Project description:The quality of the pepper fruit is significantly influenced by the properties of its surface such as color, glossiness and texture. The fruit surface is composed of a peel containing several layers including the cuticle, epidermis and the hypodermis. The peel acts as a protective barrier against biotic and abiotic stresses and is the most critical tissue affecting water loss during post harvest storage. The peel is composed of an outer epidermis with thick waxy (lipid) cuticle and few cell layers of thick-walled hypodermal cells. Despite its agronomic importance and due to the fact that the majority of studies in fruits have been conducted using flesh and peel tissues as a whole, the biochemical and genetic bases of variation in peel properties are largely unknown. In this proposal we aim to determine peel-specific gene expression in pepper by micro array hybridizations of peel and flesh RNA extracted at different developmental stages of the fruit. The cultivar Celica (Capsicum annuum) that has a large blocky fruit will be used for studying gene expression in the peel and flesh. Plants were grown in the greenhouse during the spring of 2006. Fruits were harvested at three developmental stages: young- 10 days after anthesis, mature green- 30 days after anthesis and ripe red- 45 days after anthesis. These stages were chosen because each represents a distinct phase in fruit development. At each stage, a biological replicate consists of bulked tissue from 3 fruits from each of 3 plants (a total of 9 fruits). We have a total of 4 biological replicates. For each fruit, the peel was separated from the flesh by manual dissection using thin forceps and scalpel blade. Peel and flesh samples were immediately frozen in liquid nitrogen and stored at -800C until RNA extraction. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep kit (Sigma). Keywords: Reference design
Project description:The quality of the pepper fruit is significantly influenced by the properties of its surface such as color, glossiness and texture. The fruit surface is composed of a peel containing several layers including the cuticle, epidermis and the hypodermis. The peel acts as a protective barrier against biotic and abiotic stresses and is the most critical tissue affecting water loss during post harvest storage. The peel is composed of an outer epidermis with thick waxy (lipid) cuticle and few cell layers of thick-walled hypodermal cells. Despite its agronomic importance and due to the fact that the majority of studies in fruits have been conducted using flesh and peel tissues as a whole, the biochemical and genetic bases of variation in peel properties are largely unknown. In this proposal we aim to determine peel-specific gene expression in pepper by micro array hybridizations of peel and flesh RNA extracted at different developmental stages of the fruit. The cultivar Celica (Capsicum annuum) that has a large blocky fruit will be used for studying gene expression in the peel and flesh. Plants were grown in the greenhouse during the spring of 2006. Fruits were harvested at three developmental stages: young- 10 days after anthesis, mature green- 30 days after anthesis and ripe red- 45 days after anthesis. These stages were chosen because each represents a distinct phase in fruit development. At each stage, a biological replicate consists of bulked tissue from 3 fruits from each of 3 plants (a total of 9 fruits). We have a total of 4 biological replicates. For each fruit, the peel was separated from the flesh by manual dissection using thin forceps and scalpel blade. Peel and flesh samples were immediately frozen in liquid nitrogen and stored at -800C until RNA extraction. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep kit (Sigma). Keywords: Reference design 12 hybs total
Project description:Pepper fruits at four different developmental stages were collected: early fruit [EF; 1 cm long; 7 days after pollination (dap)], mature green fruit (MG; 6-7 cm length; 20 dap), breaking or turning red fruit (BR; fruit are partially red; 35 dap), and red ripe fruit (RR; fully red; 40 dap). Tomato fruits at corresponding developmental stages were also collected: EF (less than 1cm; 7 dap), MG (40 dap), BR (50 dap), and RR (55 dap). For the monitoring of fruit-specific and fruit ripening-related genes, we did array hybridization by using the leaves as a common reference and each corresponding fruit developmental stage sample.
Project description:Fruit ripening is a very important physiological process which gains relevance in crop species due to their economical and nutritional repercussions. During fruit ripening enormous metabolic changes occur in a genetically-controlled scenario affecting the physiology of most cell compartments. Peroxisomes are single-membrane bounded organelles present in all eukaryotes which display a noteworthy nitro-oxidative metabolism, and harbor catalase as one of the major antioxidant enzymes in cells. Quantitative proteomics analysis by isobaric tags for relative and absolute quantification (iTRAQ) was used to investigate the ripening process in sweet pepper (Capsicum annuum L.) fruits. Out of 2,574 quantified proteins, 692 were found to be significantly more abundant in immature green fruits compared to red ones, but 497 showed a lower expression as the ripening proceeded. Overall, about 50% of the detectable proteins were estimated to modify their expression due to the ripening process. Data obtained from the Gene Ontology algorithms (AgriGO platform) showed that from all the identified proteins, 46 (2%) were only predicted to have a peroxisomal origin, with a high number of them displaying up-expression tendency during ripening. Catalase was framed within the group which showed lower expression in ripe fruits, but this enzyme was also susceptible to undergo posttranslational modifications (PTMs) promoted by reactive nitrogen and oxygen species (RNS and ROS) through nitration, S-nitrosylation and oxidation events which provoked its inhibition. The biochemical characterization of catalase indicates that it has atypical native molecular mass (125-135 kDa), since it behaves as a homodimer, and isoelectric point of 7.4, which is higher than that of the majority of plant catalases reported so far. Taking together, these data suggest that ROS and RNS could be essential to modulate the role of catalase for the maintenance of basic cellular peroxisomal functions during pepper fruit ripening where nitro-oxidative stress occur.