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Milk thistle, Silybum marianum (L.) Gaertn. (Asteraceae), is an economically important medicinal plant utilized for silymarin production. Moreover, the species has been positively evaluated for vegetable oil and biomass production. Despite these positive characteristics, milk thistle is still marked by traits that are typical of undomesticated species (most importantly natural fruit dispersal at maturity) and requires further genetic improvement for its complete exploitation. This manuscript summarizes all the information collected through time about a collection of nine milk thistle wild and mutant lines and it discusses the possible further utilization of these genotypes. The accessions are characterized by interesting traits related to: fruit silymarin composition (S. marianum chemotype A and B), fruit fatty acid composition (high oleic and high stearic acid lines), fruit condensed tannins content, vegetative biomass composition (modification of xylans or lignin content), vegetative biomass structure (dwarf and tall lines), modifications of leaf variegation (hypervareigated line) and different types of fruit shatter resistance at maturity. All the lines underwent subsequent generations of selfing and are stable for all the described traits. The accessions will be made available at the Genebank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Gatersleben) and may prove to be a useful genetic material for the improvement of qualitative fruit traits (silymarin quality, fatty acid composition) and for the further development of shatter-resistant S. marianum genotypes towards the complete domestication of this promising species.

Milk thistle [Silybum marianum (L.) Gaertn.] is used as a medicinal plant for the production of silymarin. Other than silymarin, the fruit also contains valuable compounds such as oil and protein, and the lignocellulosic biomass of this species has been positively evaluated for biogas, syngas, and energy production. Despite these positive characteristics, the plant is still marked by traits that are typical of undomesticated species. Specifically, fruit shattering at maturity severely hampers its possible utilization as a crop. Therefore, for the first time, an ethyl methanesulfonate mutagenesis program was implemented to identify milk thistle lines with reduced fruit shattering, and its effectiveness was evaluated through genome-wide analysis. The candidate nonshattering lines were multiplied up to M₆ for the validation of the favorable traits. The lignocellulosic biomass of the selected shatter resistant lines was analyzed according to compositional analysis. The natural milk thistle fruit dispersal mechanism was shown to be modulated by changing air relative humidity. Three vigorous, stable, shatter-resistant lines were identified. This is the first report that describes milk thistle nonshattering lines. At this stage in the three lines, the morphological description and the compositional analysis of the lignocellulosic biomass indicated that shatter resistance was concomitant to changes in vegetative biomass structure and composition. This observation is compatible with the described natural fruit dispersal mechanism, and it is of interest for the design of new milk thistle crop ideotypes for the complete domestication of the species.

Silybum marianum (L.) Gaertn. is an important medicinal plant belonging to Mediterranean flora. The medicinal properties of the species are mainly due to silymarin, a combination of different flavonolignans contained in the fruit. As for silymarin, so far a wide variability of possible S. marianum chemotypes has been described. In the present study the flavonolignan profile of 40 different S. marianum wild accessions was analysed at both population and single plant level, further extending the analysis to progenies derived from crosses between parental lines with different chemotypes. The results of this work indicate that S. marianum wild populations can be composed either of individuals with the same chemotype, or heterogeneous mixtures of individuals characterized by different chemotypes. Only three chemotypes (A, B and C) have been identified among Italian wild populations. Based on data collected we furthermore propose that chemotype C is the result of the hybridization between A and B chemotypes. If assessed at single plant level, chemotypes are extremely stable therefore evidencing a strong genetic control of silymarin biosynthetic pathway. Chemotypes A and B are present in all the analysed regions and no clear correlation between chemotypes and geographic features has been found. In conclusion, this work provides a general procedure for the characterization of different and stable chemotypes, for a deeper understanding of silymarin biosynthetic pathway, and in order to implement S. marianum breeding programmes aiming to improve silymarin quality.

Quantitative reverse transcription PCR is a sensitive technique for evaluating transcriptional profiles in different experimental datasets. To obtain a reliable quantification of the transcripts level, data normalization with stable reference genes is required. Stable reference genes are identified after analysis of their transcripts profile in every new experiment and species of interest. In Silybum marianum, a widely cultivated officinal plant, only few gene expression studies exist, and reference genes for RT-qPCR studies in the diverse plant tissues have never been investigated before. In this work, the expression stability of 10 candidate reference genes was evaluated in leaves, roots, stems and fruits of S. marianum grown under physiological environmental condition. The stability values for each candidate reference gene were calculated by four canonical statistical algorithms GeNorm, NormFinder, Bestkeeper and ΔCt method in different subsets of samples, then they were ranked with RefFinder from the most to the least suitable for normalization. Best combinations of reference genes are finally proposed for different experimental data sets, including all tissues, vegetative, and reproductive tissues separately. Three target genes putatively involved in important biosynthetic pathway leading to key metabolites in the fruits of milk thistle, such as silymarin and fatty acids, were analyzed with the chosen panels of reference genes, in comparison to the ones used in previous papers. To the best of our knowledge, this is the first report on a reliable and systematic identification and validation of the reference genes for RT-qPCR normalization to study gene expression in S. marianum.

Silybum marianum (L.) Gaertn (Asteraceae) is a valuable medicinal plant utilized for silymarin production. However, only fragmentary and contradictory information about silymarin localization within S. marianum fruit are available. In this work, a twofold research approach was adopted in order to investigate the distribution and quantification of silymarin and of other phenolic compounds within the different fruit regions (pericarp, seed integument, cotyledon). Two S. marianum wild accessions with contrasting silymarin chemotype (A and B) and a mutant line (C) with an altered fruit colour were analysed. Fruits of Cynara cardunculus were studied as a reference. Firstly, the fruit morpho-anatomy was reviewed by means of light microscopy digital imaging and, secondly, a comprehensive histolocalization of the different classes of polyphenols within the fruit was carried out. The experimental evidences confirmed that silymarin, and its precursor taxifolin, are only accumulated in the seed integuments. The dark colour of fully-ripened fruits is due to the accumulation of condensed tannins in the pericarp subepidermal cell layer. On the contrary, the studied mutant line shows reduced condensed tannin content that probably result from impairment at the level of flavonoid biosynthetic pathway. Condensed tannins content is comparatively low in S. marianum fruits and very low in the identified mutant line. This could represent an advantage for the possible employment of S. marianum fruits and of silymarin extraction by-products in the feed and food sector.

Silybum marianum (milk thistle) is currently utilised as a medicinal plant which provides raw material for the production of silymarin. Silymarin is composed of a group of flavonolignans that are well known and studied for their medicinal properties. However, the biomass yield potential and the chemical composition of its biomass suggest opportunities for much broader utilisation of S. marianum. This paper reviews the available literature about S. marianum biomass productivity and composition and about properties of products and byproducts of silymarin extraction. Alternative uses of whole plant biomass include fodder, bioenergy production and phytoremediation. Byproducts of silymarin extraction such as oil and flour have possible applications in food, feed and cosmetics. Moreover, the review explores potential alternative applications of silymarin. We conclude that a pivotal issue for further utilisation of S. marianum is the development of improved cultivars suited for the different possible utilisations.

Silymarin is the phytochemical with medicinal properties extracted from Silybum marianum (L.) Gaertn. fruits. Yet, little information is available about silymarin biosynthesis. Moreover, the generally accepted pathway, formulated thus far, is not in agreement with actual experimental measurements on flavonolignan contents. The present work analyses flavonolignan and taxifolin content in 201 S. marianum samples taking into consideration a wide phenotypic variability. Two stable chemotypes were identified: one characterized by both high silychristin and silybin content (chemotype A) and another by a high silydianin content (chemotype B). Through the correlation analysis of samples divided according to chemotype, it was possible to construct a simplified silymarin biosynthetic pathway that is sufficiently versatile in explaining experimental results responding to the actually unresolved questions about this process. The proposed pathway highlights that three separate and equally sized metabolite pools exist, namely: diastereoisomers A (silybin A plus isosilybin A), diastereoisomers B (silybin B plus isosilybin B) and silychristin. In both A and B diastereoisomers pools, isosilybin A and isosilybin B always represent a given amount of the metabolite flux through the specific metabolite pool suggesting the possible involvement of dirigent protein-like enzymes. We suggest that chemotype B possesses a complete silymarin biosynthetic pathway in which silydianin biosynthesis is enzymatically controlled. On the contrary, chemotype A is probably a natural mutant unable to biosynthesize silydianin. The present simplified pathway for silymarin biosynthesis will constitute an important tool for the further understanding of the reactions that drive flavonolignan biosynthesis in S. marianum .

Silybum marianum is a medicinal plant cultivated for silymarin production in central Europe and Asia. It is also considered a weed of both arable lands and pastures. Although S. marianum still exhibits traits that are typical of wild species; both its fruit and biomass productivity are remarkable. Therefore, the species is gaining interest as a potential novel commercial crop for multiple uses (i.e. medicinal plant, bioenergy, and vegetable oil production). Despite the intriguing characteristics of this plant and its economic importance, a detailed description of the phenological growth stages of S. marianum has never been developed. A precise and univocal description of the growth stages of this species will offer a useful tool for several agronomic and research activities, weed management practices and for further genetic improvement programmes. In the present manuscript, the description of the phenological development of S. marianum is proposed utilising the extended BBCH (Biologische Bundesantalt, Bundessortenamt and Chemische Industrie) scale. The phenological growth stages are described utilising both the 2- and 3-digit BBCH coding system. Pictures of the most representative stages supplement the manuscript.

BACKGROUND: Camelina sativa (CS) is an oilseed crop used for biofuel production. By-products from oil extraction are high in protein and can be used in ruminant rations; more information about their nutritive value is required also considering the antinutrional factor content of the by-products. The aim of this study was to evaluate the nutritive value of CS meal genotypes in comparison with canola. RESULTS: TenCSgenotypesandonecanolacultivarwereevaluated.Mealswereobtainedfromseedsaftersolventoilextraction. CSaveragecrudeprotein(CP)content(gkg −1 drymatter)was457.Numericaldifferencesinlysineandsulfuraminoacidcontent were observed among CS genotypes. Glucosinolate (mmolkg −1 ) content was higher for CS (23.1) than canola (7.2). Sinapine content (gkg −1 ) was lower for CS (2.79) than for canola (4.32). Differences were observed among CS genotypes for rumen undegraded protein (RUP). Average RUP (gkg −1 CP) was 316 for CS and 275 for canola. CONCLUSIONS: CS meal has potential for use in ruminant rations as a high-quality protein source.Invivo studies are needed to compare CS with other protein sources used in cattle rations. Implementation of breeding programs for improved meal quality is recommend. c � 2013 Society of Chemical Industry

Camelina sativa is an alternative oilseed crop that is shown to be adaptable to marginal areas and suitable for low-input cultivation. Moreover, the species shows a good productivity and interesting agronomic features. As a result of particular characteristics, the oil produced by this species has been shown to be suitable for many food and non-food applications. Despite the interesting characteristics of this plant, a detailed description of the phenological growth stages of C. sativa has never been performed. A precise and univocal description of the different growth stages of this crop will provide a useful tool for several agronomic and research activities. In this article, the description of the phenological development of C. sativa is proposed utilising the extended BBCH (Biologische Bundesantalt, Bundessortenamt and Chemische Industrie) scale (Meier et al., 2009). The different phenological growth stages are described, utilising both the two- and three-digit BBCH coding system. This article is supplemented by drawings of the most representative stages.

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.

Oleander (Nerium oleander L.) is an evergreen shrub of great ornamental interest which, in recent times, has been increasingly used as a flowering pot plant. Plants grown in pots undergo more frequent water stress conditions than those grown in the soil, due to the limited volume of substrate available for the roots. Oleander is a species adaptable to dry conditions and able to survive long periods of drought. It is well known that under water stress conditions all plants reduce photosynthetic activity, resulting in reduced plant growth. In case of severe water stress, leaves undergo strong wilting and senescence resulting in the loss of ornamental value. In the present work, a study was conducted to evaluate the ecophysiological response to water stress in four oleander cultivars previously recognised (on the basis of traits such as size, habit, earliness, abundance and duration of flowering, aptitude for cutting propagation and rapidity of growth) as suitable for pot plant production. Our data confirm the high drought tolerance of oleander. In the studied cultivars, plants submitted to water stress showed only minor variations in leaf gas exchange parameters [transpiration (E), stomatal conductance (gs) and CO2 net assimilation (A)] for at least 10 days without a change in leaf water content [assessed as relative water content (RWC)] for 22 days from the beginning of the stress treatment. During this period, non-irrigated plants maintained the same water status as control plants and were visually undistinguishable from them. Moreover, plants survived without water supply for one month. Following the supply of water again, they were able to restore RWC, gas exchange parameters and instantaneous water use efficiency [A/E ratio (WUEinst)] to the values of control plants. Furthermore, if at the end of the stress period plants appeared withered and were pale green in colour, they regained their normal appearance after they were irrigated again. Although the four studied cultivars showed some minor differences in leaf gas exchange parameters and in the manner in which the latter parameters changed after irrigation was stopped, the response to water stress was essentially the same. Therefore, as far as drought tolerance is concerned, all these cultivars have a good aptitude for use as flowering pot plants.

Summary Accumulation of soluble carbohydrates during dehydration stress is thought to be a very important mechanism for the acquisition of desiccation tolerance. Despite the proposed importance of soluble carbohydrate accumulation (especially sucrose), nothing is known about the cellular localization of carbohydrates in desiccation-tolerant plants. The present study proposes a novel and selective method for the in situ localization of sucrose and glucose in the desiccation-tolerant plant Sporobolus stapfianus . The detection of sucrose and glucose is based on a series of coupled enzymatic reactions leading to the formation of NADH. Iodonitrotetrazolium (INT) reacts with NADH, thereby providing the red-colored insoluble INT-formazan. Stained tissue sections were immediately visualized using light microscopy. Localization of the respective sugars was site specific. Sucrose was visualized in all leaf cell types during dehydration: vascular bundles, bundle sheath cells, mesophyll cells and epidermal cells. Similarly, glucose was shown to be localized in the same leaf compartments as reported for sucrose. This is the first report that describes sucrose localization in dehydrating leaf tissues of a “resurrection” plant. We conclude that, during dehydration stress, sucrose accumulates in all viable tissues; these results are in agreement with the previously proposed theories about its function as a cellular protectant.

Milk thistle [Silybum marianum (L.) Gaertn.] is an officinal crop species native to the Mediterranean. The species is gaining increasing interest as a multipurpose crop, but lack of information exists about the natural phenotypic variability available in the species for the traits of interest. In the present work, the fruit morphological and chemical traits of a collection of 26 milk thistle accessions have been analyzed. The results show that the most variable traits are related to single flavonolignan content. When the most abundant fatty acids present in the fruit are considered, oleic acid appears to have the higher variability between accessions. Correlations between variables highlight strong relationships between the different flavonolignans and a strong positive correlation between isosilybin A and the total silymarin content, suggesting a central role of this minor flavonolignan in silymarin biosynthetic pathway. The clustering procedure displays considerable diversity between the accessions that originate from localities where milk thistle is described as a native species.

The possible role of photorespiration as a general stress protection mechanism, and in C(4) plant metabolism, is controversial. In particular, the potential involvement of photorespiration in the acquisition of desiccation tolerance in 'resurrection' plants is unknown. An investigation was carried out into whether photorespiration is present in leaves of the C(4) resurrection plant Sporobolus stapfianus Gandoger (Poaceae) and whether it functions as a mechanism of stress resistance in the desiccation-tolerant younger leaves (YL) of this plant. It is shown that the enzymes involved in the photorespiratory pathway maintain their activity until 88% relative water content (RWC) in both YL and desiccation-sensitive older leaves (OL). In subsequent stages of dehydration stress, the enzymatic activity declined similarly in both YL and OL. The content of the phorespiratory metabolite, serine, and ethanolamine, a direct product of serine decarboxylation, is higher in the early stages of dehydration (88% RWC) in OL, suggesting a transiently enhanced photorespiratory activity in these leaves. This was confirmed by simultaneous gas exchange and fluorescence measurements, showing suppression of the electron transport rate in OL exposed to non-photorespiratory conditions (2% O(2)) at 85% RWC. It is concluded that a higher photorespiratory electron transport occurs in desiccation-sensitive OL, and it is therefore proposed that the capacity to scavenge excess electrons through photorespiration does not contribute to protect leaves of the desiccation-tolerant YL of S. stapfianus during the stress.

The present study analyses changes in nitrogen compounds, amino acid composition, and glutamate metabolism in the resurrection plant Sporobolus stapfianus during dehydration stress. Results showed that older leaves (OL) were desiccation-sensitive whereas younger leaves (YL) were desiccation-tolerant. OL lost their soluble protein more rapidly, and to a larger extent than YL. Enzymes of primary nitrogen assimilation were affected by desiccation and the decrease in the glutamine synthetase (GS, EC 6.3.1.2) and ferredoxin-dependent GOGAT (Fd-GOGAT, EC 1.4.7.1) activities was higher in OL than in YL, thus suggesting higher sensibility to dehydration. Moreover, YL showed higher total GS enzyme activity at the end of the dehydration stress and was shown to maintain high chloroplastic GS protein content during the entire stress period. Free amino acid content increased in both YL and OL between 88% and 6% relative water content. Interestingly, OL and YL did not accumulate the same amino acids. OL accumulated large amounts of proline and gamma-aminobutyrate whereas YL preferentially accumulated asparagine and arginine. It is concluded (i) that modifications in the nitrogen and amino acid metabolism during dehydration stress were different depending on leaf development and (ii) that proline and gamma-aminobutyrate accumulation in S. stapfianus leaves were not essential for the acquisition of desiccation tolerance. On the contrary, the accumulation of large amounts of asparagine and arginine in the YL during dehydration could be important and serve as essential nitrogen and carbon reservoirs useful during rehydration. In this context, the role of GS for asparagine accumulation in YL is discussed.

Both sucrose and amino acids accumulate in desiccation-tolerant leaf material of the C(4) resurrection plant, Sporobolus stapfianus Gandoger (Poaceae). The present investigation was aimed at examining sucrose phosphate synthase (SPS) activity and various metabolic checkpoints involved in the co-ordination of carbon partitioning between these competing pathways during dehydration. In the initial phase of dehydration, photosynthesis and starch content declined to immeasurable levels, whilst significant increases in hexose sugars, sucrose, and amino acids were associated with concomitant significant increases in SPS and pyruvate kinase (PK) activities, and maximal activity levels of phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and NADH-dependent glutamate synthase (NADH-GOGAT). The next phase of dehydration was characterized by changes in metabolism coinciding with net hexose sugar phosphorylation. This phase was characterized by a further significant increase in sucrose accumulation, with increased rates of net sucrose accumulation and maximum rates of SPS activity measured under both saturating and limiting (inhibitory) conditions. SPS protein was also increased. The stronger competitive edge of SPS for carbon entering glycolysis during hexose phosphorylation was also demonstrated by the further decrease in respiration and the simultaneous, significant decline in both PEPCase and PK activities. A decreased anabolic demand for 2-oxoglutarate (2OG), which remained constant, was shown by the co-ordinated decrease in GOGAT. It is proposed that the further increase in amino acids in this phase of dehydration may be in part attributable to the breakdown of insoluble proteins.

The agronomic performance of Camelina sativa (L.) Crantz (camelina or false flax) sown in autumn and spring over two consecutive years was tested in northern Italy. Seven C. sativa genotypes were tested and compared with rapeseed (Brassica napus L.). The main phenological stages and biometric traits were recorded, along with seed yield. In general, camelina showed a seed yield similar to that of rapeseed cultivated in the same locality and in the last decade in Italy. On average, the grain yields of camelina and rapeseed grown in the same location and conditions were ~1340 and 1625 kg ha–1, respectively. The agronomic performance of camelina varied, with climatic events having a greater effect than sowing season or genotypes. Among the investigated genotypes, however, C. sativa accession CAM 40 was the most adaptable to unfavourable environmental conditions and CAM 172 to favourable conditions. With regard to the two sowing seasons, autumn planting allowed for better performance than spring planting during the second cultivation year. The phenotypic plasticity of camelina was estimated for the first time in the present work. Branching capability was the most plastic trait under favourable yielding conditions. Among the tested genotypes, CAM 40 showed limited yield plasticity and CAM 172 demonstrated high plasticity for the same trait, offering a greater potential for future genetic improvement.