No description available.

Understanding the mechanisms plants utilize for seed storage protein (SSP) synthesis, transport and deposition have the potential rewards of enabling high yields of modified or foreign proteins. Hayashi et al. (1999) indicated that the machinery devoted to the synthesis of protein storage vacuoles in cotyledon cells can be induced in vegetative tissue by the constitutive expression of a pumpkin 2S albumin phosphinothricin-acetyl-transferase gene fusion (pumpkin 2S-PAT) resulting in the biogenesis of precursor-accumulating (PAC) vesicles in Arabidopsis leaves. This discovery was the impetus behind the work described which sought to examine this phenomenon further by ectopically evoking SSP trafficking and vesicle biogenesis machinery in leaves. With the aim of elucidating the mechanisms necessary to evoke PAC vesicle biogenesis, a suite of constructs including the pumpkin 2S-PAT and analogous napin-PAT and napin-GFP variants were synthesized. Analysis of these transgenes in Arabidopsis revealed that the pumpkin 2S albumin has a capacity unique from napin peptides to result in fusion protein accumulation. Further, the truncated pumpkin 2S albumin peptide and the pumpkin 2S albumin C-terminus were found to direct deposition to vesicles; however, the C-terminus alone was not enough to direct deposition to vesicles unless combined with a significantly shortened napin peptide. An increased ER protein throughput was correlated to trafficking of the fusion protein by Golgi-independent mechanisms resulting in stable accumulation of the unprocessed protein whereas less ER throughput indicated passage through the Golgi-dependent pathway resulting in accumulation of a processed variant. At the level of gene expression, as examined by a microarray study, both inducible and constitutive ectopic expression of pumpkin 2S-PAT resulted in substantial perturbations of the endomembrane system affecting protein folding, flowering time and ER-associated biosynthetic functions which indicated that modulation of flowering time and photoperiodism are highly dependent on protein trafficking and vacuolar biogenesis mechanisms and that high ER protein throughput occurs at the expense of biosynthesis and cessation of ER functioning.

Sorghum is a valuable grain crop and a principle source of food of particular importance in human and animal nutrition in the semi-arid regions of Africa and Asia. Despite its value, sorghum grain quality is a major limitation to its productivity and profitability. Sorghum grain is usually discounted as feed grain when compared to wheat and barley, predominantly because of its poor digestibility. The sorghum endosperm is composed of a complex starch protein matrix, whereby the starch is physically bound within the storage proteins, the kafirins. The kafirins are synthesised on the membrane bound polysomes and have a signal peptide which targets them to the lumen of the endoplasmic reticulum. Growth of protein bodies occur as - kafirins fill in the interior with  and γ kafirins occupy the periphery. Despite the -kafirins being more digestible and composing of 60- 80% of the kafirins, they are not easily accessible due to  and γ kafirins which have a high content of intermolecular disulphide bonds (S-S), rendering them highly resistant to proteases. Alteration of the structure of the protein bodies and change of the location of the-kafirins could result in a higher digestibility of sorghum proteins. This could be achieved by upregulating or downregulating the -kafirins. The improvement of grain quality, both in increased protein and starch digestibility would substantially enhance the digestibility of sorghum as animal feed as well as for human consumption. Various techniques have been utilised to classify the kafirins according to their mobility on SDS PAGE electrophoresis, Reverse Phase High Performance Liquid Chromatography (RP-HPLC), Free Zone Capillary Electrophoresis (FZCE) and Lab on Chip. Until recently the characterisation and classification of the kafirins generally have relied on the characterisation of zeins from maize. Zeins have about 70% homology to the kafirins both at the nucleotide and amino acid level. Based on the high similarity of the -kafirins to the -zeins, the - kafirins have been classified as 19 and 22 kDa. Despite their 70% homology the migration of the - kafirins on SDS PAGE is quite different to that of the zeins. Hence, I propose a new classification of the -kafirins as 23 kDa and 25 kDa based on their mobility on SDS PAGE Characterisation and cloning of the 23 and 25 kDa genes was performed using QL41 the Queensland inbred line of sorghum. Ten positive clones were isolated from a cDNA library for the 25 kDa and two clones for the 23 kDa -kafirins. The isolated clones of the 25 kDa -kafirins showed 98-99% homology with each other and also with the GenBank sequences. The major finding was the characterisation of the 23 kDa -kafirins. The two clones obtained showed 100% homology to each other as well as to the published sequences on the GenBank, and were full-length sequences. Also a partial sequence was obtained that lacked the signal peptide and was different to the other two clones. Whilst characterising the 23 kDa a second group of the 25 kDa -kafirins was identified from the genomic DNA, of all the three genotypes (QL41, 296B and QL12), which was unique from the previously isolated clones. This group of -kafirins was not among the cluster but was 5’ upstream of the cluster. This group had a higher content of the glutamine compared with the other 25 kDa group. The expression level was studied to show how each gene family contributed to the level of - kafirins. QL41 and 296B were used for this study. From the studies it was shown that the 23 kDa - kafirins genes were 20% more expressed than the 25 kDa. An attempt to identify suitable markers for the -kafirins was investigated using RFLP and SSR analysis. Thirty-two different genotypes were utilised for this study. The observed variation indicated by cluster analysis (4-38%) clearly showed variation of the -kafirins in genotype and within the kafirin genes as elucidated by the sequences in Chapter 4. Markers able to identify this variation could help in the selection of highly digestible mutants. Hence, there is potential for sorghum grain improvement using marker-assisted breeding. The need to identify a tissue specific promoter was essential, especially for a strong promoter that could drive expression in the endosperm of the monocots. A vector construct consisting of the - kafirin promoter driving the GUS reporter genes was used for transient expression from QL41. This was assessed in the sorghum and barley calli, sorghum endosperm and leaves and corn endosperm. Tissue specific expression as well as higher levels of transient expression were seen using the - kafirin promoter, compared with the ubiquitin promoter. Preliminary experiments have illustrated the potential use of a gene silencing mechanism that could enhance the digestibility of sorghum grain. The 25 kDa -kafirin gene was used as the target for gene silencing using the mechanism of iRNA. Transformation constructs were developed using the throughput vector pSTARGATE in an effort to silence the 25 kDa -kafirins. The characterisation of the -kafirins has provided valuable information for future sorghum improvement research.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

ssp-1 is a seed storage protein mutant which is deficient in one of the major seed storage proteins in Arabidopsis thaliana, the 12S cruciferins. To determine if this mutant can drive a higher level expression of a transgene than that found in wild type, the mutant was transformed with the phytohemagglutinin (PHA) gene and single copy PHA homozygotes were identified. These PHA transformants were crossed to wild type so that each PHA gene would be in the same copy number and chromosomal context in a wild type background. Immunoblotting was employed to compare the PHA levels of the single copy transformants in both genetic backgrounds. PHA levels ranged from 4.52% to 7.7% of the total protein in transformants. Two of the transformants showed 30.33% and 44.18% more PHA than that of their backcross. Therefore, a mutant such as ssp-1 may provide a means for overexpression of foreign proteins.

ssp-1 is a seed storage protein mutant which is deficient in one of the major seed storage proteins in Arabidopsis thaliana, the 12S cruciferins. To determine if this mutant can drive a higher level expression of a transgene than that found in wild type, the mutant was transformed with the phytohemagglutinin (PHA) gene and single copy PHA homozygotes were identified. These PHA transformants were crossed to wild type so that each PHA gene would be in the same copy number and chromosomal context in a wild type background. Immunoblotting was employed to compare the PHA levels of the single copy transformants in both genetic backgrounds. PHA levels ranged from 4.52% to 7.7% of the total protein in transformants. Two of the transformants showed 30.33% and 44.18% more PHA than that of their backcross. Therefore, a mutant such as ssp-1 may provide a means for overexpression of foreign proteins.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

<p>The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The <i>napA</i> gene encodes for a <i>Brassica napus</i> 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The <i>napA</i> promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to <i>napA</i> through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster.</p><p>We also show that the RY/G cluster is responsible for repression of <i>napA</i> expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the <i>napA</i> RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the <i>napA</i> RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.</p>

The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The napA gene encodes for a Brassica napus 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The napA promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to napA through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster. We also show that the RY/G cluster is responsible for repression of napA expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the napA RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the napA RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.