Amounts Of Plant Tissue Research Articles

Plant adenylate cyclases have come full circle.

In bacteria, fungi and animals, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylate cyclases (ACs), enzymes that catalyse the formation of 3',5'-cAMP from ATP, are recognized as key signalling components. In contrast, the presence of cAMP and its biological roles in higher plants have long been a matter of controversy due to the generally lower amounts in plant tissues compared with that in animal and bacterial cells, and a lack of clarity on the molecular nature of the generating and degrading enzymes, as well as downstream effectors. While treatment with 3',5'-cAMP elicited many plant responses, ACs were, however, somewhat elusive. This changed when systematic searches with amino acid motifs deduced from the conserved catalytic centres of annotated ACs from animals and bacteria identified candidate proteins in higher plants that were subsequently shown to have AC activities in vitro and in vivo. The identification of active ACs moonlighting within complex multifunctional proteins is consistent with their roles as molecular tuners and regulators of cellular and physiological functions. Furthermore, the increasing number of ACs identified as part of proteins with different domain architectures suggests that there are many more hidden ACs in plant proteomes and they may affect a multitude of mechanisms and processes at the molecular and systems levels.

Expression of an extremophilic xylanase in Nicotiana benthamiana and its use for the production of prebiotic xylooligosaccharides

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

BackgroundVirus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection.ResultsIn this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a “Y- type” asymmetric structure and contained an axillary bud that was about 1–3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening.ConclusionsOverall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

A comparison between the function of Serendipita indica and Sinorhizobium meliloti in modulating the toxicity of zinc oxide nanoparticles in alfalfa (Medicago sativa L.).

Zinc oxide nanoparticles (ZnO-NPs) are among the most commonly used nano-fertilizers (NF). However, elevated levels of ZnO-NPs in soil may affect plant growth and development due to its potential toxicity when accumulated in large amounts in plant tissues. This research was conducted using an in situ rhizobox system with the aims of evaluating zinc uptake from nano-zinc oxide amended rhizosphere soil by alfalfa plant and the effect of plant growth-promoting microorganisms on alleviating the phytotoxicity of ZnO-NPs. Treatments included microbial inoculations (Sinorhizobium meliloti, Serendipita indica) and different ZnO-NP concentrations (0, 400, and 800 mg kg-1) with three replications. The results indicated that S. indica minimized the phytotoxicity of ZnO-NPs to alfalfa by enhancing growth rate and decreasing zinc (Zn) translocation from root to shoot. Compared with plants inoculated with S. meliloti, co-inoculation with S. indica increased the shoot dry weight by 18.33% and 8.05% at 400 and 800 mg kg-1 ZnO-NPs, respectively. However, at the highest level of ZnO-NPs (800 mg kg-1), root inoculation of S. indica and S. indica + S. meliloti decreased Zn translocation factor by 60.2% and 44.3% compared to S. meliloti, respectively. Furthermore, a distinct relation between tolerance of S. indica-colonized plant to ZnO-NPs and the ability of S. indica in inhibiting or retarding degradation of polyunsaturated lipids through prevention of excess reactive oxygen species formation was observed. Malondialdehyde content of inoculated plants with S. indica either alone or in combination with S. meliloti was significantly lower than non-inoculated plants (p< 0.01). Zn-induced oxidative stress was mitigated by S. indica through enhanced activities of catalase and peroxidase enzymes. The findings of the present study indicate the potential use of endophytes fungus S. indica for ensuring food safety and security, and human health in heavy metal-polluted soil by reducing the phytoavailability of heavy metals in the aerial parts of the host plants.

Production of plant-derived anticancer precursor glucoraphanin in chromosomally engineered Escherichia coli

Glucoraphanin is a methionine-derived glucosinolate that imparts numerous health-benefits with broad bioactivity. Low amounts in plant tissues and high cost of extraction have limited the production of glucoraphanin. Metabolic engineering in heterologous microorganisms is an attractive approach to achieve efficient production of valuable natural products. In this study, a microbial fermentation process for glucoraphanin production was demonstrated. The engineered bacterial strain stably expressed 10 allogeneic enzymes in E. coli chromosome, including nine heterologous genes from Arabidopsis and Brassica and one from fungus Neurospora crassa, which could produce the specialized glucosinolate compound glucoraphanin with a titer of 0.675 μg/L by fermentation from glucose. The cofactor supplements and individual gene overexpression for glucoraphanin production were also investigated. This work highlights the possibility of supplying specialized plant glucosinolates by microbial fermentation process, instead of chemical extraction. Additionally, the limiting step enzyme, UDP-glucose-thiohydroximate glucosyltransferase, identified in this study also laid a foundation for further optimizing the glucoraphanin-producing cell factory.

Multiple-nutrient limitation of upland rainfed rice in ferralsols: a greenhouse nutrient-omission trial

To optimize the nutrient management of upland rice production on tropical ferralsols, a greenhouse experiment was established using the multi-nutrient omission approach. A reciprocal soil origin-rice cultivar transplant experiment was also conducted to better understand the relative contributions of the soil origin and the rice cultivar in rice nutrient limitation. We tested the deficiency of seven major and secondary nutrients [nitrogen (N), phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium (Mg), and silicon (Si)] and a solution of six micronutrients (B, Mn, Cu, Co, Na, Mo) likely to limit the growth of two upland rice cultivars, Chhomrong Dan and Nerica 4, on two Ferralsols from the highlands of Madagascar. We found severe multiple nutrient deficiencies. For both cultivars, P, Ca, N, Mg omission significantly depressed the shoot and root dry biomass and their amounts in plant tissues. However, the main limiting nutrients were not the same in both soils. We conclude that the multinutrient deficiencies observed for rice growth in the Ferralsols are site-specific, even though P limitation appears to be in common, and requires a holistic consideration of the mineral fertility, including micronutrients.

Acidity stress for the systemic elicitation of glyceollin phytoalexins in soybean plants

ABSTRACTGlyceollins are the major pathogen- and stress-inducible natural products (phytoalexins) of soybean that possess broad-spectrum anticancer and neuroprotective properties. Yet like other phytoalexins, glyceollins are difficult to obtain because they are typically biosynthesized only transiently and in low amounts in plant tissues. We recently identified acidity stress (pH 3.0 growth medium) as an elicitor that exerted prolonged (week-long) inductive effects on glyceollin biosynthesis and identified the NAC family TF gene GmNAC42-1 that activates glyceollin biosynthesis in response to acidity stress or WGE from the soybean pathogen Phytophthora sojae. GmNAC42-1 was annotated as an SAR gene and SAR genes were statistically overrepresented in the transcriptomic response to acidity stress suggesting that acidity stress triggers the systemic elicitation of glyceollin biosynthesis. Here, we demonstrate that acidity stress acts as a systemic elicitor when provided to soybean roots. Acidity stress preferentially elicited specific glyceollins in different soybean organs with exceptionally high yields of glyceollin I in root tissues.

The Role of Antioxidants in the Protection of Plants against Inhibitors of Protoporphyrinogen Oxidase

Protoporphyrin IX (proto), a photodynamic chlorophyll synthesis intermediate, is present in trace amounts in plant tissues. However, plants treated with protoporphyrin IX oxidase (PPO) inhibitors (e.g., acifluorfen-methyl) accumulate high levels of proto leading to the formation of reactive oxygen species (ROS) responsible for rapid light-dependent loss of cellular membrane integrity and subsequent cell death. We show that increasing the level of certain antioxidants protects plants against the herbicidal effect of PPO inhibitors. In particular, hydrophilic antioxidants such as reduced glutathione and ascorbic acid (ascorbate) had superior protective effects than the lipophilic antioxidant ?-tocopherol. Conversely, inhibiting glutathione biosynthesis with 5 mM l-buthionine sulfoximine rendered plants more sensitive to acifluorfen-methyl. These reducing agents protect plants by quenching the ROS generated by the photoactivation of proto. The quenching of superoxide by ascorbate and reduced glutathione provided superior protection than quenching of hydrogen peroxide by ascorbate. Additionally, the absolute amounts of proto present in the tissues were also reduced in the presence of the more active antioxidants, suggesting that these molecules further protect plants by enhancing the degradation of proto.

A High-Sensitivity, Microtiter-Based Plate Assay for Plant Pattern-Triggered Immunity.

The first step in the plant immune response to pathogen challenge involves the perception of conserved epitopes, called microbe-associated molecular patterns (MAMPs), by cell-surface pattern recognition receptors (PRRs). Given the key roles that MAMPs and PRRs play in plant innate immunity, great effort has been expended to identify these molecules. Current methods for assaying these immune responses are often limited in their resolution and throughput, and consequently, there is a need for medium- to high-throughput methodologies. Here, we describe the development of a 96-well microtiter plate-based assay for plant pattern-triggered immunity that measures the activity of plant peroxidase (POX) enzymes produced in response to treatment with bacterial MAMPs. The system has been optimized to minimize both the amount of plant tissue and MAMPs required and displays up to three orders of magnitude greater sensitivity than the traditional luminol-based reactive oxygen species assay when measuring the plant response to treatment with the bacterial MAMP flg22, reaching detection limits in the picomolar range. This high sensitivity opens the possibility of evaluating the immune-eliciting effects of weaker elicitors. The throughput and material requirements of the assay make it ideal for screens involving quantitative measurement of the plant innate immune response to MAMPs.

Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications

BackgroundSeveral high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications.ResultsWe developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection.ConclusionTo facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.

Highly Sensitive Salicylic Acid Quantification in Milligram Amounts of Plant Tissue.

Technical advances in mass spectrometry constantly raise the bar for analyzing trace amounts of plant hormones in only very small amounts of tissue. Here, a highly sensitive and accurate method is described for the quantitative analysis of the plant hormone salicylic acid not only in the model plant Arabidopsis thaliana but also in other plant species. The presented method is optimized for the working up of as little as 20 to 50 mg of plant tissue. The discussed protocol and the utilized laboratory equipment facilitate the implementation of the method into other laboratories that possess access to adequate state-of-the-art gas chromatography-mass spectrometry (GC-MS) equipment.

Diversidade de aranhas de solo em cultivos de milho (Zea mays)

Studies carried out on the diversity and abundance of spiders may provide a rich information base on the degree of integrity of agricultural systems where they are found. In transgenic corn, Bacillus thuringiensis proteins are expressed in great amounts in plant tissues and may affect arthropod communities. Thus, the main goal of this work was to identify the spider diversity associated to transgenic and conventional corn hybrids. Pitfall collections were performed in conventional and transgenic corn plots during the 2010/2011 crop season, at the experimental field of the Agronomy Course of the University of Cruz Alta, RS. A total of 559 spiders were collected, from which 263 were adults and 296 young individuals. In the transgenic corn 266 spiders were collected and in the conventional one 293. Eleven families were determined and the adult individuals grouped in 27 morphospecies. Families with the largest number of representatives were Linyphiidae (29.70%), Theridiidae (5.72%) and Lycosidae (5.01%). The most abundant morphospecies were Lyniphiidae sp. with 77 individuals, Erigone sp. with 40 individuals, Lynyphiidae sp. with 33 individuals, Theridiidae sp. with 21 individuals, Lycosa erythrognatha with 14 individuals and Lycosidae sp. with 13 individuals. The Shannon Diversity Index was higher for transgenic corn (H” =1.01) in February and smaller (H’=0.54) in the December collection in the conventional corn, and the Margaleff Richness Index showed higher diversity in December and February for the conventional corn (M=18.3), and smaller diversity for the transgenic corn in November (M=11.3). Families were classified in five guilds; two weavers: Irregular web builders and sheet web builders, and three hunter guilds: Night soil runners, ambush spiders and aerial night runners. The relative proportion of the spiders morphospecies found in this research, as well as the guilds, suggest that this group may not have been affected by the genetically modified corn.

Crescimento e nutrição mineral de Sida rhombifolia

O experimento foi conduzido com o objetivo de analisar a produção e a distribuição de massa seca e os teores e os acúmulos dos macronutrientes em plantas de guanxuma (Sida rhombifolia) cultivadas em condições padronizadas de nutrição mineral. As plantas cresceram em vasos de sete litros com substrato de areia, que foram irrigados diariamente com solução nutritiva e mantidos em condições de casa de vegetação. Os tratamentos corresponderam às épocas de avaliação (21, 35, 49, 63, 77, 91, 105, 119 e 133 dias após a emergência - DAE) e foram arranjados em delineamento experimental inteiramente casualizado com quatro repetições. As plantas de guanxuma apresentaram pequeno acúmulo de massa seca (0,3 g por planta) e de macronutrientes (6,9 mg N por planta; 0,7 mg P por planta; 8,6 mg K por planta; 4,9 mg Ca por planta; 2,6 mg Mg por planta; e 0,3 mg S por planta) na fase vegetativa (&lt;49DAE). Esses acúmulos se intensificaram, principalmente, após 63 DAE; a taxa diária de acúmulo foi crescente até 94 DAE (massa seca - MS), 89 DAE (N e P), 98 DAE (K), 95 DAE (Ca e S) e 93 DAE (Mg), quando houve acúmulo de 26,3 g MS por planta, 402,6 mg N por planta, 45,6 mg P por planta, 359,3 mg K por planta, 337,6 mg Ca por planta, 71,9 mg Mg por planta e 20,9 mg S por planta. N e K foram os macronutrientes com maiores teores e, consequentemente, mais requeridos e acumulados em maior quantidade nos tecidos vegetais das plantas de guanxuma.

Plant Cell Cultures as Source of Cosmetic Active Ingredients

The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

Accumulation of dry mass and macronutrients by sourgrass plants

The experiment was carried out aiming to analyze the dry mass production and distribution and the content and accumulation of macronutrients in sourgrass (Digitaria insularis) plants cultivated under mineral nutrition standard conditions. Plants grew in 7-liter pots filled with sand substrate and daily irrigated with nutrient solution, being maintained under greenhouse conditions. Treatments consisted of times of evaluation (21, 35, 49, 63, 77, 91, 105, 119, and 133 days after emergence - DAE) and were arranged in a completely randomized design with four replicates. Sourgrass showed small accumulation of dry mass (0.3 g per plant) and macronutrients (3.7 mg of N per plant, 0.4 mg of P per plant, 5.6 mg of K per plant, 0.9 mg of Ca per plant, 0.7 mg of Mg per plant, and 0.3 mg of S per plant) at vegetative growth stage (&lt; 49 DAE). Those accumulations increased mainly after 77 DAE, reaching the maximum theoretical value at 143, 135, 141, 129, 125, 120, and 128 DAE, for dry mass (12.4 g per plant), N (163.2 mg per plant), P (27.1 mg per plant), K (260.5 mg per plant), Ca (47.6 mg per plant), Mg (30.9 mg per plant), and S (13.7 mg per plant), respectively. K and N were found with higher rates and, as a consequence, they were required and accumulated in greater amounts in plant tissues of sourgrass.

Development of a rapid, reliable and simple multiplex PCR assay for early detection of transgenic plant materials

Multiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify corresponding genes simultaneously. In this study, a reliable multiplex PCR analysis protocol was established for simple and fast detection of transgenes in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase gene and 1-aminocyclopropane-1-carboxylate synthase gene, were selected for target and resident gene respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato and kumquat materials. The method facilitates early identification of transgenic buds when they are still quite small.

A simple method for isolating import-competent Arabidopsis chloroplasts

We present a simple, rapid and low-cost method for isolating a high yield of Arabidopsis chloroplasts that can be used to study chloroplast protein import. Efficiency of chloroplast isolation was dependent upon the ratio between amount of plant tissue and the buffer volume, the size and speed of the homogenisation equipment, and the size of the homogenisation beaker. The import method proved useful when characterising different precursor proteins, developmental stages and import-defective mutants. Time-course experiments enabled the measurement of import rates in the linear range. Compared to protoplastation, this isolation method has significant time and cost savings (∼80% and ∼95%, respectively), and yields chloroplasts with a higher capacity to import proteins.

Activities of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase, and Oxygen Evolution in Transgenic Tobacco Plants

Three clones of tobacco transformed with the T-DNA of Agrobacterium rhizogenes Ri plasmid A4b cultivated in vitro on a solid agar medium neither showed pronounced morphological diversity nor significantly differed in chlorophyll (Chl) contents from control plants. However, the transformation caused a 27 to 83 % decay in leaf oxygen evolution and in both ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC) activities. Therefore, the transformation brought about a reduction of active PEPC as well as activable RuBPC amounts in plant tissues. Individual clones did not mutually differ. In tobacco transformed with A. rhizogenes strain TR101 and grown in soil only the mean leaf area tended to reduce. Chl contents, Chl a/b ratio, oxygen evolution, and activities of both RuBPC and PEPC were insignificantly affected by the transformation.

A microtechnique for the analysis of free and conjugated indole-3-acetic acid in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer.

A microtechnique was developed for the quantification of indole-3-acetic acid (IAA) in plant samples of one milligram fresh weight or less. The method permitted quantification of both free and conjugated IAA using a benchtop gas chromatograph-mass spectrometer. New methods for sample purification with high recovery at microscale levels, together with simple changes that result in enhanced sensitivity of the instrumentation, allowed for a significant reduction in the amount of plant material required for analysis. Single oat (Avena sativa L.) coleoptile tips could be studied with this method and were found to contain free and total IAA levels of 137 and 399 pg.mg-1 fresh weight, respectively. A single 5-d-old Arabidopsis thaliana (L.) Heynh. seedling was shown to contain 61 pg.mg-1 fresh weight free IAA and 7850 pg.mg-1 fresh weight of total IAA following basic hydrolysis. This microtechnique provides a way to accurately measure IAA levels in very small structures and individual seedlings, thus making it a valuable research tool for elucidating the role and distribution of auxin in relation to growth and development.

Subtractive hybridization of cDNA from small amounts of plant tissue.

A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used to screen a specific library.