Cloning and characterization of a glucosyltransferase from Crocus sativus stigmas involved in flavonoid glucosylation

Background  

Flavonol glucosides constitute the second group of secondary metabolites that accumulate in Crocus sativus stigmas. To date there are no reports of functionally characterized flavonoid glucosyltransferases in C. sativus, despite the importance of these compounds as antioxidant agents. Moreover, their bitter taste makes them excellent candidates for consideration as potential organoleptic agents of saffron spice, the dry stigmas of C. sativus.     

New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC–DAD and the volatile products by GC/MS. β-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9′,10′) bonds and generating β-ionone, although β-cyclocitral resulting from a 7,8 (7′,8′) cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.     

Callus Induction from Explants of Crocus sativus

Saffron calli were induced from ovary explants on Murashige-Skoog (MS) medium supplemented with beyzyladenine (BA) and naphthalene acetic acid (NAA) as growth factors. MS medium with 5 mg l^?1 BA and 10 mg l^?1 NAA was selected for calli induction and undifferentiated calli growth, while MS medium with 1 mg l^?1 BA and 1 mg l^?1 NAA was the most appropriate for stigma differentiation. On this medium, stigma-like structures measuring 0.5–1.5 cm were obtained. Initially they were colourless, but yellow pigmentation, due to the presence of crocin, progressively increased with calli growth. Extracts of stigma-like structures were analysed by HPLC and the presence of saffron secondary metabolites was demonstrated. In addition, calli also showed yellow pigmentation.     

Plant growth promoting bacteria from Crocus sativus rhizosphere

Present study deals with the isolation of rhizobacteria and selection of plant growth promoting bacteria from Crocus sativus (Saffron) rhizosphere during its flowering period (October–November). Bacterial load was compared between rhizosphere and bulk soil by counting CFU/gm of roots and soil respectively, and was found to be ~40 times more in rhizosphere. In total 100 bacterial isolates were selected randomly from rhizosphere and bulk soil (50 each) and screened for in-vitro and in vivo plant growth promoting properties. The randomly isolated bacteria were identified by microscopy, biochemical tests and sequence homology of V1–V3 region of 16S rRNA gene. Polyphasic identification categorized Saffron rhizobacteria and bulk soil bacteria into sixteen different bacterial species with Bacillus aryabhattai (WRF5-rhizosphere; WBF3, WBF4A and WBF4B-bulk soil) common to both rhizosphere as well as bulk soil. Pseudomonas sp. in rhizosphere and Bacillus and Brevibacterium sp. in the bulk soil were the predominant genera respectively. The isolated rhizobacteria were screened for plant growth promotion activity like phosphate solubilization, siderophore and indole acetic acid production. 50 % produced siderophore and 33 % were able to solubilize phosphate whereas all the rhizobacterial isolates produced indole acetic acid. The six potential PGPR showing in vitro activities were used in pot trial to check their efficacy in vivo. These bacteria consortia demonstrated in vivo PGP activity and can be used as PGPR in Saffron as biofertilizers.This is the first report on the isolation of rhizobacteria from the Saffron rhizosphere, screening for plant growth promoting bacteria and their effect on the growth of Saffron plant.     

番红花侧芽中的新蒽醌化合物

从番红花（ＣｒｏｃｕｓｓａｔｉｖｕｓＬ．）侧芽的氯仿萃取部分分离到４个蒽醌类化合物（Ⅰ、Ⅱ、Ⅲ、Ⅳ）：Ⅰ和Ⅱ分别为大黄素和２羟基大黄素;Ⅲ和Ⅳ为两个新的蒽醌类化合物,分别为１甲基３甲氧基８羟基蒽醌２羧酸和１甲基３甲氧基６,８二羟基蒽醌２羧酸,通过光谱和化学方法确定了它们的化学结构。     

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The amplification of the seven Crocus species DNAs with twenty-one primers provided 217 repeatable and interpretable fragments, which were scored for presence/absence and employed for a cluster analysis. Results indicated that C. sativus is very closely related to C. cartwrightianus and also similar to C. thomasii. This result, concurring with part of the previous evidence, would rule out the hypothesis of close relationships between C. sativus and C. pallasii.     

EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.     

In vitro cormlet development in Crocus sativus

An improved protocol for generation of viable cormlets from tissue culture derived shoots of saffron has been developed. Multiple shoots were generated from apical buds, small corms and in vitro developed single shoots. Bunches of two to three shoots when cultured on half strength Murashige and Skoog (MS) medium containing 3 mg dm⁻³ benzyladenine (BA) and 80 g dm⁻³ sucrose developed 1.89 cormlets per shoot bunch with an average fresh mass of 1.18 g. It took nine months from culture of apical buds to the harvest of cormlets but under field conditions 22 months. Sucrose appeared to be essential for cormlet induction as no cormlets were developed in the medium devoid of sucrose and only 0.29 per shoot in medium containing mannitol. In vitro derived cormlets sprouted from apical and axillary buds on MS medium containing 12 mg dm⁻³ BA, 3 mg dm⁻³ indolebutyric acid and 30 g dm⁻³ sucrose. Daughter cormlet formation from in vitro derived cormlets was also observed.     

Crocus sativus and its allies (Iridaceae)

Crocus sativus is an autumn-flowering species, unknown as a wild plant but long-cultivated for its scarlet style branches which yield Saffron, the dye and flavouring agent. There are several naturally-occurring related species from southern Europe and south western Asia which form a natural group within the genus.C. niveus from Greece is similar to these morphologically and is included here but is less closely related. The characters of the group are defined, a key to the taxa is provided and their relationships discussed.     

Heme-containing dioxygenases involved in tryptophan oxidation

Heme iron is often used in biology for activation of oxygen. The mechanisms of oxygen activation by heme-containing monooxygenases (the cytochrome P450s) are well known, and involve formation of a Compound I species, but information on the heme-containing dioxygenase enzymes involved in tryptophan oxidation lags far behind. In this review, we gather together information emerging recently from structural, mechanistic, spectroscopic, and computational approaches on the heme dioxygenase enzymes involved in tryptophan oxidation. We explore the subtleties that differentiate various heme enzymes from each other, and use this to piece together a developing picture for oxygen activation in this particular class of heme-containing dioxygenases.     

Characterization of the platelet aggregation inducer and inhibitor isolated from Crocus sativus

Bulbs of Crocus sativus variety Cartwrightianus were found to contain both a platelet aggregation inducer and inhibitor. The aggregating factor has a Mr of 42 kDa estimated by a Sephadex G75 column and SDS-polyacrylamide slab gel electrophoresis. It was found to lack enzymatic activity such as proteinase, esterase and acid or alkaline phosphatase. The inhibitory factor was also purified to homogeneity by different chromatographic techniques and shows a Mr of 27 kDa as it was estimated by Biogel P30 column and SDS-polyacrylamide slab gel electrophoresis. It was found to possess strong proteinase activity.     

藏红花花瓣和雄蕊挥发油化学成分GC-MS分析及比较

采用水蒸气蒸馏法从藏红花花瓣和雄蕊中提取挥发油,用GC-MS技术结合计算机检索对其二者化学成分进行分离和鉴定,用色谱峰面积归一化法计算各组分的相对含量.花瓣中共鉴定出16种化合物,主要成分为正二十六烷(11.60％)、正十五烷(11.31％)、棕榈酸甲酯(10.82％)、油酸甲酯(10.35％)、2,4-二叔丁基苯酚(9.63％)、亚油酸甲酯(7.18％)、藏红花醛(5.66％);雄蕊中共鉴定出20种化合物,主要成分为油酸甲酯(30.83％)、亚油酸甲酯(24.12％)、环已醇(16.80％)、硬脂酸甲酯(12.88％)、棕榈酸甲酯(8.97％)、花生酸甲酯(1.18％)、苯并噻唑(1.01％).     

Cloning and Characterization of FLOWERING LOCUS T-Like Genes from the Perennial Geophyte Saffron Crocus (Crocus sativus)

The transition to flowering is one of the most important developmental decisions made by plants. At the molecular level, many genes coordinate this transition. Among these, genes encoding for phosphatidylethanolamine-binding proteins (PEBPs) play important roles in regulating flower time and the fate of inflorescence meristem. To investigate the role of PEBPs in an industrially important crop cultivated for its nutritional and medicinal properties, the monocotyledonous species Crocus sativus L., we have isolated three FLOWERING LOCUS T (FT)-like genes designated as CsatFT1-like, CsatFT2-like, and CsatFT3-like. The isolated genes maintain the exon/intron organization of FT-like genes and encode proteins similar to the members of the PEBP family. Phylogenetic and amino acid analysis at critical positions confirmed that the isolated sequence belongs to the FT clade of the PEBP family phylogeny distinctly from the TERMINAL FLOWER 1 (TFL1) and MOTHER OF FT AND TFL1 clades. Expression analysis indicated differences in the expression of the three FT-like genes in different organs and different expressions during the day–night diurnal clock. Additionally, analysis of isolated promoter sequences using computational methods reveals the preservation of common binding motifs in FT-like promoters from other species, thus suggesting their importance among plant species.     

In Vitro Regeneration of Style-stigma-like Structure from Stamens of Crocus sativus

Style-stigma-like structures were regenerated from stamens of Crocus sativus L. The age of the stamen explant has an obvious effect on the induction rate. Auxin NAA has larger effect on the induction of filament style-stigma-like structure. Auxin NAA of higher concentration can lead to higher induction rate. Temperature and light have different effects on the induction of style-stigma-like structure from anther's filament of C． sativus with exogenous hormones at different levels. Ultraviolet tests show that style-stigma-like structure from anther's filament of C． sativus contains crocin, safranal and picrocrocin, contents of which are obviously more than those contained in the style-stigma-like from style. Floral reversion was observed in the induction of style-stigma-like structure from petals, ovaries and styles.     

番红花雄蕊柱头状物的离体再生

以番红花（Crocus sativus L.)雄蕊为材料诱导培养出花柱柱状结构,诱导率可达30％,起源于花丝基部。影响雄蕊柱头状物诱导的主要因素为外植体的发育期和生长素NAA的使用浓度。幼嫩浅黄色雄蕊适于诱导柱头状物。温度和光照在不同激素水平下对雄蕊柱头状物诱导的影响不同。紫外检测表明,由雄蕊诱导出的柱头状物含有番红花甙、番红花醛和番红花苦甙。其含量明显高于由药柱诱导出的柱头状结构。在诱导花魔王、子房、花瓣的柱头状物的过程中,观察到成药逆转现象。     

Variation in Saffron (Crocus sativus L.) accessions and Crocus wild species by RAPD analysis

Crocus, a genus of Iridaceae is mostly grown in areas with Mediterranean environment as the best region. Saffron (C. sativus) is a perennial plant and is cultivated as an industrial crop in several regions of Iran. In this research, five accessions of cultivated Saffron from five areas in Khorasan, and Esfahan including Gonabad, Ferdows, Torbat-e-Heyidariye, Estahbanat, Gopayegan were used. Other nine species of saffron were grown naturally in Iran; so we collected two wild species (C. caspius and C. speciosus) from north of Iran (Gilan Province). RAPD markers were used to classify these species and to find their relationships. In the results of this study, the cluster analysis showed two distinct groups. Also, the maximum similarity was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33). Finally, this method as a convenience procedure could be used to separate different accessions and species of Crocus as well.