Bacterial N-Acyl Homoserine Lactone Priming Enhances Leaf-Rust Resistance in Winter Wheat and Some Genomic Regions Are Associated with Priming Efficiency.

Leaf rust (Puccinia triticina) is a common disease that causes significant yield losses in wheat. The most frequently used methods to control leaf rust are the application of fungicides and the cultivation of resistant genotypes. However, high genetic diversity and associated adaptability of pathogen populations hamper achieving durable resistance in wheat. Emerging alternatives, such as microbial priming, may represent an effective measure to stimulate plant defense mechanisms and could serve as a means of controlling a broad range of pathogens. In this study, 175 wheat genotypes were inoculated with two bacterial strains: Ensifer meliloti strain expR+ch (producing N-acyl homoserine lactone (AHL)) or transformed E. meliloti carrying the lactonase gene attM (control). In total, 21 genotypes indicated higher resistance upon bacterial AHL priming. Subsequently, the phenotypic data of 175 genotypes combined with 9917 single-nucleotide polymorphisms (SNPs) in a genome-wide association study to identify quantitative trait loci (QTLs) and associated markers for relative infection under attM and expR+ch conditions and priming efficiency using the Genome Association and Prediction Integrated Tool (GAPIT). In total, 15 QTLs for relative infection under both conditions and priming efficiency were identified on chromosomes 1A, 1B, 2A, 3A, 3B, 3D, 6A, and 6B, which may represent targets for wheat breeding for priming and leaf-rust resistance.

PEG treatment is unsuitable to study root related traits as it alters root anatomy in barley (Hordeum vulgare L.).

BACKGROUND: The frequency and severity of abiotic stress events, especially drought, are increasing due to climate change. The plant root is the most important organ for water uptake and the first to be affected by water limitation. It is therefore becoming increasingly important to include root traits in studies on drought stress tolerance. However, phenotyping under field conditions remains a challenging task. In this study, plants were grown in a hydroponic system with polyethylene glycol as an osmotic stressor and in sand pots to examine the root system of eleven spring barley genotypes. The root anatomy of two genotypes with different response to drought was investigated microscopically. RESULTS: Root diameter increased significantly (p < 0.05) under polyethylene glycol treatment by 54% but decreased significantly (p < 0.05) by 12% under drought stress in sand pots. Polyethylene glycol treatment increased root tip diameter (51%) and reduced diameter of the elongation zone (14%) compared to the control. Under drought stress, shoot mass of plants grown in sand pots showed a higher correlation (r = 0.30) with the shoot mass under field condition than polyethylene glycol treated plants (r = -0.22). CONCLUSION: These results indicate that barley roots take up polyethylene glycol by the root tip and polyethylene glycol prevents further water uptake. Polyethylene glycol-triggered osmotic stress is therefore unsuitable for investigating root morphology traits in barley. Root architecture of roots grown in sand pots is more comparable to roots grown under field conditions.

Non-invasive assessment of cultivar and sex of Cannabis sativa L. by means of hyperspectral measurement.

Cannabis sativa L. is a versatile crop attracting increasing attention for food, fiber, and medical uses. As a dioecious species, males and females are visually indistinguishable during early growth. For seed or cannabinoid production, a higher number of female plants is economically advantageous. Currently, sex determination is labor-intensive and costly. Instead, we used rapid and non-destructive hyperspectral measurement, an emerging means of assessing plant physiological status, to reliably differentiate males and females. One industrial hemp (low tetrahydrocannabinol [THC]) cultivar was pre-grown in trays before transfer to the field in control soil. Reflectance spectra were acquired from leaves during flowering and machine learning algorithms applied allowed sex classification, which was best using a radial basis function (RBF) network. Eight industrial hemp (low THC) cultivars were field grown on fertilized and control soil. Reflectance spectra were acquired from leaves at early development when the plants of all cultivars had developed between four and six leaf pairs and in three cases only flower buds were visible (start of flowering). Machine learning algorithms were applied, allowing sex classification, differentiation of cultivars and fertilizer regime, again with best results for RBF networks. Differentiating nutrient status and varietal identity is feasible with high prediction accuracy. Sex classification was error-free at flowering but less accurate (between 60% and 87%) when using spectra from leaves at early growth stages. This was influenced by both cultivar and soil conditions, reflecting developmental differences between cultivars related to nutritional status. Hyperspectral measurement combined with machine learning algorithms is valuable for non-invasive assessment of C. sativa cultivar and sex. This approach can potentially improve regulatory security and productivity of cannabis farming.

Association Mapping of Drought Tolerance Indices in Ethiopian Durum Wheat (Triticum turgidum ssp. durum).

Ethiopia is a major producer of durum wheat in sub-Saharan Africa. However, its production is prone to drought stress as it is fully dependent on rain, which is erratic and unpredictable. This study aimed to detect marker-trait associations (MTAs) and quantitative trait loci (QTLs) related to indices. Six drought tolerance indices, i.e., drought susceptibility index (DSI), geometric mean productivity (GMP), relative drought index (RDI), stress tolerance index (STI), tolerance index (TOL), and yield stability index (YSI) were calculated from least-square means (lsmeans) of grain yield (GY) and traits significantly (p < 0.001) correlated with grain yield (GY) under field drought stress (FDS) and field non-stress (FNS) conditions. GY, days to grain filling (DGF), soil plant analysis development (SPAD) chlorophyll meter, seeds per spike (SPS), harvest index (HI), and thousand kernel weight (TKW) were used to calculate DSI, GMP, RDI, STI, TOL, and YSI drought indices. Accessions, DW084, DW082, DZ004, C037, and DW092 were selected as the top five drought-tolerant based on DSI, RDI, TOL, and YSI combined ranking. Similarly, C010, DW033, DW080, DW124-2, and C011 were selected as stable accessions based on GMP and STI combined ranking. A total of 184 MTAs were detected linked with drought indices at -log10p ≥ 4.0,79 of which were significant at a false discovery rate (FDR) of 5%. Based on the linkage disequilibrium (LD, r 2 ≥ 0.2), six of the MTAs with a positive effect on GY-GMP were detected on chromosomes 2B, 3B, 4A, 5B, and 6B, explaining 14.72, 10.07, 26.61, 21.16, 21.91, and 22.21% of the phenotypic variance, respectively. The 184 MTAs were clustered into 102 QTLs. Chromosomes 1A, 2B, and 7A are QTL hotspots with 11 QTLs each. These chromosomes play a key role in drought tolerance and respective QTL may be exploited by marker-assisted selection for improving drought stress tolerance in wheat.

The genetic diversity of Ethiopian barley genotypes in relation to their geographical origin.

Ethiopia is recognized as a center of diversity for barley, and its landraces are known for the distinct genetic features compared to other barley collections. The genetic diversity of Ethiopian barley likely results from the highly diverse topography, altitude, climate conditions, soil types, and farming systems. To get detailed information on the genetic diversity a panel of 260 accessions, comprising 239 landraces and 21 barley breeding lines, obtained from the Ethiopian biodiversity institute (EBI) and the national barley improvement program, respectively were studied for their genetic diversity using the 50k iSelect single nucleotide polymorphism (SNP) array. A total of 983 highly informative SNP markers were used for structure and diversity analysis. Three genetically distinct clusters were obtained from the structure analysis comprising 80, 71, and 109 accessions, respectively. Analysis of molecular variance (AMOVA) revealed the presence of higher genetic variation (89%) within the clusters than between the clusters (11%), with moderate genetic differentiation (PhiPT = 0.11) and five accessions were detected as first-generation migrants using Monte Carlo resampling methods. The Mantel test revealed that the genetic distance between accessions is poorly associated with their geographical distance. Despite the observed weak correlation between geographic distance and genetic differentiation, for some regions like Gonder, Jimma, Gamo-Gofa, Shewa, and Welo, more than 50% of the landraces derived from these regions are assigned to one of the three clusters.

QTL for induced resistance against leaf rust in barley.

Leaf rust caused by Puccinia hordei is one of the major diseases of barley (Hordeum vulgare L.) leading to yield losses up to 60%. Even though, resistance genes Rph1 to Rph28 are known, most of these are already overcome. In this context, priming may promote enhanced resistance to P. hordei. Several bacterial communities such as the soil bacterium Ensifer (syn. Sinorhizobium) meliloti are reported to induce resistance by priming. During quorum sensing in populations of gram negative bacteria, they produce N-acyl homoserine-lactones (AHL), which induce resistance in plants in a species- and genotype-specific manner. Therefore, the present study aims to detect genotypic differences in the response of barley to AHL, followed by the identification of genomic regions involved in priming efficiency of barley. A diverse set of 198 spring barley accessions was treated with a repaired E. meliloti natural mutant strain expR+ch producing a substantial amount of AHL and a transformed E. meliloti strain carrying the lactonase gene attM from Agrobacterium tumefaciens. For P. hordei resistance the diseased leaf area and the infection type were scored 12 dpi (days post-inoculation), and the corresponding relative infection and priming efficiency were calculated. Results revealed significant effects (p<0.001) of the bacterial treatment indicating a positive effect of priming on resistance to P. hordei. In a genome-wide association study (GWAS), based on the observed phenotypic differences and 493,846 filtered SNPs derived from the Illumina 9k iSelect chip, genotyping by sequencing (GBS), and exome capture data, 11 quantitative trait loci (QTL) were identified with a hot spot on the short arm of the barley chromosome 6H, associated to improved resistance to P. hordei after priming with E. meliloti expR+ch. Genes in these QTL regions represent promising candidates for future research on the mechanisms of plant-microbe interactions.

The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress.

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but protein levels increased in response to the treatment, particularly in the root tip. Expression of HvHorcH in Arabidopsis thaliana root tips increased salt tolerance. Hence, we conclude that this protein is involved in the adaptation of plants to salinity.

Immunomodulatory Properties of Blackberry Anthocyanins in THP-1 Derived Macrophages.

An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.

Antioxidant Effects of Elderberry Anthocyanins in Human Colon Carcinoma Cells: A Study on Structure-Activity Relationships.

SCOPE: Glycosylation is a way to increase structure-stability of anthocyanins, yet compromises their bioactivity. The study investigates the antioxidant activity of purified cyanidin (Cy)-based anthocyanins and respective degradation products in Caco-2 clone C2BBe1 aiming to identify structure-activity relationships. RESULTS AND METHODS: Cyanidin 3-O-glucoside (Cy-3-glc) and cyanidin 3-O-sambubioside (Cy-3-sam) proved to be most potent regarding antioxidant properties and protection against hydrogen peroxide (H2 O2 )-induced reactive oxygen species (ROS)-levels measured with the dichloro-fluorescein (DCF) assay. Cyanidin 3-O-sambubioside-5-O-glucoside (Cy-3-sam-5-glc) and cyanidin 3-O-rutinoside (Cy-3-rut) were less efficient and not protective, reflecting potential differences in uptake and/or degradation. Following ranking in antioxidant efficiency is suggested: (concentrations ≤10 × 10-6  M) Cy-3-glc ≥ Cy-3-sam > Cy-3-sam-5-glc ≈ Cy-3-rut ≈ Cy; (concentrations ≥50 × 10-6  M) Cy-3-glc ≈ Cy-3-sam ≥ Cy > Cy-3-sam-5-glc ≈ Cy-3-rut. Cy and protocatechuic acid (PCA) reduced ROS-levels as potent as the mono- and di-glycoside, whereas phloroglucinol aldehyde (PGA) displayed pro-oxidant properties. None of the degradation products protected from oxidative stress. Gene transcription analysis of catalase (CAT), superoxide-dismutase (SOD), glutathione-peroxidase (GPx), heme-oxygenase-1 (HO-1), and glutamate-cysteine-ligase (γGCL) suggest no activation of nuclear factor erythroid 2-related factor 2 (Nrf2). CONCLUSION: More complex residues and numbers of sugar moieties appear to be counterproductive for antioxidant activity. Other mechanisms than Nrf2-activation should be considered for protective effects.

Genome-wide association study reveals the genetic complexity of fructan accumulation patterns in barley grain.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

Function and Regulation of Chloroplast Peroxiredoxin IIE.

Peroxiredoxins (PRX) are thiol peroxidases that are highly conserved throughout all biological kingdoms. Increasing evidence suggests that their high reactivity toward peroxides has a function not only in antioxidant defense but in particular in redox regulation of the cell. Peroxiredoxin IIE (PRX-IIE) is one of three PRX types found in plastids and has previously been linked to pathogen defense and protection from protein nitration. However, its posttranslational regulation and its function in the chloroplast protein network remained to be explored. Using recombinant protein, it was shown that the peroxidatic Cys121 is subjected to multiple posttranslational modifications, namely disulfide formation, S-nitrosation, S-glutathionylation, and hyperoxidation. Slightly oxidized glutathione fostered S-glutathionylation and inhibited activity in vitro. Immobilized recombinant PRX-IIE allowed trapping and subsequent identification of interaction partners by mass spectrometry. Interaction with the 14-3-3 υ protein was confirmed in vitro and was shown to be stimulated under oxidizing conditions. Interactions did not depend on phosphorylation as revealed by testing phospho-mimicry variants of PRX-IIE. Based on these data it is proposed that 14-3-3υ guides PRX­IIE to certain target proteins, possibly for redox regulation. These findings together with the other identified potential interaction partners of type II PRXs localized to plastids, mitochondria, and cytosol provide a new perspective on the redox regulatory network of the cell.

Fructans Are Differentially Distributed in Root Tissues of Asparagus.

Inulin- and neoseries-type fructans [fructooligosaccharides (FOS) and fructopolysaccharides] accumulate in storage roots of asparagus (Asparagus officinalis L.), which continue to grow throughout the lifespan of this perennial plant. However, little is known about the storage of fructans at the spatial level in planta, and the degree of control by the plant is largely uncertain. We have utilized mass spectrometry imaging (MSI) to resolve FOS distribution patterns in asparagus roots (inner, middle, and outer tissues). Fructan and proteome profiling were further applied to validate the differential abundance of various fructan structures and to correlate observed tissue-specific metabolite patterns with the abundance of related fructan biosynthesis enzymes. Our data revealed an increased abundance of FOS with higher degree of polymerization (DP > 5) and of fructopolysaccharides (DP11 to DP17) towards the inner root tissues. Three isoforms of fructan:fructan 6G-fructosyltransferase (6G-FFT), forming 6G-kestose with a ß (2-6) linkage using sucrose as receptor and 1-kestose as donor, were similarly detected in all three root tissues. In contrast, one ß-fructofuranosidase, which likely exhibits fructan:fructan 1-fructosyltransferase (1-FFT) activity, showed very high abundance in the inner tissues and lower levels in the outer tissues. We concluded a tight induction of the biosynthesis of fructans with DP > 5, following a gradient from the outer root cortex to the inner vascular tissues, which also correlates with high levels of sucrose metabolism in inner tissues, observed in our study.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development.

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-ß-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.

Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

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.

Protein Composition and Baking Quality of Wheat Flour as Affected by Split Nitrogen Application.

Baking quality of wheat flour is determined by grain protein concentration (GPC) and its composition and is highly influenced by environmental factors such as nitrogen (N) fertilization management. This study investigated the effect of split N application on grain protein composition and baking quality of two winter wheat cultivars, Tobak and JB Asano, belonging to different baking quality classes. Bread loaf volumes in both cultivars were enhanced by split N application. In contrast, GPC was only significantly increased in JB Asano. Comparative 2-DE revealed that the relative volumes of 21 and 28 unique protein spots were significantly changed by split N application in Tobak and JB Asano, respectively. Specifically, the alterations in relative abundance of certain proteins, i.e., globulins, LMW-GS, α-, and γ-gliadins as well as α-amylase/trypsin inhibitors were more sensitive to split N application. Furthermore, certain proteins identified as globulins and alpha-amylase inhibitors were changed in both wheat cultivars under split N application. These results implied that the functions of these unique proteins might have played important roles in affecting baking quality of wheat flour, especially for cultivars (i.e., Tobak in the present study) the baking quality of which is less dependent on GPC.

Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures.

Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes. We have used tobacco lines which constitutively produce high levels of cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside or a novel anthocyanin, acylated cyanidin 3-O-(coumaroyl) rutinoside to generate cell suspension cultures. The cell lines are stable in their production rates and superior to conventional plant cell cultures. Scale-up of anthocyanin production in small scale fermenters has been demonstrated. The cell cultures have also proven to be a suitable system for production of 13C-labelled anthocyanins. Our method for anthocyanin production is transferable to other plant species, such as Arabidopsis thaliana, demonstrating the potential of this approach for making a wide range of highly-decorated anthocyanins. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

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.