Potato (Solanum tuberosum L.) is the world's fourth largest food crop and large financial losses are incurred each year from wound and bruise related injuries. However, little is known about the coordinate induction of genes that may be associated with, or mark major wound-healing and periderm maturation events. Also, one of the key defense mechanisms for potato tubers is the robust barrier provided by the phellem (skin) of the native periderm. Many biological processes are involved in the formation of this stout tissue. However, little is known about induction of genes that may be associated with this process. The objectives of this research were to molecularly assess the processes of wound periderm development and maturation, and native periderm maturation in potato tubers. In this study, these processes were determined in coordination with expression profiles of selected genes. The cell cycle, cell wall protein, and pectin methyl esterase genes were determined from two diverse potato genotypes and two harvests NDTX4271-5R (ND) and Russet Burbank (RB) tubers; 2008 and 2009 harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB), and cyclin-dependent kinase regulatory subunit (StCKS1At) expression profiles were coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) expression profiles suggested involvement with closing layer formation and subsequent phellem cell layer formations. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine- and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and lastly cell wall thickening in nonmeristematic phellogen cells. StPME and StPrePME expression increased during periderm development, implicating involvement in modifications for closing layer and phellem cell formation. Collectively, these results indicate that the genes monitored were involved in and their expression profiles markedly coordinated with periderm formation and the on-set of periderm maturation; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall strengthening or thickening after cessation of cell division.

Dry bean breeding programs have made significant advances in combating both abiotic and biotic stresses as well as improving plant architectural traits via selective breeding. Flooding can cause complete crop loss in dry bean. On the other hand, breeding for an upright architecture in dry bean has been a breeding target in several programs. However, the stem cell wall components underlying this change have yet to be studied. This research focused on analyzing the cell wall components that might be involved in dry bean architecture as well as pre-germination flooding tolerance in dry bean. For the plant architecture study, two significant genomic regions were identified on Pv07 and Pv08 associated with lignin accumulation in dry bean. For the pre-germination flooding study, one unpigmented seed coat genotype (Verano) and three pigmented seed coat genotypes (Indeterminate Jamaica Red, Durango, and Midnight) had germination rates similar to that of the tolerant check.

No description available.

No description available.

No description available.

Lignocellulosic biomass is one of the most abundant raw materials on earth that can be utilized to created carbon-neutral biofuels as a replacement for conventional fossil fuels. In order to create ideal energy crops, the regulation and deposition of cell wall polysaccharides must first be fully understood. Improved understanding of cell wall regulation will enable selection of traits that can optimize biofuel feedstocks. Herein, I utilize the grass model system Brachypodium distachyon in order to understand the transcriptional regulation of secondary cell wall deposition. Gene expression profiling was used to elucidate transcription factors that regulate secondary cell wall biosynthesis. Through this method, WALL REGULATOR INTERACTING bHLH (WRIB) was identified and its role as a secondary cell wall regulator was tested. Yeast-one- and yeast-two-hybrid assays showed that WRIB is capable of binding to promoters of secondary cell wall biosynthesis genes, as well as interacting with known secondary cell wall transcription factor proteins and also Phytochrome B. These results suggest that WRIB plays an important role in the secondary cell wall regulatory network and could perhaps be modulated by Phytochrome B. Discovery of this novel and interesting gene furthers the overall understanding of secondary cell wall development with the goal of improving our ability to engineer biofuel feedstocks.

<i>Pinus taeda</i> L. suspension culture cells were used to develop a model system to study the process of lignification occurring during the early stages of cell wall formation and maturation. Chemical, biochemical and histochemical analyses of the <i>P. taeda</i> suspension cultures grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the growth regulator did not provide conclusive evidence for lignin deposition. On the other hand, cultures in which 2,4-D was substituted with α-naphthaleneacetic acid (NAA) were shown to lignify. During this induction of lignification, limited cell wall thickening occurred since transmission electron microscopy of the 2,4-D grown cells showed only primary walls while the average cell wall thickness of the NAA-grown cells was consistent with secondary (S₁) layer formation. Despite the possibility of only limited lignin deposition in the 2,4-0 grown cells, secondary metabolism had occurred as evidenced by reversed-phase and chiral chromatographic separations which revealed the ability of these cells to produce enantiomerically pure (-)-matairesinol. Administrations of [1-¹³C], [2-¹³C ] and [3-¹³C ] specifically labeled phenylalanines to the <i>P. taeda</i> suspension cultures in medium containing NAA allowed the determination of lignin bonding patterns <i>in situ</i> by solid-state ¹³C NMR spectroscopy of the resulting ¹³C enriched cells. Aqueous and organic solvent extractions and protease treatment yielded ¹³C enriched cell walls for solid-state ¹³C NMR spectroscopic analyses of the cell wall bound lignin component. Subsequently, an isolated lignin derivative from these cell walls was analyzed by solution-state ¹³C NMR spectroscopy and verified the assignments made in the solid-state. Accordingly, the above experiments represent the first demonstration of lignin bonding patterns <i>in situ</i> in a <i>Pinus</i> species as well as a suspension culture. This culture system possesses great potential as a model to thoroughly study the early stages of lignification. / Ph. D.

A resistência das paredes celulares vegetais à hidrólise enzimática é um dos grandes gargalos tecnológicos para a obtenção do etanol celulósico. Acredita-se que as modificações nas paredes celulares em processos como a mobilização de reservas, formação de aerênquima, amadurecimento de frutos e senescência, por exemplo, envolvam a ativação de módulos funcionais que culminam em alterações nas paredes celulares. Estes módulos são: 1) recepção de um sinal para início do processo; 2) Morte Celular Programada (PCD); 3) separação celular; 4) expansão celular; 5) hidrólise de hemiceluloses e 6) hidrólise de celulose. No caso da formação de aerênquimas lisígenos o processo que se inicia com a PCD e é seguido pela liberação de glicosil hidrolases que atuam a na degradação e/ou modificação da parede celular, formando espaços de ar no córtex radicular. A formação de aerênquima nas raízes de cana de açúcar é constitutiva e pouco se sabe sobre os mecanismos de modificação que ocorrem na parede celular durante este processo. Este estudo buscou compreender os padrões de variação expressão gênica, proteínas e de atividades enzimáticas associados à formação do aerênquima em raízes de cana de açúcar, com ênfase no papel das hidrolases de parede celular e em algumas proteínas relacionadas à PCD. Foram utilizados 5 segmentos de raízes de 1 cm cada, a partir do ápice radicular. No material coletado observou-se a formação gradual de aerênquima. Foram realizadas análises transcricional, proteômica e atividade enzimática das glicosil hidrolases e outras proteínas que atuam na modificação da parede celular, os quais foram identificados e quantificados ao longo da formação do aerênquima. As glicosil hidrolases pertencentes às famílias Cazy GH1, GH3, GH17, GH18 bem como expansinas, celulose sintase, lacase, calreticulina, calmodulina e proteínas relacionadas a degradação de pectinas, foram encontradas ao longo dos segmentos, principalmente após o segmento 2. De acordo com a atividade transcricional e dados da proteômica, sugere-se que os polissacarídeos seriam atacados por enzimas nos estágios iniciais da formação do aerênquima (seg 2 e 3). O ataque ocorre principalmente sobre as pectinas e o &beta;-glucano. Contudo, os dados apontam para a deposição de xiloglucano, xilanos e celulose (após seg 3), que formam um compósito ao redor dos espaços de ar. Isto sugere que parte dos polissacarídeos das paredes não sejam degradados ao longo do processo, embora enzimas específicas detectadas possam atuar na modificação dos mesmos, como verificado para algumas pectinases e membros de GH17. Além disso, nos pré-tratamentos com água foi possível observar que há maior sacarificação da parede nos seg. 1 e 2. Contudo quando retira-se a maior parte das pectinas e hemiceluloses após pré-tratamento com NaOH, a sacarificação é maior nos segmentos 2, 3 e 4, devido ao maior acesso e a maior quantidade de celulose. As glicosil hidrolases encontradas neste trabalho sugerem que estas atacam a parede de um específico conjunto de células do córtex que dá origem ao aerênquima. Já no fim do processo, quando há lise celular, algumas paredes de células remanescentes são recalcitrante à hidrólise, provavelmente devido a sua arquitetura e composição. Este trabalho traz informações para o desenvolvimento de futuras tecnologias para a produção do etanol do etanol celulósico de cana-de-açúcar / The resistance of plant cell walls to enzymatic hydrolysis is one of the main bottlenecks of the development of technology of production of cellulosic ethanol. It is believed that the modifications in cell walls related to processes of storage mobilization, aerenchyma formation, fruit ripening and senescence, for instance, involve the activation of functional moduli that culminate in alterations of cell walls. These moduli are: 1) signal perception to start the process; 2) Programmed Cell Death (PCD); 3) cell separation; 4) cell expansion; 5) hydrolysis of hemicelluloses and 6) hydrolysis of cellulose. In the case of the formation of lysigenous aerenchyma, the process starts with PCD and is followed by the release of glycosil hydrolases that act on the degradation and/or cell wall modifications, forming air spaces in the cortex of the root. The formation of aerenchyma in the roots of sugarcane is a constitutive phenomenon and little is known about the mechanisms of modification that occur in cell walls during its development. Thus, the present study focused on the visualization of the patterns of variation of gene expression, proteins and enzyme activities associated to the formation of aerenchyma in roots of sugarcane in order to understand the role of the cell wall hydrolases and some proteins related to PCD in cell wall modifications along the process. Five root segments of 1cm each, starting from root apex, were used. A gradual centripetal formation of aerenchyma was recorded in the cortex of developing roots. Analyses of the transcriptional, proteomic and enzyme activity profiles during the process revealed that several enzymes act on cell wall modifications. The glycosil hydrolases belonging to the Cazy families GH1. GH3, GH17, GH18, as well as expansins, cellulose synthase, laccase, calreticulin, calmodulin and other proteins related to pectin degradation have been found along the segments, mainly after segment 2. According to the data on transcriptomics and proteomics, it is suggested that enzymes attack polysaccharides during the initial stages of aerenchyma formation (seg. 2 and 3). The attack of the enzymes occurs mainly on pectins and &beta;-glucan. Conversely, the data point out to the deposition (or maintenance) of xyloglucan, xylan and cellulose (after seg. 3), which form a composite that surrounds the air spaces. This suggests that part of the polysaccharides present in cell walls are not degraded during the process, although specific enzymes have been detected that could act on polysaccharide mobilization, such as the GH17 family. Further, under pretreatment with water, it has been observed that cell wall saccharification was higher at segments 1 and 2. On the other hand, when most of the pectins and hemicelluloses are retrieved by pretreatment with NaOH, saccharification is higher of segments 2, 3 and 4, probably due to the higher access to the wall and also to the higher proportion of cellulose. The profiles related to the glycosil hydrolases found in this work, suggest that these enzymes attack the cell wall. Initially, they are probably kept within a group of cells that will originate the aerenchyma. At the end of the process, when there is cell lysis, the remaining walls of some cells are recalcitrant to hydrolysis probably due to changes in their architecture and composition. Our findings bring promising information that could be used in the future to improve efficiency of hydrolysis for cellulosic ethanol production from sugarcane

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

A resistência das paredes celulares vegetais à hidrólise enzimática é um dos grandes gargalos tecnológicos para a obtenção do etanol celulósico. Acredita-se que as modificações nas paredes celulares em processos como a mobilização de reservas, formação de aerênquima, amadurecimento de frutos e senescência, por exemplo, envolvam a ativação de módulos funcionais que culminam em alterações nas paredes celulares. Estes módulos são: 1) recepção de um sinal para início do processo; 2) Morte Celular Programada (PCD); 3) separação celular; 4) expansão celular; 5) hidrólise de hemiceluloses e 6) hidrólise de celulose. No caso da formação de aerênquimas lisígenos o processo que se inicia com a PCD e é seguido pela liberação de glicosil hidrolases que atuam a na degradação e/ou modificação da parede celular, formando espaços de ar no córtex radicular. A formação de aerênquima nas raízes de cana de açúcar é constitutiva e pouco se sabe sobre os mecanismos de modificação que ocorrem na parede celular durante este processo. Este estudo buscou compreender os padrões de variação expressão gênica, proteínas e de atividades enzimáticas associados à formação do aerênquima em raízes de cana de açúcar, com ênfase no papel das hidrolases de parede celular e em algumas proteínas relacionadas à PCD. Foram utilizados 5 segmentos de raízes de 1 cm cada, a partir do ápice radicular. No material coletado observou-se a formação gradual de aerênquima. Foram realizadas análises transcricional, proteômica e atividade enzimática das glicosil hidrolases e outras proteínas que atuam na modificação da parede celular, os quais foram identificados e quantificados ao longo da formação do aerênquima. As glicosil hidrolases pertencentes às famílias Cazy GH1, GH3, GH17, GH18 bem como expansinas, celulose sintase, lacase, calreticulina, calmodulina e proteínas relacionadas a degradação de pectinas, foram encontradas ao longo dos segmentos, principalmente após o segmento 2. De acordo com a atividade transcricional e dados da proteômica, sugere-se que os polissacarídeos seriam atacados por enzimas nos estágios iniciais da formação do aerênquima (seg 2 e 3). O ataque ocorre principalmente sobre as pectinas e o &beta;-glucano. Contudo, os dados apontam para a deposição de xiloglucano, xilanos e celulose (após seg 3), que formam um compósito ao redor dos espaços de ar. Isto sugere que parte dos polissacarídeos das paredes não sejam degradados ao longo do processo, embora enzimas específicas detectadas possam atuar na modificação dos mesmos, como verificado para algumas pectinases e membros de GH17. Além disso, nos pré-tratamentos com água foi possível observar que há maior sacarificação da parede nos seg. 1 e 2. Contudo quando retira-se a maior parte das pectinas e hemiceluloses após pré-tratamento com NaOH, a sacarificação é maior nos segmentos 2, 3 e 4, devido ao maior acesso e a maior quantidade de celulose. As glicosil hidrolases encontradas neste trabalho sugerem que estas atacam a parede de um específico conjunto de células do córtex que dá origem ao aerênquima. Já no fim do processo, quando há lise celular, algumas paredes de células remanescentes são recalcitrante à hidrólise, provavelmente devido a sua arquitetura e composição. Este trabalho traz informações para o desenvolvimento de futuras tecnologias para a produção do etanol do etanol celulósico de cana-de-açúcar / The resistance of plant cell walls to enzymatic hydrolysis is one of the main bottlenecks of the development of technology of production of cellulosic ethanol. It is believed that the modifications in cell walls related to processes of storage mobilization, aerenchyma formation, fruit ripening and senescence, for instance, involve the activation of functional moduli that culminate in alterations of cell walls. These moduli are: 1) signal perception to start the process; 2) Programmed Cell Death (PCD); 3) cell separation; 4) cell expansion; 5) hydrolysis of hemicelluloses and 6) hydrolysis of cellulose. In the case of the formation of lysigenous aerenchyma, the process starts with PCD and is followed by the release of glycosil hydrolases that act on the degradation and/or cell wall modifications, forming air spaces in the cortex of the root. The formation of aerenchyma in the roots of sugarcane is a constitutive phenomenon and little is known about the mechanisms of modification that occur in cell walls during its development. Thus, the present study focused on the visualization of the patterns of variation of gene expression, proteins and enzyme activities associated to the formation of aerenchyma in roots of sugarcane in order to understand the role of the cell wall hydrolases and some proteins related to PCD in cell wall modifications along the process. Five root segments of 1cm each, starting from root apex, were used. A gradual centripetal formation of aerenchyma was recorded in the cortex of developing roots. Analyses of the transcriptional, proteomic and enzyme activity profiles during the process revealed that several enzymes act on cell wall modifications. The glycosil hydrolases belonging to the Cazy families GH1. GH3, GH17, GH18, as well as expansins, cellulose synthase, laccase, calreticulin, calmodulin and other proteins related to pectin degradation have been found along the segments, mainly after segment 2. According to the data on transcriptomics and proteomics, it is suggested that enzymes attack polysaccharides during the initial stages of aerenchyma formation (seg. 2 and 3). The attack of the enzymes occurs mainly on pectins and &beta;-glucan. Conversely, the data point out to the deposition (or maintenance) of xyloglucan, xylan and cellulose (after seg. 3), which form a composite that surrounds the air spaces. This suggests that part of the polysaccharides present in cell walls are not degraded during the process, although specific enzymes have been detected that could act on polysaccharide mobilization, such as the GH17 family. Further, under pretreatment with water, it has been observed that cell wall saccharification was higher at segments 1 and 2. On the other hand, when most of the pectins and hemicelluloses are retrieved by pretreatment with NaOH, saccharification is higher of segments 2, 3 and 4, probably due to the higher access to the wall and also to the higher proportion of cellulose. The profiles related to the glycosil hydrolases found in this work, suggest that these enzymes attack the cell wall. Initially, they are probably kept within a group of cells that will originate the aerenchyma. At the end of the process, when there is cell lysis, the remaining walls of some cells are recalcitrant to hydrolysis probably due to changes in their architecture and composition. Our findings bring promising information that could be used in the future to improve efficiency of hydrolysis for cellulosic ethanol production from sugarcane