Comparison of Profiling of Hairy Root of Two Tibetan Medicinal Plants Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim.

BACKGROUND: Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry. METHODS: To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions. RESULTS: The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings. CONCLUSION: Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.

Isolation of a novel alginate lyase-producing Bacillus litoralis strain and its potential to ferment Sargassum horneri for biofertilizer.

Algae have long been used to augment plant productivity through their beneficial effects. Alginate oligosaccharide is believed to be one of the important components to enhance growth and crop yield. In this study, we isolated and characterized a Bacillus litoralis strain, named Bacillus M3, from decayed kelps. We further demonstrated that the M3 strain could secrete alginate lyase to degrade alginate. The crude enzyme exhibited the highest activity (33.74 U/mg) at pH 7.0 and 50°C. The M3 strain was also able to ferment the brown alga Sargassum horneri. Fermentation results revealed that a fermentation period of 8-12 hr was the best harvest time with the highest level of alginate oligosaccharides. Plant growth assay showed that the seaweed fermentation extract had an obvious promotion effect on root and seedling growth of Lycopersicon eseulentum L. Our results suggest that fermentation extract of Sargassum horneri by the novel strain of Bacillus litoralis M3 has significant development potential for biofertilizer production and agriculture application.

Androgenesis in Solanaceae.

The Solanaceae is one of the most important families for global agriculture. Among the different solanaceous species, tobacco (Nicotiana tabacum), potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum) are five crops of outstanding importance worldwide. In these crops, maximum yields are produced by hybrid plants created by crossing pure (homozygous) lines with the desired traits. Pure lines may be produced by conventional breeding methods, which is time consuming and costly. Alternatively, it is possible to accelerate the production of pure lines by creating doubled haploid (DH) plants derived from (haploid) male gametophytes or their precursors (androgenesis). In this way, the different steps for the production of pure lines can be reduced to only one generation, which implies important time and cost savings. This and other advantages make androgenic DHs the choice in a number of important crops where any of the different experimental in vitro techniques (anther culture or isolated microspore culture) is well set up. The Solanaceae family is an excellent example of heterogeneity in terms of response to these techniques, including highly responding species such as tobacco, considered a model system, and tomato, one of the most recalcitrant species, where no reliable and reproducible methods are yet available. Interestingly, the first evidence of androgenesis, particularly through in vitro anther culture, was demonstrated in a solanaceous species, Datura innoxia. In this chapter, we report the state of the art of the research about androgenic DHs in Solanaceae, paying special attention to datura, tobacco, potato, tomato, eggplant, and pepper.

Correlação entre floração, frutificação e variáveis ambientais em Solanum lycocarpum. A. St. Hil, Solanaceae / Correlation between flowering, fructification and environmental variables in Solanum lycocarpum A. St.-Hil, Solanaceae

A lobeira (S. lycocarpum) é uma espécie típica e abundante do Cerrado, que ocupa principalmente ambientes antropizados. Possui características interessantes do ponto de vista da biologia reprodutiva, que provavelmente estão favorecendo a ampla ocupação pela espécie. Tendo em vista que a planta floresce e frutifica durante todo o ano, o presente estudo teve por objetivo verificar se existe correlação entre floração, frutificação e variáveis ambientais (temperatura, umidade e precipitação), como suporte para estudos futuros referentes à biologia reprodutiva e ecologia de espécies de plantas do Cerrado. Foi escolhida uma população de lobeira situada no município de Morrinhos, Sul do estado de Goiás, composta de 34 plantas em fase reprodutiva. Todas as plantas foram georreferenciados com um aparelho GPS. Foram feitas observações mensais durante 13 meses (junho de 2005 a julho de 2006) e quantificados flores abertas e frutos produzidos nos intervalos entre observações. Pode-se inferir alta conversão de flores hermafroditas em frutos. Por meio da correlação de Spearman, pode-se constatar correlação positiva entre floração e precipitação e umidade, o mesmo, aparentemente, não ocorre para frutificação.

Solanum lycocarpum (lobeira) is a typical and abundant species of brazilian Cerrado, which occupies mainly surrounding disturbed areas. It has interesting characteristics from the point of view of reproductive biology, that probably are favoring the large occupation of habitats by the species. Based on the fact that the species produces flowers and fruit during all the year, the present study had the purpose to verify the association between flower and fruit production with environmental variables (temperature, relative humidity and precipitation), aiming to support future studies referring to reproductive biology and ecology of plant species from Cerrado biome. A population of S. lycocarpum composed of 34 plants in reproductive phase, situated in Morrinhos, south of the State of Goiás, Brazil, , was evaluated. All the plants were geographically referenced with a GPS receptor. Observations were made monthly during 13 months (June, 2005 to July, 2006) quantifying open flowers and fruits produced in the intervals between the observations. It was possible suggest high conversion of flowers in fruits. The Spearman rank correlation showed positive correlation of flower number with precipitation and relative humidity. Fruit number was not correlated with the environmental variables.

Solasodine accumulation in regenerated plants of Solanum torvum Sw / Acúmulo de solasodina em plantas micropropagadas de Solanum torvum Sw

A nodal segment culture was developed in order to assess Solanum torvum Sw. regeneration and solasodine levels. The influence of auxins (indoleacetic acid, 1-Naphthaleneacetic acid) and benzyl adenine on S. torvum growth in micropropagation was investigated. A nodal segment culture was initiated with seeds germinated in MS basal medium added of GA3 and grown in different concentrations of IAA, IAA + BAP and NAA + BAP. Sixty-day-old plants from the in vitro culture were collected, frozen and lyophilized; then, the methyl orange method was used to quantify solasodine for the spectrophotometric assay. The best results regarding plant regeneration and solasodine accumulation were obtained by using the MS basal medium without addition of plant growth regulators; however, there was great production of calluses presenting friable bases. Based on these results, cell cultures can be initiated from such calluses with application of other auxins and cytokinins to enhance solasodine production, besides different elicitors, light intensities and sucrose concentrations.

A regeneração de Solanum torvum e a avaliação do conteúdo de solasodina foram os objetivos de cultura de segmentos nodais. A influência de auxinas (ácido 3-indolacético, ácido naftalenoacético) e de 6-benzilaminopurina no crescimento de S. torvum na micropropagação foi investigado. Cultura de segmentos nodais foi iniciada por sementes germinadas em meio básico MS acrescido de GA3 e cultivadas em diferentes concentrações de AIA, AIA + BAP e ANA + BAP. Plantas da cultura in vitro com 60 dias foram coletadas, congeladas e liofilizadas e o método de alaranjado de metila foi utilizado para quantificação de solasodina para o ensaio espectrofotométrico. Os melhores resultados para regeneração vegetal e acúmulo de solasodina foram alcançados no meio MS sem adição de reguladores de crescimento havendo, porém grande produção de calos de base friáveis. Esses resultados mostram que a partir desses calos, cultura de células pode ser iniciada com aplicação de outras auxinas e citocininas para o aumento da produção de solasodina além de diferentes eliciadores, intensidades luminosas e concentrações de sacarose.

MPF2-like-a MADS-box genes control the inflated Calyx syndrome in Withania (Solanaceae): roles of Darwinian selection.

The Chinese lantern, which is the inflated calyx syndrome (ICS) of Physalis, is formed by MPF2 in the presence of the plant hormones, cytokinin and gibberellin. MPF2 knockdown mutants of Physalis have small leaves, no ICS, and are male sterile, thus, revealing three MPF2-related functions. Of the close relatives of Physalis, Tubocapsicum has only a rudimentary calyx, whereas others, like the Withania species, have ICS. From all Withania samples tested, two classes of MPF2-like orthologs, MPF2-like-A and MPF2-like-B, were isolated, whereas only the latter class was obtained from tetraploid Tubocapsicum. Though distinct differences can be observed between MPF2-like-A and MPF2-like-B proteins, that is MPF2-like-A proteins have an aberrant structure in that they have a three amino acid deletion in their C-domain and an eight amino acid extension at the C-terminal end, MPF2-like-A genes are phylogenetically closer to the Physalis MPF2-like genes. Unlike MPF2-like-B, the overexpression of MPF2-like-A in Arabidopsis revealed extra large sepals thus suggesting that MPF2-like-A genes are very likely responsible for the ICS formation in Withania. This correlated with the expression pattern of MPF2-like-A in vegetative and flower tissues, whereas MPF2-like-B is expressed only in vegetative tissues of Withania. In Tubocapsicum, however, MPF2-like-B RNA is detectable in all tissues tested. Finally, positive Darwinian selection was observed in the branch leading to Physalis MPF2-like and Withania MPF2-like-A proteins, followed by purifying selection once the trait had evolved. By contrast, purifying selection was detected for all other MPF2-like proteins tested. The contribution of the MPF2-like gene duplication to subfunctionalization is discussed.

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

Weed management in Solanaceae crops in Portugal.

Portugal has very good climatic-edafic conditions for Solanaceae crops, regarding to either yield quality or quantity. Tomato (Lycopersicon esculentum Miller.), potato (Solanum tuberosum L.) and pepper (Capsicum annuum L.) are the most social-economically important Solanaceae and aubergine (Solanum melongena L.) area of cultivation is increasing. Tomato is cultivated for fresh consumption and, primarily, for industrial processing. Is one of the most profitable vegetable crop and the main vegetable for industry. Potato is the annual vegetable crop with the largest cultivated area. Pepper is one of the main crops for vegetable frozen industry. Tomato, pepper and aubergine are cultivated in the field (outdoor) in Spring-Summer season. In greenhouses, they're also grown during other months and, at the southmost region (Algarve), during the whole year. Potato is cultivated almost the whole year through. Weed management is essential to achieve yield rentability and, for crops growing in the field, herbicides play an important role, due to their efficacy or inherent limitations of other control measures. This paper presents the state of art, in Portugal, regarding to some cultural and social-economical aspects of these crops (e.g., cultivated areas, productions, main producer regions), main weeds, weed control methods and, in particular, registered herbicides, with indication of their usage conditions (application timings and spectrum of weeds controlled) according to the principles of Good Plant Protection Practice and Integrated Weed Management.

Induced defense in Nicotiana attenuata (Solanaceae) fruit and flowers.

Plants protect themselves against herbivory using a continuum of strategies, ranging from constitutive defenses to intermittent induced responses. Induced defenses may not provide immediate and maximum protection, but could be advantageous when continuous defense is either energetically or ecologically costly. As such, induced defenses in flowers could help defend relatively valuable tissue while keeping reproductive structures accessible and attractive to pollinators. Thus far, no one has demonstrated the efficacy of induced defenses against floral herbivores (florivores) in the field. Here we show that mechanical leaf damage in wild tobacco, Nicotiana attenuata (Solanaceae), reduced both flower and fruit herbivory in the field and that exogenous application of methyl jasmonate, a potent elicitor of induced responses, reduced both leaf and floral damage in natural populations. This result is consistent with a survey of damage in the field, which showed a negative relationship between leaf damage and flower and fruit damage. Although optimal defense theory predicts that induced defenses should be rare in reproductive tissues, owing to their high fitness value, our results suggest otherwise. Induced defenses in leaves and reproductive tissues may allow plants to respond effectively to the concomitant pressures of defending against herbivory and attracting pollinators.

Tobacco to tomatoes: a phylogenetic perspective on fruit diversity in the Solanaceae.

The Solanaceae contains many species of agricultural importance. Several of these are cultivated for their fruits, such as the tomato, the pepper and the aubergine. The family is very diverse in fruit type with capsules, drupes, pyrenes, berries, and several sorts of dehiscent non-capsular fruits occurring in the 90+ genera. In this paper, recent work on fruit type evolution in angiosperms is reviewed in relation to dispersal agents and habitat ecology. Defining fruit types in the Solanaceae in a simple five state system, then mapping them onto a previously published molecular phylogeny based on chloroplast DNA allows discussion of the evolution of these fruit types in a phylogenetic framework. Capsules are plesiomorphic in the family, and although berries are a synapomorphy (shared derived character) for a large clade including the genus Solanum (tomatoes and aubergines), they have arisen several times in the family as a whole. Problems with homology of drupes and pyrenes are discussed, and areas for future investigation of fruit structure homology identified. The distribution of fruit types in the large and diverse genus Solanum is also discussed in the light of monophyletic groups identified using chloroplast gene sequences. This variety is related to recent advances in the understanding of the molecular biology of fruit development. Finally, several key areas of future comparative, phylogenetic investigation into fruit type evolution in the family are highlighted.

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates.

Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.

Ethylene response to pollen tube growth in Nicotiana tabacum flowers.

In flowers of Nicotiana tabacum L., pollination induces a transient increase in ethylene production by the pistil. The characteristic dynamics of the increase in ethylene correspond to the main steps of the pollen-tube journey into the pistil: penetration into the stigma, growth through the style, entry into the ovary and fertilization. Ethylene is synthesized de novo in the pistil, and its production is reduced in the dark. Ethylene production was monitored in tobacco flowers after pollination with incongruous pollen from three different Nicotiana species, N. rustica, N. repanda and N. trigonophylla, and with pollen from Petunia hybrida. Pollen from all of these different sources can germinate on the stigma surface but each pollen type shows a different behavior and efficiency in penetrating the pistil tissues. Thus, these different crosses provided a model with which to study the response of the pistil to pollination and fertilization. Ethylene evolution upon pollination in tobacco differed in each cross, suggesting that ethylene is correlated with the response to pollen tube growth in the tobacco flower.

Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara.

The gene for a thermal hysteresis (antifreeze) protein (sthp-64) from the bittersweet nightshade, Solanum dulcamara, was cloned and characterized. An expression cDNA library prepared from November S. dulcamara was screened using a polyclonal antibody generated against a previously purified 67 kDa thermal hysteresis protein, and positive clones were identified and sequenced. The full-length thermal hysteresis protein gene was cloned into an Escherichia coli expression vector and expressed as a fusion protein. The putative thermal hysteresis protein (STHP-64) contains two conserved regions 56 and 57 amino acids in length which have the C-X4-C-X22-23-H-X1-H zinc finger motif which is present in WRKY proteins, a family of transcription factors which play a role in regulating expression of pathogenesis-related proteins in plants. Additional features of transcription factors, such as an acidic domain between the two zinc-fingers and a glutamine-rich region upstream of the first zinc-finger are also present in STHP-64. A DNA binding assay showed that the expressed STHP-64 fusion protein has specific DNA-binding ability. A unique feature of STHP-64 is that the C-terminus contains 10 consecutive 13-mer repeats. Such repeats are a common feature of animal antifreeze proteins. The expressed STHP-64 fusion protein had low levels of thermal hysteresis activity, but this activity was considerably increased by addition of citrate, which is known as an enhancer of certain insect antifreeze proteins. Northern blots demonstrated that the STHP-64 transcript was not present in leaves until November and December, suggesting that cold acclimation induces STHP-64 production.

Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots.

Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.

Plant-growth inhibitory activity of cedrelanolide from Cedrela salvadorensis.

The effect of cedrelanolide, the most abundant limonoid isolated from Cedrela salvadorensis (Meliaceae), was assayed as a plant-growth inhibitory compound against monocotyledonous and dicotyledonous seeds. This compound inhibited germination, seed respiration, and seedling dry weights of some plant species (Lolium multiflorum, var. Hercules, Triticum vulgare, var. Salamanca, Physalis ixocarpa, and Trifolium alexandrinum). Our results indicate that cedrelanolide interferes with monocot preemergence properties, mainly energy metabolism of the seeds at the level of respiration. In addition, the compound inhibits photophosphorylation, H+ uptake, and noncyclic electron flow. This behavior might be responsible for its plant-growth inhibitory properties and its possible role as an allelopathic agent.

In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity.

Withania somnifera plantlets were produced in vitro from the shoot-tip of aseptically germinated seedlings. Culture conditions were optimized using different plant growth regulators which gave rise to 120 shoots from a single bud. The plantlets were then transferred to pots and maintained in greenhouse for 4 months. 90% of these in vitro propagated plantlets survived and showed normal growth. Leaves from these plants were used for isolation of the withanolides. Methanolic extract of leaves from plantlets growing in tissue culture and those transferred to the greenhouse were evaluated for immunomodulatory activity. While the extract from greenhouse samples showed potent immunosuppressive activity, those from tissue cultures samples did not show any activity. Fractionation and characterization of withanolides, using HPLC, NMR, MS methods revealed the presence of withaferin A in the greenhouse samples. Our results indicate that Withania species may require longer time and better differentiation and also natural environment for the production of withaferin A.

Effects of CO2 on stomatal conductance: do stomata open at very high CO2 concentrations?

Potato and wheat plants were grown for 50 d at 400, 1000 and 10000 micromoles mol-1 carbon dioxide (CO2). and sweetpotato and soybean were grown at 1000 micromoles mol-1 CO2 in controlled environment chambers to study stomatal conductance and plant water use. Lighting was provided with fluorescent lamps as a 12 h photoperiod with 300 micromoles m-2 s-1 PAR. Mid-day stomatal conductances for potato were greatest at 400 and 10000 micromoles mol-1 and least at 1000 micromoles mol-1 CO2. Mid-day conductances for wheat were greatest at 400 micromoles mol-1 and least at 1000 and 10000 micromoles mol-1 CO2. Mid-dark period conductances for potato were significantly greater at 10000 micromoles mol-1 than at 400 or 1000 micromoles mol-1, whereas dark conductance for wheat was similar in all CO2 treatments. Temporarily changing the CO2 concentration from the native 1000 micromoles mol-1 to 400 micromoles mol-1 increased mid-day conductance for all species, while temporarily changing from 1000 to 10000 micromoles mol-1 also increased conductance for potato and sweetpotato. Temporarily changing the dark period CO2 from 1000 to 10000 micromoles mol-1 increased conductance for potato, soybean and sweetpotato. In all cases, the stomatal responses were reversible, i.e. conductances returned to original rates following temporary changes in CO2 concentration. Canopy water use for potato was greatest at 10000, intermediate at 400, and least at 1000 micromoles mol-1 CO2, whereas canopy water use for wheat was greatest at 400 and similar at 1000 and 10000 micromoles mol-1 CO2. Elevated CO2 treatments (i.e. 1000 and 10000 micromoles mol-1) resulted in increased plant biomass for both wheat and potato relative to 400 micromoles mol-1, and no injurious effects were apparent from the 10000 micromoles mol-1 treatment. Results indicate that super-elevated CO2 (i.e. 10000 micromoles mol-1) can increase stomatal conductance in some species, particularly during the dark period, resulting in increased water use and decreased water use efficiency.