Evaluation of Protein Concentration, Amino Acid Profile and Antinutritional Compounds in Hempseed Meal from Dioecious and Monoecious Varieties

Hempseed meal from three dioecious and three monoecious varieties has been evaluated for content and quality of the protein and for the concentration of antinutritional compounds. Hemp seeds were obtained from plants grown in two experimental fields for two consecutive years (2011-2012). For all the varieties, hempseed meal resulted in a rich source of protein (34% mean content) with an amino acid profile extremely rich in arginine and slightly poor in lysine. Differences between dioecious and monoecious varieties were observed in the content of antinutritional compounds. They were more concentrated in monoecious varieties in comparison with those dioecious. The concentration of phytic acid in hempseed meal deserves attention in both groups, being 63 and 75.4 g·kg-1 of dry matter in dioecious and monocieous varieties, respectively. The results show that, besides the recognized value of hemp oil, also the hempseed cake could find application in animal feed as a substitute of other cakes (soybean, rapeseed). From this point of view, the dioecious varieties showing lower contents of antinutritional compounds with respect to the monoecious varieties would be preferred.

Variation in the Content of Cyanogenic Glycosides in Flaxseed Meal from Twenty-One Varieties

Flaxseed meal from twenty-one varieties has been evaluated for content of cyanogenic glycosides (linamarin, linustatin and neolinustatin). Varieties belonging to three different groups of productive attitude (oil, fiber and intermediate) were considered. The total content of cyanogenic glycosides ranged from 0.74 to 1.60 g?Kg?1 CN?. As expected, linamarin was a minority component accounting for only 2% - 14% of total glycosides. Linustatin resulted statistically lower in the intermediate group than that in the other groups and, in particular, it was the lowest in the Festival variety. Neolinustatin resulted lower in the fiber group although the variety Ventimiglia (belonging to oil group) showed a negligible level of this compound. Neolinustatin was positively correlated to total cyanogenic glycosides (r = 0.709) and inversely correlated to linustatin (r = ?0.326). The variability observed in the content of the various forms of glycosides could be exploited in breeding programs to reduce the content of these toxic compounds in flaxseed meal.

Genomic Fingerprinting of <i>Camelina</i>Species Using cTBP as Molecular Marker

Interest on the genus Camelina has recently increased due to the biofuel, or jet fuel, potential of the oil extracted from seeds of the cultivated species Camelina sativa (L.) Crantz. While our knowledge on C. sativa is constantly augmenting, only few studies have been performed on the other species of the genus, which could be a potentially useful material for the genetic improvement of C. sativa. The genus Camelina consists of 11 species, but only six (C. sativa, C. microcarpa, C. alyssum, C. rumelica, C. hispida and C. laxa) could be retrieved from germplasm banks to carry out genomic fingerprinting studies based on the use of the cTBP molecular marker. Each species, with the exception of C. alyssum that is proposed to be a subspecies of C. sativa, shows a distinct cTBP profile resulting from multiple DNA length polymorphisms present in the second intron of the members of the β-tubulin gene family. In contrast to the high level of genetic diversity detected among the six Camelina species, low variability is observed among and within the accessions of the same species, except for C. hispida that is characterized by an intra-accession high number of cTBP polymorphic bands. In addition, cTBP is also able to identify incorrectly classified accessions and provide information on the ploidy level of each species.

Variability in Glucosinolate Content among <i>Camelina</i>Species

Glucosinolate content in Camelina sativa (L.) Crantz (false flax, gold-of-pleasure) and its relatives C. microcarpa, C. alyssum, C. rumelica and C. hispida was investigated. With the exception of C. hispida in which GSL3 was absent, in all remaining species, three characteristic glucosinolates (GSL1, GSL2 and GSL3) were identified. Camelina genotypes of spring type (C. sativa CAM134, C. alyssum CAM21) showed a typical pattern of glucosenolates with GSL1 > GSL3. GSL1 was present in traces in C. microcarpa and at low levels in C. rumelica and C. alyssum subsp. alyssum. In C. hispida, the GSL1 content was greater than GSL2 and, only in this specie, GSL2 represented less than 50% of total glucosinolates. These differences in the glucosinolate pattern among Camelina species could be exploited to reduce the total content of glucosinolates in C. sativa.

A Simplified Approach for Olive (<i>Olea europaea</i>L.) Genotyping and Cultivars Traceability

The Tubulin Based Polymorphism (TBP) method was used to genotype olive cultivars of different origin and to produce short-size cultivar-specific molecular probes. Both the first and the second intron of the members of the olive β-tubulin gene family were exploited as sources of DNA polymorphism. Compared with the data obtained with the use of a set of 11 SSR markers selected from an Olea europea L. database, TBP is shown to provide similar, if not better, information about the polymorphic content of the olive genomes, releasing, at the same time, a simple and discriminatory DNA barcode specific for any of the analyzed cultivars. Such a barcode is the source for the preparation of variety specific molecular probes.

Antinutritive Compounds in Twelve <i>Camelina sativa </i>Genotypes

Camelina sativa is an oilseed crop becoming important in North America and Europe for biodiesel production. The use of Camelina flours in animal diet may be limited by antinutritive compounds. The content of glucosinolates, phytic acid, sinapine and condensed tannins was evaluated in twelve accessions of Camelina sativa. All compounds showed significant differences among genotypes. Only the concentration of glucosinolates in the flour deserves attention, while the content of phytic acid, sinapine and condensed tannins are to acceptable levels. Camelina showed the presence of three different glucosinolates (GSL1, GSL2 and GSL3) in the flour, with differences among genotypes regarding the relative abundance of each glucosinolate. The content of glucosinolates is inversely correlated with that of sinapine. The glucosinolate content in Camelina flour has to be reduced to increase the use of this flour in animal diet, but avoiding altering the sinapine content.

Glucosinolates and Sinapine in Camelina Meal

Forty seven accessions of camelina (Camelina sativa L. Crantz) were analyzed for glucosinolates (GSLs) and sinapine in defatted meal. These antinutritional compounds are undesirable in camelina meal for use in animal feeding and therefore we show their variability to identify the best varieties for future breeding programs. Total GSLs ranged from 19.6 to 40.3 mmol Kg-1 dry weight (DW) with an average of 30.3 mmol kg-1 DW. Great variability has also been observed in the levels of individual GSLs (GSL1, GSL2 and GSL3), so that the content of GSL1 and GSL3 were not correlated to each other in the accessions of camelina. Five out of six winter forms of camelina showed low content of GSLs. Sinapine ranged from 1.09 to 4.75 g Kg-1 DW with an average of 2.57 g kg-1 DW. The sinapine content was not correlated with that of GSLs. The use of camelina meal is only limited by the presence of GSLs while sinapine content can be ignored in camelina varieties.

<i>In Silico</i>Exploration of <i>Cannabis sativa</i>L. Genome for Simple Sequence Repeats (SSRs)

Simple sequence repeat (SSR) or microsatellite markers, are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker assisted selection, and breeding. Recent developments in sequencing technologies and bioinformatics analyses provide new opportunity to produce a high number of less costly SSRs. Here, we used for the first time a wholegenome shotgun sequencing of the nuclear genome and transcriptome of hemp to develop microsatellite markers for C. sativa L. (hemp). Hemp is an ancient crop that is widely cultivated as a source of fiber, seeds and medicine. The analysis using the MISA program revealed a total of 407,491 SSRs (from mono-nucleotide to deca-nucleotide) in the hemp genome and 15,655 SSRs in the transcriptome. Analysis of the frequency and distribution of SSRs showed that the mono-nucleotide repeats were the most abundant (55.4%) in the genome whereas the tri-nucleotide motifs (30.4%) resulted highly predominant in the transcriptome. Poly A/T was predominant over poly G/C in both genome and transcriptome sequences. Among the tri-nucleotide repeats AAG/CTT (34.5%) resulted the most abundant in the transcriptome. Repeats larger than tri-nucleotide were also observed in the hemp genome and transcriptome. Dinucleotide and tri-nucleotide repeat expansion of 8605 and 1401 times iteration were observed however, other SSR expansion more than 387 times repetition was not found. Primers were designed for amplification of few long microsatellite sequences which could be used to identify polymorphism and to study genetic diversity among hemp cultivars.

Conservation of endangered animals: From biotechnologies to digital preservation

In the recent years, the number of endangered animals, both referred to livestock and wild species, has grown enormously. The “livestock” term refers to animals domesticated for producing commodities for man such as food, fiber and draught. Livestock biodiversity is integral to our culture, history, environment, and economy. Thousands of livestock breeds have evolved over time to suit particular environments and farming systems. Conservation and analyses of these genetic resources rely on demographic characterization and correct breeding schemes. In addition, molecular genetic studies allow to identify and monitor the genetic diversity within and across breeds and to reconstruct their evolution history. The conservation of livestock variability is also a crucial element in order to preserve and valorise specific nutritional and nutraceutical properties of animal products. Efficient ex situ and in situ conservation strategies, as well as the creation of bio-banks and specific biotechnological and bioinformatics tools for genetic analyses and digital preservation, are obligatory requirements in order to implement an appropriate action for the conservation of animal biodiversity. The main issues concerning different species are summarised, with particular reference to the livestock biodiversity still existing. Some examples of ex situ conservation strategies, which mainly refer to cryoconservation of semen, ova, embryos or tissues, developed in Italy, are presented, and the different actions in defense of Animal Genetic Resources (AnGR) developed within the European Community are illustrated. Interestingly, the same strategies for biological and digital analyses and preservation of livestock biodiversity can be exported to wild endangered animals in order to plan a correct conservation and repopulation of the species. Furthermore, the European Union has set up the guidelines to safeguard the biodiversity and to combat the extinction of animal species, and has made the protection of biodiversity and ecosystems one of the main objectives of the Sixth Environment Action Programme.

Molecular Mechanism of the Specificity of Protein Import into Chloroplasts and Mitochondria in Plant Cells

Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.

Emerging Role of the Ubiquitin Proteasome System in the Control of Shoot Apical Meristem Function

The shoot apical meristem （SAM） is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant＇s life. A plethora of different families of transcription factors （TFs） play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system （UPS）. However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors.

Protein Domains Involved in Assembly in the Endoplasmic Reticulum Promote Vacuolar Delivery when Fused to Secretory GFP, Indicating a Protein Quality Control Pathway for Degradation in the Plant Vacuole

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum （ER）. Through interactions with chaperones such as the binding protein （BiP） and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein γ-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 Protein Forms an SCF Ubiquitin Ligase and Regulates Hypocotyl Elongation

The regulation of protein turnover by the ubiquitin proteasome system （UPS） is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome （CSN）, regulates ＇cullin-ring＇ E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 （COP9 INTERACTING F-BOX KELCH 1）. We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteoly- sis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFKl-depleted seedlings, while complete loss of CSN activity sup- presses the Iong-hypocotyl phenotype of CFKl-overexpressing seedlings. We propose that CFK1 （and its regulation by CSN） is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Bidirectional Electron Transfer in the Reaction Centre of Photosystem I

In the past decade light-induced electron transfer reactions in photosystem I have been the subject of intensive investigations that have led to the elucidation of some unique characteristics, the most striking of which is the existence of two parallel, functional, redox active cofactors chains. This process is generally referred to as bidirectional electron transfer. Here we present a review of the principal evidences that have led to the uncovering of bidirectionality in the reaction centre of photosystem I. A special focus is dedicated to the results obtained combining time-resolved spectroscopic techniques, either difference absorption or electron paramagnetic resonance, with molecular genetics, which allows, through modification of the binding of redox active cofactors with the reaction centre subunits, an effect on their physical-chemical properties.