Knowledge gaps on how to adapt crop production under changing saline circumstances in the Netherlands.

Salinization, the increase and accumulation of salts in water and soil, impacts productivity of arable crops and is exacerbated by climate change. The Netherlands, like several other deltas and semi-arid regions, faces increasing salinization that negatively impacts agriculture and freshwater availability. Although a lot of salinity expertise exist in the Netherlands, several knowledge gaps on the impact of salinization in the Netherlands, as well as steps to facilitate closing this knowledge gaps to improve saline agriculture in the Netherlands, still exist. This review/opinion article moves beyond existing papers on salinization in bringing together various adaptation measures by thoroughly reviewing the measures through a triple P (People, Planet, Profit) lens. Five main salinity adaptation measures of the crop-soil-water continuum are 1) breeding and selection of salt tolerant varieties, 2) increased cultivation of halophytes, 3) soil management interventions, 4) use of biostimulants, and 5) irrigation techniques. These adaptation measures are described, discussed and analysed for their compliance to the sustainable development elements People, Planet and Profit. All five adaptation measures have potential positive impact on livelihood, contribute to food security and generate revenue but on the other hand, these measures may contribute to unwarranted changes of the ecosystem. The paper ends with a concluding chapter in which the bottlenecks and knowledge gaps that need resolving are identified based on the critical, including triple P, assessment of the discussed adaptation measures. Three key knowledge gaps on breeding, agronomy, environmental sciences and socioeconomics are identified with several approaches that lead to insights elucidated. Thereby informing on future research and action plans to optimize implementation of salinity adaptation measures in the Netherlands.

Phylogenomic analysis provides insights into MADS-box and TCP gene diversification and floral development of the Asteraceae, supported by de novo genome and transcriptome sequences from dandelion (Taraxacum officinale).

The Asteraceae is the largest angiosperm family with more than 25,000 species. Individual studies have shown that MADS-box and TCP transcription factors are regulators of the development and symmetry of flowers, contributing to their iconic flower-head (capitulum) and floret. However, a systematic study of MADS-box and TCP genes across the Asteraceae is lacking. We performed a comparative analysis of genome sequences of 33 angiosperm species including our de novo assembly of diploid sexual dandelion (Taraxacum officinale) and 11 other Asteraceae to investigate the lineage-specific evolution of MADS-box and TCP genes in the Asteraceae. We compared the phylogenomic results of MADS-box and TCP genes with their expression in T. officinale floral tissues at different developmental stages to demonstrate the regulation of genes with Asteraceae-specific attributes. Here, we show that MADS-box MIKC c and TCP-CYCLOIDEA (CYC) genes have expanded in the Asteraceae. The phylogenomic analysis identified AGAMOUS-like (AG-like: SEEDSTICK [STK]-like), SEPALATA-like (SEP3-like), and TCP-PROLIFERATING CELL FACTOR (PCF)-like copies with lineage-specific genomic contexts in the Asteraceae, Cichorioideae, or dandelion. Different expression patterns of some of these gene copies suggest functional divergence. We also confirm the presence and revisit the evolutionary history of previously named "Asteraceae-Specific MADS-box genes (AS-MADS)." Specifically, we identify non-Asteraceae homologs, indicating a more ancient origin of this gene clade. Syntenic relationships support that AS-MADS is paralogous to FLOWERING LOCUS C (FLC) as demonstrated by the shared ancient duplication of FLC and SEP3.

The power of seaweeds as plant biostimulants to boost crop production under abiotic stress.

Abiotic stresses like drought and salinity are major factors resulting in crop yield losses and soil degradation worldwide. To meet increasing food demands, we must improve crop productivity, especially under increasing abiotic stresses due to climate change. Recent studies suggest that seaweed-based biostimulants could be a solution to this problem. Here, we summarize the current findings of using these biostimulants and highlight current knowledge gaps. Seaweed extracts were shown to enhance nutrient uptake and improve growth performance in crops under stressed and normal conditions. Seaweed extracts contain several active compounds, for example, polysaccharides, polyphenols and phytohormones. Although some of these compounds have growth-promoting properties on plants, the molecular mechanisms that underly seaweed extract action remain understudied. In this paper, we review the role of these extracts and their bioactive compounds as plant biostimulants. The targeted application of seaweed extract to improve crop performance and protein accumulation is also discussed.

Daily Intake of Lemna minor or Spinach as Vegetable Does Not Show Significant Difference on Health Parameters and Taste Preference.

The growing world population will increase the demand for new sustainable foods and ingredients. Here we studied the safety and tolerance of Lemna minor, a new sustainable vegetable crop from the duckweed family. Twenty-four healthy adults consumed either L. minor plant material or spinach as vegetable (170 g fresh weight) as part of a warm meal on 11 consecutively days in a randomized controlled parallel trial design. The intervention meals had a different recipe for each day of the week. All participants had to report daily if they experienced gastric complaints, feelings of hunger, fullness, desire to eat, thirst, general health, nausea, and stool consistency. Only hunger, flatulence and constipation were significantly different between both intervention groups. At the start and end of the intervention, blood and urine were sampled in order to analyze biomarkers for general health, e.g., kidney function, liver function, cardiovascular health, inflammation and iron status. Both intervention groups did not show significant differences for these biomarkers. In taste attributes the L. minor-based products showed in only a few specific cases a significant difference compared to the spinach-based products. Based on the results we conclude that 11 consecutive days intake of 170 g fresh weight L. minor plants as a cooked vegetable does not result in any adverse effect in healthy adult subjects.

A controlled human intervention trial to study protein quality by amino acid uptake kinetics with the novel Lemna protein concentrate as case study.

A human intervention trial was conducted to study amino acid uptake of the novel Lemna protein concentrate (LPC) in comparison to whey (WPC). The study was a cross-over, double-blind, controlled trial in which 12 healthy participants received 20 grams of LPC and WPC in randomised order. The LPC consumption resulted in a significant lower postprandial increase in almost all individual amino acids, total amino acid (TAA) and total essential amino acids (TEAA) compared to WPC based on area under the curve (AUC) calculations. When the AUC after WPC consumption was set at 100%, LPC showed a relative AUC of 60.4% for TAA and 66.3% for the TEAA. Interindividual variation for LPC was high with an uptake of TEAA of LPC compared to WPC ranging from 18.2 to 94.2%. Human intervention trials can partly replace animal trials as they fully reflect the human situation and provide estimates on individual variations.

Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

Azolla ferns testify: seed plants and ferns share a common ancestor for leucoanthocyanidin reductase enzymes.

Questions about in vivo substrates for proanthocyanidin (PA) biosynthesis and condensation have not been resolved and wide gaps in the understanding of transport and biogenesis in 'tannosomes' persist. Here we examined the evolution of PA biosynthesis in ferns not previously reported, asking what PAs are synthesised and how. Chemical and gene-expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fern Azolla filiculoides. In vitro assay and phylogenomics of PIP-dehydrogenases served to infer the evolution of leucoanthocyanidin reductase (LAR). Sporophyte-synthesised (epi)catechin polymers, averaging only seven subunits, accumulated to 5.3% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. filiculoides. LAR, and paralogous fern WLAR-enzymes with differing substrate binding sites, represent an evolutionary innovation of the common ancestor of fern and seed plants. The specific ecological niche of Azolla ferns, a floating plant-microbe mat massively fixing CO2 and N2 , shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.

CRISPR/Cas9 Gene Editing of Gluten in Wheat to Reduce Gluten Content and Exposure-Reviewing Methods to Screen for Coeliac Safety.

Ingestion of gluten proteins (gliadins and glutenins) from wheat, barley and rye can cause coeliac disease (CD) in genetically predisposed individuals. The only remedy is a strict and lifelong gluten-free diet. There is a growing desire for coeliac-safe, whole-grain wheat-based products, as consumption of whole-grain foods reduces the risk of chronic diseases. However, due to the large number of gluten genes and the complexity of the wheat genome, wheat that is coeliac-safe but retains baking quality cannot be produced by conventional breeding alone. CD is triggered by immunogenic epitopes, notably those present in α-, γ-, and ω-gliadins. RNA interference (RNAi) silencing has been used to down-regulate gliadin families. Recently, targeted gene editing using CRISPR/Cas9 has been applied to gliadins. These methods produce offspring with silenced, deleted, and/or edited gliadins, that overall may reduce the exposure of patients to CD epitopes. Here we review methods to efficiently screen and select the lines from gliadin gene editing programs for CD epitopes at the DNA and protein level, for baking quality, and ultimately in clinical trials. The application of gene editing for the production of coeliac-safe wheat is further considered within the context of food production and in view of current national and international regulatory frameworks.

Postprandial amino acid, glucose and insulin responses among healthy adults after a single intake of Lemna minor in comparison with green peas: a randomised trial.

A high protein content combined with its enormous growth capacity make duckweed an interesting alternative protein source, but information about postprandial responses in humans is lacking. The present study aimed to assess the postprandial serum amino acid profile of Lemna minor in healthy adults in comparison with green peas. A secondary objective was to obtain insights regarding human safety. A total of twelve healthy volunteers participated in a randomised, cross-over trial. Subjects received two protein sources in randomised order with a 1-week washout period. After an overnight fast, subjects consumed L. minor or peas (equivalent to 20 g of protein). After a baseline sample, blood samples were taken 15, 30, 45, 60, 75, 90, 120, 150 and 180 min after consumption to assess amino acid, glucose and insulin levels. Heart rate, blood pressure and aural temperature were measured before and after consumption, and subjects reported on gastrointestinal discomfort for four subsequent days. Compared with green peas, significantly lower blood concentrations of amino acids from L. minor were observed, indicating lower digestibility. L. minor consumption resulted in lower plasma glucose and insulin levels compared with peas, probably due to different glucose content. There were no significant differences concerning the assessed health parameters or the number of gastrointestinal complaints, indicating that a single bolus of L. minor - grown under controlled conditions - did not induce acute adverse effects in humans. Further studies need to investigate effects of repeated L. minor intake and whether proteins purified from L. minor can be digested more easily.

Food processing and breeding strategies for coeliac-safe and healthy wheat products.

A strict gluten-free diet is currently the only treatment for the 1-2% of the world population who suffer from coeliac disease (CD). However, due to the presence of wheat and wheat derivatives in many food products, avoiding gluten consumption is difficult. Gluten-free products, made without wheat, barley or rye, typically require the inclusion of numerous additives, resulting in products that are often less healthy than gluten-based equivalents. Here, we present and discuss two broad approaches to decrease wheat gluten immunogenicity for CD patients. The first approach is based on food processing strategies, which aim to remove gliadins or all gluten from edible products. We find that several of the candidate food processing techniques to produce low gluten-immunogenic products from wheat already exist. The second approach focuses on wheat breeding strategies to remove immunogenic epitopes from the gluten proteins, while maintaining their food-processing properties. A combination of breeding strategies, including mutation breeding and possibly genome editing, will be necessary to produce coeliac-safe wheat. Individuals suffering from CD and people genetically susceptible who may develop CD after prolonged gluten consumption would benefit from reduced CD-immunogenic wheat. Although the production of healthy and less CD-toxic wheat varieties and food products will be challenging, increasing global demand may require these issues to be addressed in the near future by food processing and cereal breeding companies.

Oats in healthy gluten-free and regular diets: A perspective.

During the 20th century, the economic position of oats (Avena sativa L.) decreased strongly in favour of higher yielding crops including winter wheat and maize. Presently, oat represents only ~1.3% of the total world grain production, and its production system is fragmented. Nonetheless, current interest is growing because of recent knowledge on its potential benefits in food, feed and agriculture. This perspective will serve as a further impetus, with special focus on the recently valued advantages of oats in human food and health. Five approved European Food Safety Authority (EFSA) health claims apply to oats. Four relate to the oat-specific soluble fibres, the beta-glucans, and concern the maintenance and reduction of blood cholesterol, better blood glucose balance and increased faecal bulk. The fifth claim concerns the high content of unsaturated fatty acids, especially present in the endosperm, which reduces the risks of heart and vascular diseases. Furthermore, oat starch has a low glycemic index, which is favourable for weight control. Oat-specific polyphenols and avenanthramides have antioxidant and anti-inflammatory properties. Thus, oats can contribute significantly to the presently recommended whole-grain diet. Next to globulins, oats contain a small fraction of prolamin storage proteins, called 'avenins', but at a much lower quantity than gluten proteins in wheat, barley and rye. Oat avenins do not contain any of the known coeliac disease epitopes from gluten of wheat, barley and rye. Long-term food studies confirm the safety of oats for coeliac disease patients and the positive health effects of oat products in a gluten-free diet. These effects are general and independent of oat varieties. In the EU (since 2009), the USA (since 2013) and Canada (since 2015) oat products may be sold as gluten-free provided that any gluten contamination level is below 20ppm. Oats are, however, generally not gluten-free when produced in a conventional production chain, because of regular contamination with wheat, barley or rye. Therefore, establishing a separate gluten-free oat production chain requires controlling all steps in the chain; the strict conditions will be discussed. Genomic tools, including a single nucleotide polymorphism (SNP) marker array and a dense genetic map, have recently been developed and will support marker-assisted breeding. In 2015, the Oat Global initiative emerged enabling a world-wide cooperation starting with a data sharing facility on genotypic, metabolic and phenotypic characteristics. Further, the EU project TRAFOON (Traditional Food Networks) facilitated the transfer of knowledge to small- and medium-sized enterprises (SMEs) to stimulate innovations in oat production, processing, products and marketing, among others with regard to gluten-free. Finally, with focus on counteracting market fragmentation of the global oat market and production chains, interactive innovation strategies between customers (consumers) and companies through co-creation are discussed.

Why Oats Are Safe and Healthy for Celiac Disease Patients.

The water-insoluble storage proteins of cereals (prolamins) are called "gluten" in wheat, barley, and rye, and "avenins" in oat. Gluten can provoke celiac disease (CD) in genetically susceptible individuals (those with human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 serotypes). Avenins are present at a lower concentration (10%-15% of total protein content) in oat as compared to gluten in wheat (80%-85%). The avenins in the genus Avena (cultivated oat as well as various wild species of which gene bank accessions were analyzed) are free of the known CD immunogenic epitopes from wheat, barley, and rye. T cells that recognize avenin-specific epitopes have been found very rarely in CD patients. CD patients that consume oats daily do not show significantly increased levels of intraepithelial lymphocyte (EIL) cells. The safety and the positive health effects of the long-term inclusion of oats in the gluten-free diet have been confirmed in long-term studies. Since 2009 (EC 41/2009) and 2013 (FDA) oat products may be sold as gluten-free in several countries provided a gluten contamination level below 20 ppm. Introduction of oats in the gluten-free diet of celiac patients is advised after the recovery of the intestine. Health effects of oat consumption are reflected in European Food Safety Authority (EFSA)- and Food and Drug Administration (FDA)-approved health claims. Oats can form a healthy, nutritious, fiber-rich, and safe complement to the gluten-free diet.

Label free targeted detection and quantification of celiac disease immunogenic epitopes by mass spectrometry.

Celiac disease (CD) is a food-related disease caused by certain gluten peptides containing T-cell stimulating epitopes from wheat, rye, and barley. CD-patients have to maintain a gluten-free diet and are therefore dependent on reliable testing and labeling of gluten-free products. So far, the R5-ELISA is the approved method to detect if food products can be labeled gluten-free. Because the R5-ELISA detects gluten in general, there is a demand for an improved detection method that quantifies specifically CD-epitopes. Therefore, we developed a new method for detection and quantification of CD-epitopes, based on liquid chromatography (LC) coupled to mass spectrometry (MS) in multiple reaction monitoring (MRM) mode. This method enables targeted label free comparative analysis of the gluten proteins present in different wheat varieties and species, and in wheat-based food products. We have tested our method by analyzing several wheat varieties that vary in CD-epitope content, as was shown before using immunoblotting and specific monoclonal antibodies. The results showed that a modern bread wheat variety Toronto contained the highest amounts of CD immunogenic peptides compared with the older bread wheat variety Minaret and the tetraploid wheat variety Dibillik Sinde. Our developed method can detect quantitatively and simultaneously multiple specific CD-epitopes in a high throughput manner.

Profiling of Nutritional and Health-Related Compounds in Oat Varieties.

The use of oats in the human diet has decreased over the past 70 years. This is an unfortunate development from the perspective of human health because oats have a high nutritional value and contain many compounds, including ß-glucan, polyphenols, vitamins, and unsaturated fatty acids that are able to maintain or may even improve consumer's health. In addition, oats fit into a gluten-free diet of celiac disease patients because they lack the T-cell stimulating epitopes from wheat, rye, and barley. We focused on the presence of health-related compounds in oats and how their levels vary among varieties in response to the type of soil. Ten oat varieties were grown in the Netherlands in sandy and clay soil and were analyzed for the presence and concentration of healthy compounds (ß-glucan, fatty acids, vitamin E, and antioxidant activity), avenin composition, total protein and starch content, and agronomical characteristics. Principal component analysis showed that genetic background influenced the levels of all analyzed components. Protein, starch, ß-glucan, and antioxidants were also affected by the type of soil. The obtained results showed that this kind of analysis can be used to profile oat varieties in general and enables the selection of specific varieties with specific compound characteristics.

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing.

BACKGROUND: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. RESULTS: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 'unique deduced protein fragments' of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. CONCLUSIONS: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.

Investigating the transport dynamics of anthocyanins from unprocessed fruit and processed fruit juice from sour cherry (Prunus cerasus L.) across intestinal epithelial cells.

Anthocyanins can contribute to human health through preventing a variety of diseases. The uptake of these compounds from food and the parameters determining uptake efficiency within the human body are still poorly understood. Here we have employed a Caco-2 cell based system to investigate the transport of key antioxidant food components from sour cherry (Prunus cerasus L.) across the intestinal epithelial barrier. Anthocyanins and (-)-epicatechin were supplied in three contrasting matrices: fruit, processed fruit cherry juice, and polyphenolic fractions obtained by solid-phase extraction. Results show that both compound types behave differently. Fruit or juice matrices display comparable transport across the epithelial cell layer. The juice supplements sucrose and citric acid, which are regularly added to processed foods, have a positive effect on stability and transport. Polyphenolic fractions display a lower transport efficiency, relative to that of the fruit or juice, indicating the importance of food matrix components for intestinal absorption of polyphenols.

Tailor-made fructan synthesis in plants: a review.

Fructan, a fructose polymer, is produced by many bacteria and plants. Fructan is used as carbohydrate reserve, and in bacteria also as protective outside layer. Chicory is a commercial fructan producing crop. The disadvantage of this crop is its fructan breakdown before harvest. Studies using genetically modification showed that fructan biosynthesis is difficult to steer in chicory. Alternatives for production of tailor-made fructan, fructan with a desired polymer length and linkage type, are originally non-fructan-accumulating plants expressing introduced fructosyltransferase genes. The usage of bacterial fructosyltransferases hindered plant performance, whereas plant-derived fructan genes can successfully be used for this purpose. The polymer length distribution and the yield are dependent on the origin of the fructan genes and the availability of sucrose in the host. Limitations seen in chicory for the production of tailor-made fructan are lacking in putative new platform crops like sugar beet and sugarcane and rice.

A diet high in resistant starch modulates microbiota composition, SCFA concentrations, and gene expression in pig intestine.

Resistant starch (RS) is highly fermentable by microbiota in the colon, resulting in the production of SCFAs. RS is thought to mediate a large proportion of its health benefits, including increased satiety, through the actions of SCFAs. The aim of this study was to investigate the effects of a diet high in RS on luminal microbiota composition, luminal SCFA concentrations, and the expression of host genes involved in SCFA uptake, SCFA signaling, and satiety regulation in mucosal tissue obtained from small intestine, cecum, and colon. Twenty adult female pigs were either assigned to a digestible starch (DS) diet or a diet high in RS (34%) for a period of 2 wk. After the intervention, luminal content and mucosal scrapings were obtained for detailed molecular analysis. RS was completely degraded in the cecum. In both the cecum and colon, differences in microbiota composition were observed between DS- and RS-fed pigs. In the colon these included the stimulation of the healthy gut-associated butyrate-producing Faecalibacterium prausnitzii, whereas potentially pathogenic members of the Gammaproteobacteria, including Escherichia coli and Pseudomonas spp., were reduced in relative abundance. Cecal and colonic SCFA concentrations were significantly greater in RS-fed pigs, and cecal gene expression of monocarboxylate transporter 1 (SLC16A1) and glucagon (GCG) was induced by RS. In conclusion, our data show that RS modulates microbiota composition, SCFA concentrations, and host gene expression in pig intestine. Combined, our data provide an enhanced understanding of the interaction between diet, microbiota, and host.

Expansion of the gamma-gliadin gene family in Aegilops and Triticum.

BACKGROUND: The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective. RESULTS: We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups. CONCLUSIONS: We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.

Sink filling, inulin metabolizing enzymes and carbohydrate status in field grown chicory (Cichorium intybus L.).

Inulin is a fructose-based polymer that is isolated from chicory (Cichorium intybus L.) taproots. The degree of polymerization (DP) determines its application and hence the value of the crop. The DP is highly dependent on the field conditions and harvest time. Therefore, the present study was carried out with the objective to understand the regulation of inulin metabolism and the process that determines the chain length and inulin yield throughout the whole growing season. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. The following characteristics were determined in taproots: concentrations of glucose, fructose and sucrose, the inulin mean polymer length (mDP), yield, gene expression and activity of enzymes involved in inulin metabolism. Inulin synthesis, catalyzed by sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT), started at the onset of taproot development. Inulin yield as a function of time followed a sigmoid curve reaching a maximum in November. Inulin reached a maximum mDP of about 15 in September, than gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three different phases in the growing season and analyzed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST that starts in June is not regulated by day length and temperature. From mid-September onwards, the mean degree of polymerization (mDP) decreased gradually although inulin yield still increased. The decrease in mDP combined with increased yield results from fructan exohydrolase activity, induced by low temperature, and the back transfer activity of 1-FFT. Overall, this study provides background information on how to improve inulin yield and quality in chicory.