Efeitos da microgravidade em plantas de cana-de-açúcar

Sugarcane (Saccharum spp.) is a plant from Poaceae family that has an impressive ability to accumulate sucrose in the stalk, making it a significant component of the economy of many countries. About 100 countries produce sugarcane in an area of 22 million hectares worldwide. For this reason, many studies have been done using sugarcane as a plant model in order to improve production. A change in gravity may be one kind of abiotic stress, since it generates rapid responses after stimulation. In this work we decided to investigate the possible morphophysiological, biochemical and molecular changes resulting from microgravity. Here, we present the contributions of an experiment where sugarcane plants were submitted to microgravity flight using a vehicle VSB-30, a sounding rocket developed by Aeronautics and Space Institute teams, in cooperation with the German Space Agency. Sugarcane plants with 10 days older were submitted to a period of six minutes of microgravity using the VSB-30 rocket. The morphophysiological analyses of roots and leaves showed that plants submitted to the flight showed changes in the conduction tissues, irregular pattern of arrangement of vascular bundles and thickening of the cell walls, among other anatomical changes that indicate that the morphology of the plants was substantially influenced by gravitational stimulation, besides the accumulation of hydrogen peroxide, an important signaling molecule in stress conditions. We carried out RNA extraction and sequencing using Illumina platform. Plants subjected to microgravity also showed changes in enzyme activity. It was observed an increased in superoxide dismutase activity in leaves and a decreased in its activity in roots as well as for ascorbate peroxidase activity. Thus, it was concluded that the changes in gravity were perceived by plants, and that microgravity environment triggered changes associated with a reactive oxygen specie signaling process. This work has helped the understanding of how the gravity affects the structural organization of the plants, by comparing the anatomy of plants subjected to microgravity and plants grown in 1g gravity

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Caracterização funcional de dois cDNAs de cana-de- açúcar : PKCI e SHAGGY

Flowering is controlled by several environmental and endogenous factors, usually associated with a complex network of metabolic mechanisms. The gene characterization in Arabidopsis model has provided much information about the genetic and molecular mechanisms that control flowering process. Some of these genes had been found in rice and maize. However, in sugarcane this processe is not well known. It is known that early flowering may reduce its production up to 60% at northeast conditions. Considering the impact of early flowering in sugarcane production, the aim of this work was to make the gene characterization of two cDNAs previously identified in subtractive cDNA libraries: scPKCI and scSHAGGY. The in silico analysis showed that these two cDNAs presented both their sequence and functional catalytic domains conserved. The results of transgenic plants containing the overexpression of the gene cassette scPKCI in sense orientation showed that this construction had a negative influence on the plant development as it was observed a decrease in plant height and leaf size. For the scPKCI overexpression in antisense orientation it was observed change in the number of branches from T1 transgenic plants, whereas transgenic T2 plants showed slow development during germination and initial stages of development. The other cDNA analyzed had homology to SHAGGY protein. The overexpression construct in sense orientation did not shown any effect on development. The only difference observed it was an increase in stigma structure. These results allowed us to propose a model how these two genes may be interact and affect floweringdevelopment.

Análise de cDNAs identificados em bibliotecas substrativas de cDNA para floração de variedades de cana-de-açúcar cultivadas no Rio Grande do Norte(RN): fator de transição NAC, Calmodulina e Fosfatidiltransferase

The flowering is a physiological process that it is vital for plants. This physiological process has been well studied in the plant model Arabidopsis, but in sugarcane this process is not well known. The transition of the shoot apical meristem from vegetative to flowering is a critical factor for plant development. At Brazil northeastern region, the transition to flowering in sugarcane has an important effect as it may reduce up to 60% its production. This is a consequence of the sugar translocation from stalks to the shoot apical meristem which is necessary during the flowering process. Therefore, the aim of this work was to explore and analyze cDNAs previously identified using subtractive cDNA libraries. The results showed that these cDNAs showed differential expression profile in varieties of sugarcane (early x late flowering). The in silico analysis suggested that these cDNAs had homology to calmodulin, NAC transcription factor and phosphatidylinositol, a SEC14, which were described in the literature as having a role in the process of floral development. To better understand the role of the cDNA homologous to calmodulin, tobacco plants were transformed with overexpression cassettes in sense and antissense orientation. Plants overexpressing the cassette in sense orientation did not flowered, while plants overexpressing the cassette in the antissense orientation produced flowers. The data obtained in this study suggested the possible role from CAM sequence, SEC14 and NAC in the induction/floral development pathway in sugarcane, this is the first study in order to analyze these genes in the sugarcane flowering process.

Prospecção e caracterização de genes associados ao processo de indução floral em cana-de-açúcar

The northeastern region is responsible to 14.32% of sugarcane national production. This lowered contribution is due to edaphoclimatic condition. Flowering is a vital process to plant which consumes lots of energy and it culminates in a process called isoporization. This one can give in a decreasing of 60% on alcohol and water production. It may consider that cropped sugarcane has a hibrid with octaploid genome, there are varieties with a flowering standard until of non flowering. Using this natural genetic potential on different croppings of sugarcane, the aim of this work was to understand as this process occurs by the usage of subtractive approaches. The total RNA was extracted using Trizol of peaks of merisematics of croppings with induced flowering and other with late flowering. From this total RNA were built four subtractives libraries (B1- induced early flowering subtracted on late flowering not induced; B2- late flowering not induced subtracted induced early flowering; B3- induced early flowering subtracted of not induced early flowering; B02- not induced early flowering subtracted from induced early flowering) using kits Super Smart cDNA synthesis and BD Clontech kit select cDNA subtraction (Clontech). This material was clone don vector pGEM T-easy(Promega) and changed in competent cells of E.coli DH10B. Given analysis sequence was carried out a program BLASTn against database of NCBI and genome of Arabidopsis thaliana, rice and maize. Clones were grouped in 9 different classes according to function. Some factors already related as couples of flower induction were identified at different libraries. And grouped proteins with cell cycle and it controls were presents, mainly kinases proteins. Related factors to proteic sinthesis, metabolism, defence, cell communication were also given in both libraries .Some identified genes did not show similarity on database or homology with hypothesis function, and it can represents new genes to be deposited in international database. These results offers that some identified on sugarcane, classified as on factors classes, cell cycle and cell communication, trough unknown genes, can be linked with genetic changing to the flowering process found in the northeastern region

Caracterização de dois cDNAs homológos e uma AP endonuclease em cana-de-açúcar

The genome of all organisms is subject to injuries that can be caused by endogenous and environmental factors. If these lesions are not corrected, it can be fixed generating a mutation which can be lethal to the organisms. In order to prevent this, there are different DNA repair mechanisms. These mechanisms are well known in bacteria, yeast, human, but not in plants. Two plant models Oriza sativa and Arabidopsis thaliana had the genome sequenced and due to this some DNA repair genes have been characterized. The aim of this work is to characterized two sugarcane cDNAs that had homology to AP endonuclease: scARP1 and scARP3. In silico has been done with these two sequences and other from plants. It has been observed domain conservation on these sequences, but the cystein at 65 position that is a characteristic from the redox domain in APE1 protein was not so conservated in plants. Phylogenetic relationship showed two branches, one branch with dicots and monocots sequence and the other branch with only monocots sequences. Another approach in order to characterized these two cDNAs was to construct overexpression cassettes (sense and antisense orientation) using the 35S promoter. After that, these cassettes were transferred to the binary vector pPZP211. Furthermore, previously in the laboratory was obtained a plant from nicotiana tabacum containing the overexpression cassette in anti-sense orientation. It has been observed that this plant had a slow development and problems in setting seeds. After some manual crossing, some seeds were obtained (T2) and it was analyzed the T2 segregation. The third approach used in this work was to clone the promoter region from these two cDNAs by PCR walking. The sequences obtained were analyzed using the program PLANTCARE. It was observed in these sequences some motives that may be related to oxidative stress response

Caracterização de um homólogo de HINT1 em cana-de-açúcar encontrado em bibliotecas subtrativas de CDNA para floração

The sugarcane is a monocot plant grown in tropical and subtropical regions, with Brazil being the largest producer. Despite its economic importance, little is known about the molecular flowering process in sugarcane. This physiological process can promote a loss up to 60% in sugar or bioethanol. Thus, this work had as objective characterize a HINT1 homologous gene previously identified in subtractive libraries of flowering. Genomic analysis of gene and promoter region structure allowed the observation that there are at least two distinct genes homologous to HINT on sugarcane. Bioinformatics analyses showed the conservation of the characteristic protein domain of HIT superfamily and indicate a phylogenetic relationship associated to cell location. Moreover, a possible relation with the SBTILISIN-like protein family through the information available in interatomas was observed. This suggests that the HINT gene of sugarcane can be related to plant development, there are several possibilities of interactions in the regulation of floral induction process, because the sequences present in regulatory regions indicate that differential expression of HINT was related to with climatic factors in the Northeast region of Brazil as well as to biotic stress and phytohormones. Furthermore, the sugarcane phenotypes indicate that the influence of HINT may happen due to product accumulation of its enzymatic activity. For these characteristics this gene can be used as a marker in the selection of new varieties.

Efeitos da microgravidade em plantas de cana-de-açúcar

Sugarcane (Saccharum spp.) is a plant from Poaceae family that has an impressive ability to accumulate sucrose in the stalk, making it a significant component of the economy of many countries. About 100 countries produce sugarcane in an area of 22 million hectares worldwide. For this reason, many studies have been done using sugarcane as a plant model in order to improve production. A change in gravity may be one kind of abiotic stress, since it generates rapid responses after stimulation. In this work we decided to investigate the possible morphophysiological, biochemical and molecular changes resulting from microgravity. Here, we present the contributions of an experiment where sugarcane plants were submitted to microgravity flight using a vehicle VSB-30, a sounding rocket developed by Aeronautics and Space Institute teams, in cooperation with the German Space Agency. Sugarcane plants with 10 days older were submitted to a period of six minutes of microgravity using the VSB-30 rocket. The morphophysiological analyses of roots and leaves showed that plants submitted to the flight showed changes in the conduction tissues, irregular pattern of arrangement of vascular bundles and thickening of the cell walls, among other anatomical changes that indicate that the morphology of the plants was substantially influenced by gravitational stimulation, besides the accumulation of hydrogen peroxide, an important signaling molecule in stress conditions. We carried out RNA extraction and sequencing using Illumina platform. Plants subjected to microgravity also showed changes in enzyme activity. It was observed an increased in superoxide dismutase activity in leaves and a decreased in its activity in roots as well as for ascorbate peroxidase activity. Thus, it was concluded that the changes in gravity were perceived by plants, and that microgravity environment triggered changes associated with a reactive oxygen specie signaling process. This work has helped the understanding of how the gravity affects the structural organization of the plants, by comparing the anatomy of plants subjected to microgravity and plants grown in 1g gravity

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3