RESUMO
The integration of cellular and molecular data is essential for understanding the mechanisms involved in the acquisition of competence by plant somatic cells and the cytological changes that underlie this process. In the present study, we investigated the dynamics and fate of Passiflora edulis Sims cotyledon explants that were committed to somatic embryogenesis by characterizing the associated ultrastructural events and analysing the expression of a putative P. edulis ortholog of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene. Embryogenic calli were obtained from zygotic embryo explants cultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Callus formation was initiated by the division of cells derived from the protodermal and subprotodermal cells on the abaxial side of the cotyledons. The isodiametric protodermal cells of the cotyledon explants adopted a columnar shape and became meristematic at the onset of PeSERK expression, which was not initially detected in explant cells. Therefore, we propose that these changes represent the first observable steps towards the acquisition of a competent state within this regeneration system. PeSERK expression was limited to the early stages of somatic embryogenesis; the expression of this gene was confined to proembryogenic zones and was absent in the embryos after the globular stage. Our data also demonstrated that the dynamics of the mobilization of reserve compounds correlated with the differentiation of the embryogenic callus.
Assuntos
Passiflora/enzimologia , Proteínas de Plantas/genética , Proteínas Quinases/genética , Sementes/enzimologia , Diferenciação Celular , Expressão Gênica , Genes de Plantas , Passiflora/crescimento & desenvolvimento , Passiflora/ultraestrutura , Proteínas de Plantas/metabolismo , Técnicas de Embriogênese Somática de Plantas , Proteínas Quinases/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/ultraestruturaRESUMO
Lipoxygenases (LOXs, EC 1.13.11.12) are a class of non-heme iron containing dioxygenases which catalyze the regiospecific and stereospecific hydroperoxidation of polyunsaturated fatty acids with 1,4-pentadiene system such as linoleic acid and linolenic acid in plants. In this work we studied the LOX activity in damaged as well as in distal leaves in response to specialist (Agraulis vanillae vanillae) or generalist (Spodoptera frugiperda) insect attack. Enzymatic assays showed that induction of LOX activity occurred locally and systemically in response to both insects' attacks. Northern blot analysis revealed that LOX expression is also insect-inducible in agreement with enzymatic assay results. In addition, northern analysis corroborated previous reports that LOX activity is wound- and methyl jasmonate-inducible. These results suggest that the herbivore-response in passion fruit is mediated by jasmonates, since a key enzyme of the biosynthetic pathway of jasmonic acid is induced upon lepidopteran insects' attacks.
Assuntos
Lepidópteros/fisiologia , Lipoxigenase/biossíntese , Passiflora/enzimologia , Animais , Ciclopentanos/metabolismo , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica de Plantas , Lipoxigenase/genética , Oxilipinas/metabolismo , Passiflora/genética , Folhas de Planta/enzimologia , RNA de Plantas/metabolismoRESUMO
BACKGROUND AND AIMS: Myo-inositol-1l-phosphate synthase (MIPS) catalyses the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. Inositol phospholipids play a vital role in membrane trafficking and signalling pathways, auxin storage and transport, phytic acid biosynthesis, cell wall biosynthesis and production of stress-related molecules. In the present study, an MIPS cDNA from developing Passiflora edulis f. flavicarpa seeds was characterized and an investigation made into its spatial and differential expression, as well as changes in its transcription during exposure of growing plants to cold and heat stresses. METHODS: The MIPS-encoding gene was isolated by polymerase chain reaction (PCR) methods, and transcript levels were examined using semi-quantitative reverse transcription-PCR (RT-PCR) during seed development and in response to heat and cold stress. In addition, the copy number of the cloned PeMIPS1 gene in the genome of Passiflora edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea was determined by Southern blot analyses. KEY RESULTS: A full-length cDNA clone of the PeMIPS1 from P. edulis was isolated and characterized. Southern blot analyses indicated that the genomic DNA might have diverse sequences of MIPS-encoding genes and one copy of the cloned PeMIPS1 gene in the genomes of P. edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea. RT-PCR expression analyses revealed the presence of PeMIPS1 transcripts in ovules, pollen grains and leaves, and during the seed developmental stages, where it peaked at 9 d after pollination. The PeMIPS1 gene is differentially regulated under cold and heat stress, presenting a light-responsive transcription. CONCLUSIONS: Experimental data suggest that PeMIPS1 transcription plays an important role in the establishment of developmental programmes and during the response of plants to environmental changes. The PeMIPS1 is differentially transcribed during cold and heat stress, presenting a light response pattern, suggesting that it is important for environmental stress response.
Assuntos
Meio Ambiente , Regulação da Expressão Gênica de Plantas , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Passiflora/enzimologia , Passiflora/genética , Clonagem Molecular , Temperatura Baixa , Regulação da Expressão Gênica no Desenvolvimento , Temperatura Alta , Luz , Dados de Sequência Molecular , Filogenia , Sementes/enzimologia , Transcrição GênicaRESUMO
Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.