Muscarinic M1 Receptor Gene Expression in Streptozotocin Induced Diabetic Rats: Regulation of Insulin Secretion By Aegle marmelose And Costus pictus Leaf Extracts

The present work is to understand the alterations of total Muscarinic and Muscarinic MI receptors in brain and pancreatic islets of Streptozotocin induced diabetic rats. The work focuses on the evaluation of the antihyperglycemic activity of aqueous extracts of Aegle marmelose and Costus pictus leaves in vivo and the changes in the total Muscarinic and Muscarinic MI receptors during diabetes and after the treatment with insulin. The insulin secretory activity of Aegle marmelose and Costus pictus leaf extracts and the effect of cholinergic receptor agonist were investigated in vitro using rat primary pancreatic islet culture. Muscarinic MI receptor kinetics and gene expression during diabetes and regulation of insulin secretion by Aegle marmelose and Costus pie/us leaf extracts will help us to elucidate the role of Muscarinic and Muscarinic MI receptors in hyperglycemia and the regulatory activity of these plant extracts on insulin secretion through Muscarinic receptors.

Serotonin- 5-HT2A and Glutamate Receptors Gene Expression in Streptozotocin induced diabetic rats:Effects of pyridoxine and Aegle marmelose leaf extract

Department of Biotechnology, Cochin University of Science and Technology

Use of microprojectile bombardment in transient expression assays to analyze protochlorophyllide reductase gene expression in greening maize seedling leaf cells

In young cells of leaf meristems the progenitors of chloroplasts are small organelles known as proplastids, which divide and differentiate into chloroplasts. However, in the absence of light, proplastids undergo a different sequence of development and become etioplasts. When light is supplied to etiolated plants during the "greening" process, etioplasts differentiate into chloroplasts containing chlorophyll. An important light dependent step in chlorophyll biosynthesis is the photoreduction of protochlorophyllide to chlorophyllide by the NADPH:protochlorophyllide reductase (PCR) enzyme. This enzyme is present at high activity only in etiolated tissue and during early stages of light-induced chlorophyll synthesis. The enzyme and its corresponding mRNAs decrease dramatically with prolonged exposure to light. We have investigated the light-dependent transcriptional regulation of a PCR gene in greening maize leaf cells using a transient expression assay based on microprojectile bombardment. The promoter region was isolated and cloned into a ?-glucuronidase (GUS) reporter gene expression plasmid. We have used this chimeric plasmid in tungsten particle bombardment of both etiolated and greening maize seedling leaves to determine whether the cloned promoter region contains regulatory sequences that control light-responsive PCR gene expression.

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [ 100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production. Copyright (C) 2008 S. Karger AG, Basel.

Effects of leaf extracts of Myrcia guianensis (Aubl.) DC. on growth and gene expression during root development of Sorghum bicolor (L.) Moench

The allelopathic potential of leaf extracts from the medicinal plant Myrcia guianensis (Aubl.) DC. was studied in Petri dish bioassays on sorghum and determined the seed germination, germination rate index (GRI), root growth, secondary root number, the genes involved in root development (SHR, PHB, PHV and REV) and microRNA 166 that regulates these genes. The hydroalcoholic extract was more inhibitory than methanol extract (moderate inhibition) and aqueous extract at 25 and 100% concentration were least inhibitory. Application of higher dose of hydroalcoholic M. guianenesis leaf extracts on sorghum seeds, inhibited the root development and changed the expression of SHR and PHB genes and microRNA 166. This suggested that the expression of these genes could be indicator of allelopathic potential for inhibition of root development in sorghum.

Senescence-related gene expression profiles of rosette leaves of Arabidopsis thaliana: Leaf age versus plant age

Senescence is a form of programmed cell death (PCD) which leads to the death of whole organs, e.g., leaves or flowers, and eventually to the death of entire plants. Like all forms of PCD, senescence is a highly regulated and energy consuming process. Senescence parameters, like protein content, chlorophyll content, expression of photosynthesis-associated genes or senescence-associated genes (SAGs), reveal that senescence occurs in old leaves derived from young plants (6 week old) as well as in young leaves derived from older plants (8 week old), indicating that it is governed by the actual age of the leaves. in order to analyse the differential gene expression profiles during leaf senescence, hybridizations of high-density genome arrays were performed with: i) individual leaves within the rosette of a 6-week-old plant and ii) leaves of the same position within the rosette but harvested from plants of different ages, ranging from 5 to 8 weeks. Cluster and genetree analyses, according to the expression pattern revealed that genes which are up-regulated with respect to the age of the entire plant, showed completely different expression profiles with respect to the age of the individual leaves within one rosette. This was observed even though the actual difference in leaf age was approximately the same. This indicates that gene expression appears to be governed by different parameters: i) the age of the individual leaf and ii) the age and developmental stage of the entire plant.

Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue.

Effects of Cor15a-IPT Gene Expression on Leaf Senescence in Transgenic Petunia x hybrida and Dendranthema x grandiflorum.

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.

Expression Studies of the Zeaxanthin Epoxidase Gene in Nicotiana plumbaginifolia1

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.

Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter.

The promoter of the bean PAL2 gene (encoding phenylalanine ammonia-lyase; EC 4.3.1.5) is a model for studies of tissue-restricted gene expression in plants. Petal epidermis is one of the tissues in which this promoter is activated in tobacco. Previous work suggested that a major factor establishing the pattern of PAL2 expression in tobacco petals is the tissue distribution of a protein closely related to Myb305, which is a Myb-like transcriptional activator from snapdragon. In the present work, we show that Myb305 expression in tobacco leaves causes ectopic activation of the PAL2 promoter. To achieve Myb305 expression in planta, a viral expression vector was used. This approach combines the utility of transient assays with the possibility of direct biochemical detection of the introduced factor and may have wider application for studying the function of plant transcription factors.

Genotypic consistency in the expression of leaf water potential in rice (Oryza sativa L.)

Early work has shown variation in the grain yield of rice cultivars grown under water stress conditions to be associated with the plant water status, mainly with the maintenance of high leaf water potential (LWP) at flowering and grain filling stage. Considerable variation for LWP among rice varieties has been recorded. The present work was designed to investigate genotypic consistency in water potential within the plant and under canopy manipulation to vary plant water requirement. In a glasshouse experiment, with six rice genotypes, a consistent water potential gradient from stem base to leaf tip has been observed. Leaf tip water potential has been found as the minimum LWP that can be recorded at any time of stress. Genotypes with similar canopy size could maintain different levels of LWP under stress conditions. In a field experiment, with four selected lines, four canopy sizes and two canopy mixture treatments were introduced prior to the imposition of control, mild and severe water stress conditions. It was found that the line differences in LWP and relative water content (RWC) were expressed under both mild and severe stress conditions, regardless of canopy size, tiller number and whether they were mixed with another line with different capacity to maintain LWP. Although there were some differences among canopy size treatments for radiation interception in three water conditions, canopy manipulation (plant size) within a line did not affect the expression of LWP and hence genotypic variation in LWP was maintained. Under both glasshouse and field conditions, lines that maintained high LWP had larger xylem diameter and stem areas than those that had low LWP. The results indicated that the size of the vascular bundles could influence the maintenance of plant water relations under water deficit. (c) 2005 Elsevier B.V. All rights reserved.

Impact de la lumière sur la production de protéines recombinantes chez Nicotiana benthamiana

La moléculture végétale est une approche prometteuse pour la production de protéines d’intérêt médical ou industriel. Considérant les variations de rendement possibles dans une plante soumise à différentes conditions culturales, nos objectifs étaient : (i) de cartographier l’accumulation d’un antigène viral d’intérêt clinique dans les feuilles du tabac sauvage Nicotiana benthamiana utilisé comme bio-usine, et (ii) d’évaluer l’impact de la lumière en période de croissance sur le rendement total en antigène. Nous avons étudié les relations entre l’âge foliaire, le régime lumineux, l’expression du transgène et le rendement final en antigène dans les feuilles. Nos données confirment l’influence de l’âge sur les variations de rendement d’une feuille à l’autre, et l’impact positif de l’intensité lumineuse sur le rendement par plante. Elles mettent aussi en relief l’importance des tiges secondaires sur le rendement et le rôle clé de la transcription du transgène sur la teneur en antigène à l’échelle cellulaire.

Effets de l’environnement lumineux et de l’âge foliaire sur la croissance, la capacité photosynthétique et la production protéique chez "Nicotiana benthamiana"

Cette étude visait à caractériser la croissance, la capacité photosynthétique, la concentration en azote et protéines totales solubles, la production de protéines recombinantes (HA) ainsi que la quantité de lumière interceptée à différents stades de développement de plants de Nicotiana benthamiana afin d’optimiser la production de vaccins. L’évolution des réponses physiologiques étudiées fut similaire chez toutes les feuilles primaires, suggérant que le processus de sénescence s’initie et progresse de façon semblable indépendamment de leur ordre d’initiation. Toutefois, la superposition des patrons temporels de sénescence et de croissance foliaire a mené à un rendement HA maximal se situant invariablement dans la partie médiane du plant lorsqu’exprimé sur une base foliaire. À l’échelle du plant entier, nos résultats suggèrent qu’il est possible d’augmenter la production de vaccins en récoltant les plants à un stade de développement plus tardif, ou en augmentant la densité de culture et en récoltant ces plants plus tôt.

Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.