Study of electron-dense thylakoids in chloroplasts of Stevia rebaudiana Bertoni in relation to the biosynthesis of diterpenoids

Using transmission electronic microscopy and mass spectrometry electron-dense thylakoids of chloroplasts of Stevia rebaudiana leaves during active vegetable growth of this plant were studied in relation to the biosynthesis of diterpenoid glycosides (DGs). It was found that these compounds are absent in these thylakoids, but they contain a water-insoluble weakly polar ent-kauren, a known biosynthetic precursor of DGs as well as gibberellins. This finding provides a base for the suggestion that similar, electron-dense, thylakoids were observed earlier by other authors in other plant species. These data allowed us to conclude that an intensive biosynthesis of ent-kauren is likely related to adaptation of the short-day plants including Stevia rebaudiana to vegetable growth under the long day conditions.     

Stevioside

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.     

Higher amount of steviol detected in the leaves of a non-toxic endangered medicinal herb, <Emphasis Type="Italic">Aconitum heterophyllum</Emphasis>

Aconitum heterophyllum is the only non-toxic species of Aconitum genus having immense therapeutic potential. To date, only roots of this plant species are used for the preparation of various herbal formulations while leaves are discarded due to lack of information on the presence of any therapeutically active compounds. To the best of our knowledge, this is the first report on the detection and quantification of diterpene steviol in the leaves of A. heterophyllum. Interestingly, the level of steviol is equivalent to Stevia rebaudiana leaves which is the richest source reported so far. Thus, A. heterophyllum leaves will not only provide a novel source for extraction of steviol but also benefit the harvesters to get additional economic returns on leaf biomass for this high-value non-toxic plant species.     

Stevioside mediated chemosensitization studies and cytotoxicity assay on breast cancer cell lines MDA-MB-231 and SKBR3

Cancer is one of the most impacting life-threatening disease for the human populace. Hence, over the years we have seen a consistent interest to study and investigate new treatments to cure and prevent this disease. Medicinal plants have played a progressive part in treatment since many years. In this research study, we have explored the cytotoxicity effect of purified bioactive compound isolated from Stevia rebaudiana leaves and the key mechanism responsible for apoptosis in human breast cancer cells. The anticancer properties of Stevia rebaudiana leaves has been suggested in earlier literature. Hence, the aim of this study was to investigate the cytotoxicity of purified stevioside in human breast cancer cell lines MDA-MB-231 and SKBR3. Results showed that purified stevioside inhibited the growth of cancerous cell lines. The IC50 obtained after treatment with stevioside on cancer cells MDA-MB-231 and SKBR3 are 55 µM and 66 µM respectively. This shows purified stevioside is capable of inducing apoptosis indicating its promising anticancer activity. However, so far chemosensitization effects of stevioside on breast cancer have not been fully explained by other studies. Hence, additionally, this study also evaluates the chemosensitization potential of stevioside in combination with 5-FU. This research study shows the importance of Stevia rebaudiana as a good source of bioactive compounds with high anti-cancer property.     

Hypothesis/review: The structural basis of sweetness perception of sweet-tasting plant proteins can be deduced from sequence analysis

Human perception of sweetness, behind the felt pleasure, is thought to play a role as an indicator of energy density of foods. For humans, only a small number of plant proteins taste sweet. As non-caloric sweeteners, these plant proteins have attracted attention as candidates for the control of obesity, oral health and diabetic management. Significant advances have been made in the characterization of the sweet-tasting plant proteins, as well as their binding interactions with the appropriate receptors. The elucidation of sweet-taste receptor gene sequences represents an important step towards the understanding of sweet taste perception. However, many questions on the molecular basis of sweet-taste elicitation by plant proteins remain unanswered. In particular, why homologues of these proteins do not elicit similar responses? This question is discussed in this report, on the basis of available sequences and structures of sweet-tasting proteins, as well as of sweetness-sensing receptors. A simple procedure based on sequence comparisons between sweet-tasting protein and its homologous counterparts was proposed to identify critical residues for sweetness elicitation. The open question on the physiological function of sweet-tasting plant proteins is also considered. In particular, this review leads us to suggest that sweet-tasting proteins may interact with taste receptor in a serendipity manner.     

Life history and host range of Sauris nr. purpurotincta,an unsuitable biological control agent for Chinese tallowtree

Foreign surveys in China discovered a defoliating insect species feeding on the leaves of Chinese tallowtree (Triadica sebifera), an invasive weed of the southeastern U.S.A. The life history of this species, Sauris nr. purpurotincta (Lepidoptera: Geometridae), was examined and larval no-choice and adult multiple-choice host range tests were conducted in quarantine to evaluate their suitability for biological control of Chinese tallowtree. The results indicated that the larvae have five instars and require approximately 22 days to complete development to the adult stage. Host range tests indicated that the larvae could not feed and complete development on most species tested. However, 40% of the larvae survived when fed leaves of Hippomane mancinella, a state-listed endangered species in Florida, and all larvae survived when fed Morella cerifera, a common native species of the southeastern U.S.A. Multiple-choice oviposition tests indicated eggs were laid on leaves of both a south Florida native plant Gymnanthes lucida and Chinese tallowtree. Considering this broad host range, this species will not be considered further for biological control of Chinese tallowtree in the U.S.A.     

Gustatory responses in primates to the sweetener aspartame and their phylogenetic implications

Two-bottle preference tests have been applied to 70 (sub-) speciesof the order of Primates and, for comparison, to the tree shrew(Tupaia belangeri) to determine their responses to aspartame(APM), the first known sweet-tasting dipeptide which has toman a sweetness potency of about 200 times that of sucrose.It was found that only the Cercopithecidae, the Hylobatidaeand the Pongidae respond like man to this dipeptide and preferit to water. The other primates tested to date, show no responseto this sweetener. From a phylogenetic point of view, we notethat APM shows species specificity similar to thaumatin. Thus,a clear dichotomy exists within the order of Primates with respectto both thaumatin and APM. The results here illustrate oncemore the gustatory diversity among primates and are a compellingargument for the existence of different sweet taste receptorsor recognition sites in primate species.     

<Emphasis Type="Italic">Colletotrichum atractylodicola</Emphasis> sp. nov.: the anthracnose pathogen of <Emphasis Type="Italic">Atractylodes chinensis</Emphasis> in China

A new anthracnose disease of Atractylodes chinensis was observed in Liaoning province in China. The causal agent was isolated from diseased leaves. Based on morphology and ITS sequence data, it was identified as a Colletotrichum species belonging to the C. destructivum species complex. A multi-locus DNA sequence analysis (ITS, GAPDH, CHS-1, ACT, TUB2) revealed that the fungus represents a new species that is described here as C. atractylodicola sp. nov. Pathogenicity tests confirmed that the isolated species is the causal agent of the observed anthracnose symptoms on A. chinensis leaves.     

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP‐glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3‐β‐d ‐glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.     

Lycianthes moziniana (solanaceae): An underutilized Mexican food plant with “new” crop potential

Lycianthes moziniana is an agrestic (weedy) perennial encouraged by traditional farmers for its edible, sweet-tasting fruits. This species is endemic to the Mexican highland plateau and Oaxaca where the ethnotaxonomic and ethnohistoric data reflect a long-standing relationship with human populations. Today, the fruits of this plant are used for domestic consumption, small-scale commercialization, and ceremonial purposes. To ensure the production of fruits, traditional farmers afford the plants a special management which has evolutionary repercussions. Recently, modern agricultural practices have placed this species in danger of extinction. Preliminary chemical analyses and yield data suggest thatL. moziniana is particularly well-suited for development as a “new” crop for Mexico.     

甜味蛋白研究进展

甜味蛋白是一类具有高甜度、低热卡、多功能的天然甜味剂。但要从几种古老的植物中提取甜味蛋白较为困难 ,且难以开发和利用。随着生物技术的发展 ,尤其是利用基因工程技术将甜味蛋白基因克隆到微生物细胞中 ,构建产甜味蛋白的基因工程菌 ,为商品化生产甜味蛋白开辟了一条快速而有效的新途径。     

Characterization of bacterial mutagenicity mediated by 13-hydroxy-ent-kaurenoic acid (steviol) and several structurally-related derivatives and evaluation of potential to induce glutathione S-transferase in mice

Stevioside is a sweet-tasting diterpene glycoside that is derived from Stevia rebaudiana (Bertoni) Bertoni (Compositae). It is used commercially in Japan and other parts of the world as a sucrose substitute. Whereas stevioside demonstrates no mutagenic activity in a variety of test systems, the aglycone, steviol (13-hydroxy-ent-kaurenoic acid), is mutagenic toward Salmonella typhimurium strain TM677 in the presence of a metabolic activating system derived from the liver of Aroclor 1254-pretreated rats. The required activating component is localized in the microsomal fraction of rat liver, suggestive of a cytochrome P-450-mediated reaction. Partially purified epoxide hydrolase does not inhibit steviol-induced mutagenicity, indicating that an active metabolite is not an epoxide that serves as a substrate for this enzyme preparation. The 13-hydroxy group of steviol is required for the expression of mutagenicity since ent-kaurenoic acid is nonmutagenic, and acetylation of steviol at this position negates mutagenicity. Similarly, diterpenes bearing a strong structural resemblance to steviol, cafestol and kahweol, were found to demonstrate no mutagenic activity toward Salmonella typhimurium TM677, as were their respective acetates and palmitic acid esters. Conversely, 19-O-beta-D-glucopyranosyl steviol, a potential hydrolysis product of stevioside, is mutagenic and bactericidal in the presence of a metabolic activating system. Additionally, in contrast to the nonmutagenic diterpenes cafestol and kahweol that are effective as inducers of glutathione S-transferase activity, evaluation by administration to mice proved steviol, isosteviol and various steviol glycosides to be inactive in this process. Thus, structural differences among these naturally occurring and semi-synthetic diterpenes appear to impart major differences in biological activity that may relate to human health upon dietary ingestion.     

甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

Rebaudioside F, a diterpene glycoside from Stevia rebaudiana

The sweet diterpenoid glycoside, rebaudioside F, was isolated from leaves of a high rebaudioside C producing line of Stevia rebaudiana, and its structure was established by chemical and spectral studies.     

Identification of miR414 and expression analysis of conserved miRNAs from Stevia rebaudiana

MicroRNAs (miRNAs) usually contain 19-24 nucleotides and have been identified as important eukaryotic gene regulators. Applications of various computational approaches have simplified the task by predicting miRNAs from available sequence data sources. In this study, we identified a conserved miR414 from a computational analysis of EST sequence data available from Stevia rebaudiana. In addition, we also identified six conserved miRNAs namely miR169, miR319, miR414, miR164, miR167 and miR398 using stem-loop RT-PCR analysis. Hence, miR414 was commonly identified using both methods. The expression analysis of these miRNAs documented their roles in growth and development of Stevia. Furthermore, the detected miRNAs were found to target genes involved in plant growth, development, metabolism and signal transduction. This is the first study reporting these conserved miRNAs and their expression in Stevia.     

Steviol glycoside biosynthesis

Steviol glycosides are found in high concentrations in the leaves of the Paraguayan perennial herb Stevia rebaudiana and their intense sweetness, as well as high concentration in Stevia leaf tissue, has made them the subject of research interest for over 100 years. Steviol glycosides are diterpenoids whose biosynthetic pathways share four steps in common with gibberellic acid formation. The convergence of genomics and plant biochemistry has led to the rapid elucidation of the genes coding for the various enzymes in the biosynthetic pathway. Functional characterization of the enzymes coded for by those genes is on-going. The first committed step in the pathway is the synthesis of the aglycone steviol and the various glycosides found in the leaf tissue result from the elaboration of steviol by a number of glucosyltransferases.     

Curculin,a sweet-tasting and taste-modifying protein,is a non-functional mannose-binding lectin

A three-dimensional model of curculin, a sweet-tasting and taste-modifying protein from the fruits of Curculigo latifolia, was built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop (Galanthus nivalis). The three mannose-binding sites present in GNA were found in curculin but are devoid of mannose-binding activity as shown by docking experiments performed with mannose. Some regions well exposed on the surface of the three-dimensional model of curculin could act as epitopes responsible for the sweet-tasting properties of this protein.     

Complete amino Acid sequence of soybean leaf p21 : similarity to the thaumatin-like polypeptides

A polypeptide structurally related to the thaumatin family of proteins has been purified from soybean (Glycine max) leaves and the complete amino acid sequence has been determined. The mature protein, which we have termed P21, has a calculated molecular weight of 21,461 and an isoelectric point of 4.6. The soybean protein shows 64% amino acid identity with thaumatin, a sweet-tasting protein found in the West African shrub Thaumatococcus danielli, and as much as 71% identity with thaumatin-like polypeptides present in tobacco and maize.     

Chromosome numbers in North and Central American Compositae

Chromosome numbers are reported for 364 collections representing 70 species and varieties in 16 genera. Of these, 35 species and varieties have not had counts published previously. Counts of 26 collections in 14 species differ from all previously published counts for the same species. One new combination,Stevia anadenotricha, and one new variety,Stevia subpubescens var.intermedia, are published.     

Seasonal dynamics in a yeast population on the Oxalis acetosella L. leaves

Analysis of an epiphytic yeast population on the leaves of the evergreen common wood sorrel Oxalis acetosella L. throughout a year showed that the density and the species composition of this population underwent regular seasonal changes. There were almost no yeasts on the young spring leaves. However, the yeast population on the mature leaves tended to increase in the autumn, reaching a maximum after the formation of continuous snow cover. Then the yeast population on the leaves tended to decrease, reaching a minimum in the spring. The species diversity of the yeasts was maximum in the autumn. The population of the epiphytic yeast species Cystofilobasidium capitatum, Rhodotorula fujisanensis, Leucosporium scottii, and Cryptococcus flavus peaked in the autumn. On the other hand, the population of the widespread epiphytic species Cryptococcus laurentii on the wood sorrel leaves peaked in January. The relative abundance of the red-pigmented phytobionts Rhodotorula glutinis and Sporobolomyces roseus virtually did not change throughout the year. The relative abundance of the euribiotic species Cryptococcus albidus showed irregular monthly variations. The data obtained show that the epiphytic microbial population of various plants can be comprehensively studied only by analyzing this population throughout the vegetative period of the plants.