indicates a better coping strategy for the tolerant genotype and a quicker reaction to WD when previously stressed with ND.

Unraveling metabolic patterns and molecular mechanisms underlying storability in sugar beet.

BACKGROUND: Sugar beet is an important crop for sugar production. Sugar beet roots are stored up to several weeks post-harvest waiting for processing in the sugar factories. During this time, sucrose loss and invert sugar accumulation decreases the final yield and processing quality. To improve storability, more information about post-harvest metabolism is required. We investigated primary and secondary metabolites of six sugar beet varieties during storage. Based on their variety-specific sucrose loss, three storage classes representing well, moderate, and bad storability were compared. Furthermore, metabolic data were visualized together with transcriptome data to identify potential mechanisms involved in the storage process. RESULTS: We found that sugar beet varieties that performed well during storage have higher pools of 15 free amino acids which were already observable at harvest. This storage class-specific feature is visible at harvest as well as after 13 weeks of storage. The profile of most of the detected organic acids and semi-polar metabolites changed during storage. Only pyroglutamic acid and two semi-polar metabolites, including ferulic acid, show higher levels in well storable varieties before and/or after 13 weeks of storage. The combinatorial OMICs approach revealed that well storable varieties had increased downregulation of genes involved in amino acid degradation before and after 13 weeks of storage. Furthermore, we found that most of the differentially genes involved in protein degradation were downregulated in well storable varieties at both timepoints, before and after 13 weeks of storage. CONCLUSIONS: Our results indicate that increased levels of 15 free amino acids, pyroglutamic acid and two semi-polar compounds, including ferulic acid, were associated with a better storability of sugar beet taproots. Predictive metabolic patterns were already apparent at harvest. With respect to elongated storage, we highlighted the role of free amino acids in the taproot. Using complementary transcriptomic data, we could identify potential underlying mechanisms of sugar beet storability. These include the downregulation of genes for amino acid degradation and metabolism as well as a suppressed proteolysis in the well storable varieties.

Effects of Arbuscular Mycorrhization on Fruit Quality in Industrialized Tomato Production.

Industrialized tomato production faces a decrease in flavors and nutritional value due to conventional breeding. Moreover, tomato production heavily relies on nitrogen and phosphate fertilization. Phosphate uptake and improvement of fruit quality by arbuscular mycorrhizal (AM) fungi are well-studied. We addressed the question of whether commercially used tomato cultivars grown in a hydroponic system can be mycorrhizal, leading to improved fruit quality. Tomato plants inoculated with Rhizophagus irregularis were grown under different phosphate concentrations and in substrates used in industrial tomato production. Changes in fruit gene expression and metabolite levels were checked by RNAseq and metabolite determination, respectively. The tests revealed that reduction of phosphate to 80% and use of mixed substrate allow AM establishment without affecting yield. By comparing green fruits from non-mycorrhizal and mycorrhizal plants, differentially expressed genes (DEGs) were found to possibly be involved in processes regulating fruit maturation and nutrition. Red fruits from mycorrhizal plants showed a trend of higher BRIX values and increased levels of carotenoids in comparison to those from non-mycorrhizal plants. Free amino acids exhibited up to four times higher levels in red fruits due to AM, showing the potential of mycorrhization to increase the nutritional value of tomatoes in industrialized production.

Purple corn extract induces long-lasting reprogramming and M2 phenotypic switch of adipose tissue macrophages in obese mice.

BACKGROUND: Obesity is a chronic and systemic inflammatory disorder and an important risk factor for the onset of several chronic syndromes. Adipose tissue (AT) plays a crucial role in the development of obesity, promoting the infiltration and accumulation of leukocytes in the tissue and sustaining adipocyte expansion. Anthocyanins exert a broad range of health benefits, but their effect in improving obesity-related inflammation in vivo has been poorly characterized. We examined the effects of a purple corn cob extract in the context of AT inflammation in a murine diet-induced obesity (DIO) model. METHODS: Male C57BL/6J mice were subjected to control diet (CTR + H2O), high fat diet (HF + H2O) or high fat diet plus purple corn extract (HF + RED) for 12 weeks. Blood glucose, AT, and liver gene expression, metabolism, biochemistry, and histology were analysed and flow cytometry was performed on AT leukocytes and Kupffer cells. RESULTS: RED extract intake resulted in lower MCP-1 mediated recruitment and proliferation of macrophages into crown-like structures in the AT. AT macrophages (ATM) of HF + RED group upregulated M2 markers (ArgI, Fizz1, TGFß), downregulating inflammatory mediators (TNF-α, IL-6, IL-1ß, COX-2) thanks to the suppression of NF-kB signalling. ATM also increased the expression of iron metabolism-related genes (FABP4, Hmox1, Ferroportin, CD163, TfR1, Ceruloplasmin, FtL1, FtH1) associated with a reduction in iron storage and increased turnover. ATM from HF + RED mice did not respond to LPS treatment ex vivo, confirming the long-lasting effects of the treatment on M2 polarization. Adipocytes of HF + RED group improved lipid metabolism and displayed a lower inflammation grade. Liver histology revealed a remarkable reduction of steatosis in the HF + RED group, and Kupffer cell profiling displayed a marked switch towards the M2 phenotype. CONCLUSIONS: RED extract attenuated AT inflammation in vivo, with a long-lasting reprogramming of ATM and adipocyte profiles towards the anti-inflammatory phenotype, therefore representing a valuable supplement in the context of obesity-associated disorders.

Hydroxycinnamic acids in sunflower leaves serve as UV-A screening pigments.

Flavonoids and hydroxycinnamic acid derivatives, which are located in the upper epidermis of plants, are well known to screen ultraviolet radiation, thus protecting the underlying tissue from these harmful wavelengths. Both classes of secondary products complement each other over the UV spectral region according to their absorption spectra: flavonoids are most efficient as UV-A attenuators while hydroxycinnamates (HCAs) screen well within the UV-B region. Analysis of epidermal transmittance revealed a substantial UV-A screen in Helianthus annuus L. cv. Peredovick. Identifying responsible pigments by HPLC-MS, we found surprisingly low amounts of flavonoids but dominant abundance of the HCA derivatives chlorogenic and di-caffeoyl quinic acid. Both display low UV-A absorbance and thus, should contribute only a little to UV-A protection. However, growth at high light led to a decrease of epidermal transmittance at 366 nm of up to 90%. Underpinning the screening role, HCA autofluorescence microscopy revealed storage to occur predominantly in vacuoles of the upper epidermis. UV-A treatment in the absence of D1-repair resulted in photosystem II inactivation proportional to epidermal UV-A transmittance. Our findings suggest that UV-A protection can be achieved solely with HCAs, apparently through accumulation of high amounts of these compounds.

Changes of soluble sugars and ATP content during DMSO droplet freezing and PVS3 droplet vitrification of potato shoot tips.

The potato's great genetic diversity needs to be maintained for future agricultural applications and can be preserved at ultra-low temperatures. To decipher detailed physiological processes, the aim of the study was to analyze the regrowth in 28 gene bank accessions and to reveal metabolite changes in a subset of four accessions that showed pronounced differences after shoot tip cryopreservation using DMSO droplet freezing and PVS3 droplet vitrification. Regrowth varied in all 28 genotypes ranging from 5% ('Kagiri') to 100% ('Karakter') and was higher after PVS3 droplet vitrification (71 ±â€¯19%) than after cryopreservation using DMSO (54 ±â€¯17%). Sucrose, glucose, and fructose were analyzed and showed significant increases after pre-culture in combination with PVS3 or DMSO and liquid nitrogen treatment and were reduced during regeneration. In contrast, adenosine triphosphate (ATP) reached its minimum concentration after cryoprotection and liquid nitrogen treatment and recovered most quickly after PVS3 droplet vitrification. A shortening of the explant pre-culture period reduced dramatically the regrowth after PVS3 vitrification. However, correlations between the shoot tip regrowth and sugar concentration were absent and significant at a low extent with ATP (r = 0.4, P < 0.01). Interestingly, several sub-cultivations of the donor plants from the previous stock affected negatively the regrowth. In conclusion, the cryopreservation protocol, genotypes, pre-culture period and number of sub-cultures affect the regrowth ability of explants, which was best estimated by the ATP concentration after low-temperature treatment. Due to the superior performance of PVS3, the routine potato cryopreservation at the Gatersleben gene bank was changed to PVS3 droplet vitrification.

Mini-Scale Isolation and Preparation of Plasma Membrane Proteins from Potato Roots for LC/MS Analysis.

Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30 g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5 g and allows studying the plasma membrane proteome by LC-MS.

Nitrogen Deficiency Induced Alterations in the Root Proteome of a Pair of Potato (Solanum tuberosum L.) Varieties Contrasting for their Response to Low N.

Improving crop nitrogen use efficiency is important both from the economic and the environmental viewpoint. Here, the aim is to highlight differences between the proteomic response of the roots of two potato cultivars contrasting in their response to nitrogen (N) deficiency, in an effort to understand which proteins and metabolic pathways contribute to the tolerance of N deprivation. The two cultivars ''Topas'' (tolerant) and ''Lambada'' (sensitive) are grown under both an N sufficient and an N deficient regime, using an in vitro-based cultivation system. Responsive proteins are identified and quantified using label-free quantitative shotgun proteomics. The contrasting cultivars differed with respect to components of the glutamine synthetase/glutamine oxoglutarate aminotransferase pathway, tricarboxylic acid cycle, the glycolysis/gluconeogenesis pathway as well as protein and amino acid synthesis machinery. Additional differences are associated with protein catabolism and defense mechanisms.

Comparative shoot proteome analysis of two potato (Solanum tuberosum L.) genotypes contrasting in nitrogen deficiency responses in vitro.

Aiming at a better understanding of the physiological and biochemical background of nitrogen use efficiency, alterations in the shoot proteome under N-deficiency were investigated in two contrasting potato genotypes grown in vitro with 60 and 7.5mM N, respectively. A gel based proteomic approach was applied to identify candidate proteins associated with genotype specific responses to N-deficiency. 21% of the detected proteins differed in abundance between the two genotypes. Between control and N-deficiency conditions 19.5% were differentially accumulated in the sensitive and 15% in the tolerant genotype. 93% of the highly N-deficiency responsive proteins were identified by MALDI TOF/TOF mass spectrometry. The major part was associated with photosynthesis, carbohydrate metabolism, stress response and regulation. Differential accumulation of enzymes involved in the Calvin cycle and glycolysis suggest activation of alternative carbohydrate pathways. In the tolerant genotype, increased abundance under N-deficiency was also found for enzymes involved in chlorophyll synthesis and stability of enzymes, which increase photosynthetic carbon fixation efficiency. Out of a total of 106 differentially abundant proteins, only eight were detected in both genotypes. Our findings suggest that mutually responsive proteins reflect universal stress responses while adaptation to N-deficiency in metabolic pathways is more genotype specific. SIGNIFICANCE: Nitrogen losses from arable farm land considerably contribute to environmental pollution. In potato, this is a special problem due cultivation on light soils, irrigation and the shallow root system. Therefore, breeding of cultivars with improved nitrogen use efficiency and stable yields under reduced N fertilization is an important issue. Knowledge of genotype dependent adaptation to N-deficiency at the proteome level can help to understand regulation of N efficiency and development of N-efficient cultivars.

Metabolite profiling of red and blue potatoes revealed cultivar and tissue specific patterns for anthocyanins and other polyphenols.

MAIN CONCLUSION: Metabolite profiling of tuber flesh and peel for selected colored potato varieties revealed cultivar and tissue specific profiles of anthocyanins and other polyphenols with variations in composition and concentration. Starchy tubers of Solanum tuberosum are a staple crop and food in many countries. Among cultivated potato varieties a huge biodiversity exists, including an increasing number of red and purple colored cultivars. This coloration relates to the accumulation of anthocyanins and is supposed to offer nutritional benefits possibly associated with the antioxidative capacity of anthocyanins. However, the anthocyanin composition and its relation to the overall polyphenol constitution in colored potato tubers have not been investigated closely. This study focuses on the phytochemical characterization of the phenolic composition of a variety of colored potato tubers, both for peel and flesh tissues. First, liquid chromatography (LC) separation coupled to UV and mass spectrometry (MS) detection of polyphenolic compounds of potato tubers from 57 cultivars was used to assign groups of potato cultivars differing in their anthocyanin and polyphenol profiles. Tissues from 19 selected cultivars were then analyzed by LC separation coupled to multiple reaction monitoring (MRM) to detect quantitative differences in anthocyanin and polyphenol composition. The measured intensities of 21 anthocyanins present in the analyzed potato cultivars and tissues could be correlated with the specific tuber coloration. Besides secondary metabolites well-known for potato tubers, the metabolic profiling led to the detection of two anthocyanins not described for potato tuber previously, which we tentatively annotated as pelargonidin feruloyl-xylosyl-glucosyl-galactoside and cyanidin 3-p-coumaroylrutinoside-5-glucoside. We detected significant correlations between some of the measured metabolites, as for example the negative correlation between the main anthocyanins of red and blue potato cultivars. Mainly hydroxylation and methylation patterns of the B-ring of dihydroflavonols, leading to the formation of specific anthocyanidin backbones, can be assigned to a distinct coloring of the potato cultivars and tuber tissues. However, basically the same glycosylation and acylation reactions occur regardless of the main anthocyanidin precursor present in the respective red and blue/purple tissue. Thus, the different anthocyanin profiles in red and blue potato cultivars likely relate to superior regulation of the expression and activities of hydroxylases and methyltransferases rather than to differences for downstream glycosyl- and acyltransferases. In this regard, the characterized potato cultivars represent a valuable resource for the molecular analysis of the genetic background and the regulation of anthocyanin side chain modification.

Proteomic comparison of Chelidonium majus L. latex in different phases of plant development.

Chelidonium majus L. (Papaveraceae) latex is used in traditinonal folk medicine to treat papillae, warts, condylomas, which are visible effects of human papilloma virus (HPV) infections. The aim of this work was to provide new insights into the biology and medicinal use of C. majus milky sap in the flowering and fruit ripening period of the plant by comparing the protein content between samples collected on respective developmental stages using LC-MS-based label-free proteome approach. For quantification, the multiplexed LC-MS data were processed using comparative chemometric approach. Progenesis LC-MS results showed that in green fruit phase (stage IV), comparing to flowering phase (stage III) of plant development, a range of proteins with higher abundance were identified as stress- and defense-related. On the other hand at stage III very intense protein synthesis, processes of transcription, protein folding and active transport of molecules (ABC transporters) are well represented. 2-DE protein maps showed an abundant set of spots with similar MWs (about 30-35 kDa) and pIs (ca. 5.5-6.5), which were identified as major latex proteins (MLPs). Therefore we suggest that biological activity of C. majus latex could be related to its protein content, which shifts during plant development from intense biosynthetic processes (biosynthesis and transport of small molecules, like alkaloids) to plant defense mechanisms against pathogens. Further studies will help to elucidate if these defense-related and pathogenesis-related proteins, like MLP, together with small-molecule compounds, could inhibit viral infection, what could be a step to fully understand the medicinal activity of C. majus latex.

Combination of transcriptomic and proteomic approaches helps to unravel the protein composition of Chelidonium majus L. milky sap.

MAIN CONCLUSION: A novel annotated Chelidonium majus L. transcriptome database composed of 23,004 unique coding sequences allowed to significantly improve the sensitivity of proteomic C. majus assessments, which showed novel defense-related proteins characteristic to its latex. To date, the composition of Chelidonium majus L. milky sap and biosynthesis of its components are poorly characterized. We, therefore, performed de novo sequencing and assembly of C. majus transcriptome using Illumina technology. Approximately, 119 Mb of raw sequence data was obtained. Assembly resulted in 107,088 contigs, with N50 of 1913 bp and N90 of 450 bp. Among 34,965 unique coding sequences (CDS), 23,004 obtained CDS database served as a basis for further proteomic analyses. The database was then used for the identification of proteins from C. majus milky sap, and whole plant extracts analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach. Of about 334 different putative proteins were identified in C. majus milky sap and 1155 in C. majus whole plant extract. The quantitative comparative analysis confirmed that C. majus latex contains proteins connected with response to stress conditions and generation of precursor metabolites and energy. Notable proteins characteristic to latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO, which could be responsible for browning of the sap after exposure to air), and enzymes responsible for anthocyanidin, phenylpropanoid, and alkaloid biosynthesis.

Proteomic analysis of two divergently responding potato genotypes (Solanum tuberosum L.) following osmotic stress treatment in vitro.

UNLABELLED: Starch potatoes (Solanum tuberosum L.) are of interest for production of starch, ethanol, and biopolymers. Due to the predicted increase in drought periods, the breeding of starch potatoes for drought tolerance is essential. This study aims to elucidate the physiological mechanisms that give rise to drought tolerance. Two genotypes contrasting in drought tolerance were compared. We applied osmotic stress which is a known component of drought stress under in vitro conditions. Shoot tips were harvested after 11days of culture on control medium and medium supplied with 0.2M sorbitol. Their proteomes were analyzed using two-dimensional isoelectric focussing sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D-IEF/SDS-PAGE). Of a total of 679 distinct protein spots, 118 and 20 spots with differential abundance were found in the sensitive and the tolerant genotype, respectively, after the application of stress. Using mass spectrometry, the proteins in 100 differentially abundant spots were identified; a majority of these proteins were from the chloroplast. For the sensitive genotype, an increase in the abundance of proteinase inhibitors and their precursors, changes in stress responsive proteins and an altered RNA/DNA-binding response were observed. The differentially abundant spots of the tolerant genotype comprised one chaperone and one hydrogen peroxide detoxifying protein. BIOLOGICAL SIGNIFICANCE: Our findings reveal that the two genotypes have different responses to osmotic stress in terms of protein degradation and reactive oxygen species (ROS) scavenging and production. Our data suggest that the tolerant genotype might adjust to the applied stress more quickly. A comparative temporal analysis might provide further insights into these rapid changes and assist in the development of biomarkers.

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

The reproducibility of liquid chromatography separation technology and its potential impact on large scale plant metabolomics experiments.

Unraveling the constituents of biological samples using HPLC is a central core technology in metabolomics experiments. Consistency in retention time across many samples is a critical criterion for judging the quality of a data set, which must be met before further analysis are possible. Here, the performance of two ultra high-performance liquid chromatography (UHPLC) systems has been compared using an established separation protocol optimized for phenylpropanoids, a class of secondary compounds found in plants displaying intermediate polarity. The two systems differed markedly with respect to their reproducibility and pressure stability. The standard deviation of the retention time of representative peaks differs up to 30-folds between the systems. Adjustments made to the gradient profiles succeeded in equalizing their level of performance. However, the modifications made to the separation protocol reduced the quality of the separation, particularly of the more rapidly eluting components, and lengthened the run time.

Arxula adeninivorans recombinant urate oxidase and its application in the production of food with low uric acid content.

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food.

Mass spectrometry based imaging techniques for spatially resolved analysis of molecules.

Higher plants are composed of a multitude of tissues with specific functions, reflected by distinct profiles for transcripts, proteins, and metabolites. Comprehensive analysis of metabolites and proteins has advanced tremendously within recent years, and this progress has been driven by the rapid development of sophisticated mass spectrometric techniques. In most of the current "omics"-studies, analysis is performed on whole organ or whole plant extracts, rendering to the loss of spatial information. Mass spectrometry imaging (MSI) techniques have opened a new avenue to obtain information on the spatial distribution of metabolites and of proteins. Pioneered in the field of medicine, the approaches are now applied to study the spatial profiles of molecules in plant systems. A range of different plant organs and tissues have been successfully analyzed by MSI, and patterns of various classes of metabolites from primary and secondary metabolism could be obtained. It can be envisaged that MSI approaches will substantially contribute to build spatially resolved biochemical networks.

Correlation of rutin accumulation with 3-O-glucosyl transferase and phenylalanine ammonia-lyase activities during the ripening of tomato fruit.

In tomato, the predominant flavonoid is quercetin-3-rutinoside (rutin). In this study, we aim to investigate the phenylalanine ammonia-lyase (PAL) and the quercetin-3-O-glucosyl transferase (3-GT) reactions in the formation of rutin during tomato fruit ripening. Tomatoes of the Moneymaker variety at different development stages (green, breaker, turning, pink, red, and deep red) were divided into flesh and peel fractions. In each sample, both the content of rutin and the enzymatic activities for PAL and 3-GT were recorded. The highest activities of PAL were recorded in the peel of turning fruit (3,000 µkat/mg fresh weight). In fruit flesh, maximal activity was observed in red fruit (917.3 µkat/mg). For both tissues, PAL activity strongly decreased at the final (deep red) fruit stage. The activity of 3-GT in peel peaked in the turning fruit stage (50.7 pkat/mg), while in flesh maximal activity (33.4 pkat/mg) was observed in green fruit, which rapidly declined at the turning stage. Higher levels of rutin were detected in the tomato peel compared to the flesh part with the highest level being found at the green stage. The relation of PAL and 3-GT activities to rutin content is also evaluated.