The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess.) Rydb. of Artemisia L. (Asteraceae) in Turkey / Uma investigação sobre o cloroplasto e os genomas nucleares de táxons pertencentes ao subgênero Dracunculus (Bess.) Rydb. de Artemisia L. (Asteraceae) na Turquia

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and subspecific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn't been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Artemisia é um dos maiores gêneros da família Asteraceae, com cerca de 500 a 600 táxons em níveis específicos e subespecíficos e organizados em cinco subgêneros. Em razão do grande número de táxons, muitos taxonomistas estão tentando resolver o problema de sua classificação e filogenia, mas sua classificação natural ainda não foi alcançada. Nesta pesquisa, 60 indivíduos pertencentes a quatro táxons do subgênero Dracunculus de Artemisia L. na Turquia foram examinados. Para todos os indivíduos examinados de populações iguais e diferentes pertencentes aos táxons do subgênero Dracunculus, foram determinadas as sequências das regiões psbA-trnH do DNA do cloroplasto e ITS do DNA nuclear. Além disso, as regiões gênicas obtidas foram registradas no banco de dados do NCBI GenBank e um número de acesso foi obtido. Foi constatado que não houve fluxo gênico nem hibridização entre os quatro táxons estudados do subgênero Dracunculus, os quais também completaram sua especiação. De acordo com os resultados deste estudo molecular, A. campestris var. campestris, A. campestris var. marschalliana e A. campestris var. araratica foram propostos para ser elevados do nível de variedade para o nível de espécie. Esta pesquisa é importante porque é o primeiro estudo de base molecular relacionado com o subgênero Dracunculus em crescimento na Turquia.

Plastid transformation: advances and challenges for its implementation in agricultural crops

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.

Photosynthesis, growth, and survival in seedlings of four tropical fruit-tree species under intense radiation

In the Amazon region, agroforestry systems (AFSs) are recommended as a sustainable production alternative for local communities. A common component in Amazonian AFSs are tropical fruit trees, which can form the canopy or grow in the understory. In this study, we evaluated the effect of high radiation on photosynthesis, growth and seedling survival of four Amazonian fruit-tree species: Theobroma cacao, Eugenia stipitata, Inga edulis and Psidium guajava. Growth, chlorophyll fluorescence, gas exchange, and leaf pigments were measured in seedlings of each species grown for 12 months inside shade houses with low (8%), medium (30%) and high relative illumination (100%). Eugenia stipitata and T. cacao had the lowest acclimation capacity to high solar radiation, followed by I. edulis. Therefore, these species must be grown under intermediate light levels in early growth stages, to protect them from direct sunlight. In contrast, P. guajava seedlings demonstrated high tolerance to elevated radiation, therefore, this species can be planted under full sunlight. (AU)

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Effects of changes in micro-weather conditions on structural features, total protein and carbohydrate content in leaves of the Atlantic rain forest tree golden trumpet (Tabebuia chrysotricha) / Efeito de alterações nas condições micro-climáticas sobre características estruturais, e proteína e carboidratos histoquimicamente marcados, de folhas da árvore da floresta Atlântica ipê amarelo

Abstract Golden trumpet, Tabebuia chrysotricha, is a native tree from the Brazilian Atlantic rain forest, with a broad latitudinal distribution. In this study, we investigated the potential effects of short-term changes in micro-weather conditions on structural features, and total protein and carbohydrate content of golden trumpet leaves, using structural and histochemical approaches. Leaves were harvested in four different micro-weather conditions: 1. Afternoon, after a hot, sunny day; 2. at dawn, after a previously hot, sunny day; 3. at noon, of a hot, sunny day; and 4. at noon, of a cold, cloudy day. Leaflets exposed to low light irradiance showed flattened chloroplasts, uniformly distributed within the cells, throughout the palisade parenchyma. Conversely, leaflets exposed to high light irradiance presented flattened and rounded chloroplasts, in the upper and lower palisade parenchyma cells, respectively. The strongest protein staining was found for leaves harvested at the coldest period, whereas the weakest protein staining was found for leaves harvested after a hot, sunny day. The largest and most numerous starch grains were found for leaves harvested in the afternoon, after a hot, sunny day. Conversely, the smallest and less numerous starch grains were found for leaves harvested at dawn. Analysis of the data reported herein suggests that the leaflet responses to transient changes in micro-weather conditions are likely to contribute to the golden trumpet successful establishment in the broad latitudinal distribution in which the species is found.

Resumo Ipê amarelo é uma árvore nativa da floresta Atlântica brasileira, encontrada em uma ampla distribuição latitudinal. Neste estudo, nós investigamos os efeitos potenciais de alterações de curto prazo nas condições micro-climáticas sobre características estruturais, proteína e carboidratos histoquimicamente marcados, de folhas de ipê amarelo, usando estratégias de análise estrutural e histoquímicas. As folhas foram marcadas em quatro condições microclimáticas distintas: 1. Tarde, após um dia quente e ensolarado; 2. Amanhecer, após um dia quente e ensolarado; 3. Ao meio-dia, de um dia quente e ensolarado; e 4. Ao meio-dia, de um dia frio e nublado. Folíolos expostos à baixa irradiância luminosa apresentaram cloroplastos achatados, uniformemente distribuídos no interior das células, por todo o parênquima paliçádico, enquanto que folíolos expostos à alta irradiância apresentaram cloroplastos achatados e arredondados, nas células superiores e inferiores do parênquima paliçádico, respectivamente. A marcação mais intensa para proteína foi observada para folhas coletadas no momento mais frio de coleta, enquanto que a marcação mais fraca foi observada para folhas coletadas após um dia quente e ensolarado. Os grãos de amido maiores e mais numerosos foram observados em folhas coletadas durante a tarde de dia quente e ensolarado, enquanto que os menores e menos numerosos grãos de amido foram observados em folhas coletadas ao amanhecer.

Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.

Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

Background: Transgenic plants inhabiting single Bt gene are prone to develop insect resistance and this resistance has been reported in case of some important yield-devastating insect larvae of commercial crops, such as cotton and rice. Therefore, it has become essential to adapt new strategies to overcome the problem of insect resistance and these new strategies should be sophisticated enough to target such resistant larvae in broad spectrum. Among these, plants may be transformed with Bt gene tagged with some fusion-protein gene that possesses lectin-binding capability to boost the binding sites for crystal protein gene within insect mid-gut in order to overcome any chances of insect tolerance against Bt toxin. Enhanced chloroplast-targeted Bt gene expression can also help in the reduction of insect resistance. Results: In the present investigation, a combined effect of both these strategies was successfully used in cotton (G. hirsutum). For this purpose, plant expression vector pKian-1 was created, after a series of cloning steps, carrying Cry1Ac gene ligated with chloroplast transit peptide towards N-terminal and Ricin B-Chain towards C-terminal, generating TP-Cry1Ac-RB construct. Conclusions: Efficacy of pKian-1 plasmid vector was confirmed by in-planta Agrobacterium-mediated leaf GUS assay in tobacco. Cotton (G. hirsutum) local variety MNH-786 was transformed with pKian-1 and the stable integration of TP-Cry1Ac-RB construct in putative transgenic plants was confirmed by PCR; while fusion-protein expression in cytosol as well as chloroplast was substantiated by Western blot analysis. Whereas, confocal microscopy of leaf-sections of transgenic plants exposed that hybrid-Bt protein was expressing inside chloroplasts.

Efeito de sistemas de consórcio e inseticida na formação dos estômatos em plântulas de erva-doce (Foeniculum vulgare Mill. ) / Effects of intercropping systems and insecticide in formation of stomata in fennel seedlings

Foeniculum vulgare Mill., pertencente à família Apiacea, é conhecida como erva-doce e apresenta grande importância medicinal e comercial, tanto no Brasil como em vários outros países. Objetivou-se com esta pesquisa, estudar o desenvolvimento dos estômatos em plântulas de F. vulgare oriundas de sementes produzidas em sistemas de consórcio erva-doce X algodão e com aplicação do inseticida monocrotofós. A erva-doce foi cultivada em consórcio com algodão colorido cultivar BRS Safira, sendo utilizados os seguintes tratamentos: 1A2E, uma fileira de algodão e duas de erva-doce; 2A1E, duas fileiras de algodão e uma de erva-doce; ES, erva-doce solteira; onde foram distribuídos com e sem aplicação de inseticida, totalizando seis tratamentos. As sementes colhidas foram semeadas em areia e mantidas em casa de vegetação por 25 dias. Partes das plântulas (zona de transição, caule, cotilédones e folhas) foram seccionadas à mão livre, coradas e montadas em lâminas com glicerina para observação em microscópio. Foram avaliadas as seguintes características: número de estômatos, diâmetro polar e equatorial dos estômatos e número de cloroplastos nas células-guarda. Os dados foram analisados em delineamento inteiramente casualizado e distribuídos em arranjo fatorial 3X2; sendo realizado teste de Tukey a 5% de probabilidade. Na zona de transição e no caule observou-se aumento do número e do diâmetro polar dos estômatos quando foram utilizados sistemas de consórcio. Nos cotilédones, a erva-doce solteira proporcionou maior número de estômatos, porém com menor diâmetro e com menor quantidade de cloroplastos. Já na folha, os consórcios influenciaram positivamente o número de estômatos e de cloroplastos. De forma geral, os sistemas de consórcio e o inseticida influenciaram positivamente o desenvolvimento dos estômatos das plântulas de erva-doce.

Foeniculum vulgare Mill., belonging to the family Apiaceae, is known as fennel and has great medicinal and commercial importance, both in Brazil and in several other countries. The objective of this research was to study the development of stomata of F. vulgare seedlings grown from seeds produced in intercropping systems fennel and cotton, with application of insecticide monocrotophos. The fennel was grown in association with colored cotton BRS Safira, with the following treatments: 1A2E, one rows of cotton and two fennel; 2A1E, two rows of cotton and one fennel; ES, fennel single; were distributed with and without application of insecticide, total six treatments. The seeds were sown in sand and kept in a greenhouse for 25 days. Parts of seedlings (transition zone, stem, cotyledons and leaves) were cut freehand, stained and mounted on slides with glycerol for observation under microscope. Were evaluated the following characteristics: stomata number, polar and equatorial diameter of the stomata and chloroplasts number in guard cells. The data were analyzed in completely randomized and distributed in factorial 3x2, being conducted Tukey test at 5% probability. The transition zone and stem showed an increase of the stomata number and polar diameter the when consortium systems were used. In cotyledons, fennel single provided the highest stomata number, but with smaller diameter and fewer chloroplasts. In leaf, the consortia have positively influenced the stomata and chloroplasts number. In general, the intercropping systems and insecticide positively influenced the development of stomata in fennel plants.

Adaptações fisiológicas e anatômicas de melissa officinalis L. (Lamiaceae) cultivadas sob malhas termorrefletoras em diferentes intensidades luminosas / Physiological and morphological adaptations of melissa officinalis L. (Lamiaceae) cultivated under thermo-reflector shading nets at different luminous intensities

Objetivou-se, com a realização da pesquisa, avaliar modificações fisiológicas e anatômicas em plantas de melissa, cultivadas sob malhas termorrefletoras (Aluminet®), em diferentes níveis de sombreamento, visando conhecer a plasticidade fenotípica em resposta de adaptação a diferentes quantidades de luz. Os tratamentos foram caracterizados por plantas submetidas a pleno sol e a 20 e 60 por cento de intensidade luminosa, e arranjados conforme o delineamento inteiramente casualizado (DIC). As quantificações de clorofila foram feitas em quatro repetições, as medições das epidermes e parênquimas foram repetidas 15 vezes e utilizou-se 10 repetições para as avaliações das características de cloroplastos e grãos de amido destes. Plantas submetidas a 20 por cento de intensidade luminosa apresentaram maior quantidade de clorofila a e, portanto, maior razão clorofila a/b. Comparativamente, as folhas de melissa a pleno sol e a 60 por cento de luz apresentaram células da epiderme adaxial mais espessas, mas as células da epiderme abaxial mostraram características encontradas em folhas de sombra, ou seja, mais finas. Quanto maior a intensidade luminosa, maior o número de cloroplastos, porém, a pleno sol mostraram-se mais finos e com menor área. Os grãos de amido de plantas cultivadas sob ambientes sombreados tiveram maior área e ocuparam maior parte nos cloroplastos de plantas a 60 por cento de intensidade luminosa. Assim, plantas de melissa, quando submetidas ao sombreamento, tiveram plasticidade fenotípica.

The aim of this study was to evaluate physiological and anatomical modifications in lemon balm plants, cultivated under thermo-reflector nets (Aluminet®) at different levels of shading, in order to understand the phenotypic plasticity in adaptation response to different light quantities. The treatments were characterized by plants subjected to full sun and 20 and 60 percent of luminous intensity, and arranged in completely randomized design (CRD). The quantifications of chlorophylls were done in four replicates, the measurements of epidermis and parenchymas were repeated 15 times and 10 replicates were used to evaluate characteristics of chloroplasts and their starch grains. Plants subjected to 20 percent of luminous intensity showed higher quantity of chlorophyll a and, therefore, higher chlorophyll a/b ratio. Lemon balm leaves under full sun and 60 percent of light showed thicker adaxial epidermis cells, but the abaxial epidermis cells showed characteristics found in shaded leaves, i.e., they were slender. The higher the light intensity, the larger the number of chloroplasts; however, under full sun, they were slender and had smaller area. The starch grains of leaves grown under shaded environments showed larger area and, at 60 percent of luminous intensity, occupied the largest part of chloroplasts. Thus, lemon balm plants, subjected to shading conditions, showed phenotypic plasticity.

Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis

The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15 percent SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

Fine structural study of the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta)

The present study analyzed several characters of the red seaweed Gymnogongrus torulosus, such as cellular structure of the thallus, cuticle, pit plug and cell wall ultrastructure, and morphology of some organelles like plastids, Golgi bodies and mitochondria. Also, anomalous chloroplasts with thylakoid disorganization were found in medullary cells. The significance of this thylakoid disposition is still unclear. This is one of the first studies focused on the fine structure of a red alga recorded in Argentina.

Evolución molecular y genómica en angiospermas / Molecular and genomic evolution in angiosperms

Se revisaron, sintetizaron y compararon aspectos de la evolución de los genomas en las angioespermas. Las plantas con flores tres genomas: el del cloroplasto, el de la mitocondria y el nuclear. El genoma del cloroplasto tiene una estructura muy conservada (120-217kb) que incluye 110-113 genes. El genoma de la mitocondria es mayor (300-600kb) y contiene cerca de 60 genes, es un genoma muy dinámico ya que gana y pierde fácilmente secuencias nucleares y del cloroplasto. Los genomas nucleares de las angioespermas se encuentran entre los más grandes conocidos. El genoma nuclear de Arabidopsis thailiana tiene 125Mb que comprenden 25498 genes. Del arroz (oryza sativa) se han secuenciado dos variedades y su genoma es casi 4 veces más grande que el de A. thaliana y comprenden de 32000 a 55615 genes. Para el maíz (Zea mays), aunque no secuenciado, se tiene mucha información y se estima que 60 a 80 por ciento de su genoma nuclear está constituido por elementos móviles. Las substituciones sinónimas de los genes de cloroplasto son de 2 a 3 veces más rápidas que en las mitocondrias, y los genes nucleares cambian de 10 a 15 veces más rápido que los mitocondriales. Sin embargo, las tasas de substitución de los sitios no sinónimos son similares entre mitocondria y cloroplasto, y son un poco más altas en el núcleo. Los estudios detallados de estos tres genomas permiten avanzar en el entendimiento de las complejidades genéticas y evolutivas de este grupo de plantas

Mitochondrial and chloroplast localization of FtsH-like proteins in sugarcane based on their phylogenetic profile

A phylogenetic analysis of plant FtsH-like proteins was performed using protein sequences from the GENEBANK database and five groups of plant FtsH-like proteins were identified by neighbor-joining analysis. Prediction of the subcellular location of the proteins suggested that two (FtsH-m1 & FtsH-m2) were mitochondrial and three (FtsH-p1, FtsH-p2, FtsH-p3) were plastid targeting. The phylogenetic profile of plant FtsH-like proteins was used to search sugarcane expressed sequence tag (EST) clusters in the SUCEST database. Initially, 153 clusters presenting homology with FtsH-like proteins were recovered, of which 23 were confirmed by a BLAST search in the GENEBANK database and by comparison of their hidropathy index with that of previously described FtsH-like proteins. Sugarcane presented EST clusters in all phylogenetic groups. In silico expression analysis showed that the groups are differentially expressed in sugarcane tissues, with FtsH-p2 and FtsH-m1 presenting increased levels of expression.

Morphogenesis in short-term and long-term anther derived calli of Oenothera hookeri de vries

Anther culture of O. hookeri on Murashige and Skoog (1962) medium supplemented with 2 mg l-1 2,4-dichlorophenoxyacetic acid and 2 mg-1 1-naphthaleneacetic acid produced callus formation. When subcultured onto medium lacking auxin, the callus regenerated through the organogenic pathway. Non-organogenic and organogenic callus was observed using histological methods after 2, 3 and 24 weeks in culture. Three types of calli were recognized: non-organogenic friable calli, organogenic friable calli with roots and organogenic hard calli with shoots. The microscopical sections showed striking differences in tissue organization among friable and compact calli. Vascular bundles were prominent in compact calli, but were not found in friable calli. Calli sections showed at light microscopy cells at two developmental stages; differentiated highly vacuolated cells and meristematic small isodiametric cells with densely stained cytoplasm. At electron microscopy level abnormal chloroplasts were present in non-organogenic calli, while chloroplasts were well developed in organogenic hard calli. Peroxisomes with paracrystalline protein bodies were abundant in both types of calli.

Imaging oscillations in Gonyzaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.