RESUMO
The discovery of plant DNA recombination techniques triggered the development of a wide range of genetically modified crops. The transgenics were the first generation of modified plants; however, these crops were quickly questioned due to the artificial combination of DNA between different species. As a result, the second generation of modified plants known as cisgenic and/or intragenic crops arose as an alternative to genetic plant engineering. Cisgenic and/or intragenic crops development establishes the combination of DNA from the plant itself or related species avoiding the introduction of foreign genetic material, such as selection markers and/or reporter genes. Nowadays it has been made successful cisgenic and/or intragenic modifications in crops such as potato and apple. The present study shows the possibility of reaching similar approach in corn plants. This research was focused on achieve intragenic overexpression of the maize Rubisco activase (Rca) protein. The results were compared with changes in the expression of the same protein, in maize plants grown after 23 cycles of conventional selection and open field planting. Experimental evidence shows that maize intragenic modification is possible for increasing specific gene expression, preserving plant genome free of foreign DNA and achieving further significant savings in time and man labor for crop improvement.
Assuntos
Engenharia Genética/métodos , Genoma de Planta/genética , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Produtos Agrícolas/genética , DNA de Plantas/genética , Proteínas de Plantas/genéticaRESUMO
The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.
Assuntos
Receptor de Insulina/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Somatomedinas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima , Zea mays/metabolismo , Ligação Competitiva , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Fosforilação/efeitos dos fármacos , Células Vegetais/efeitos dos fármacos , Células Vegetais/enzimologia , Células Vegetais/metabolismo , Proteínas de Plantas/agonistas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptor de Insulina/agonistas , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Zea mays/citologia , Zea mays/crescimento & desenvolvimentoRESUMO
Rubisco activase (RCA) is a molecular chaperone present in maize as 43 kDa and 41 kDa polypeptides. They are encoded by two different genes comprising an identical ORF that corresponds to the 43 kDa RCA polypeptide, and their transcripts do not show putative splicing sites. To determine the origin of the 41 kDa polypeptide, leaf poly A(+) mRNA was in vitro translated. Results demonstrated de novo synthesis only for the 43 kDa RCA. Antibodies developed against peptides from either the carboxy- or the amino-terminal end of 43 kDa RCA showed by western blot that the 43 kDa polypeptide amino-terminal region is missing in the 41 kDa polypeptide, whereas both RCA polypeptides shared the carboxy-end region. Regulation of RCA polypeptide ratios was determined in plant leaves at different developmental stages and under stressing environmental conditions. Increased levels of 43/41 kDa RCA ratio were found in leaves under low light exposure, whereas this ratio declined under water stress. Measurements of chaperone activity either on each RCA polypeptide alone or in a mixture showed the functional relevance of different 43/41 kDa RCA polypeptide ratios. Greater chaperone activity was found for the 41 kDa than for the 43 kDa polypeptide. Taken together, these results indicate that 41 kDa RCA polypeptide formation is regulated by limited proteolysis of the 43 kDa RCA at its amino-terminal region. This pathway is sensitive to developmental and environmental signals, and seems to play a relevant function during plant stress.
Assuntos
Chaperonas Moleculares/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Zea mays/metabolismo , Ritmo Circadiano , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Luz , Fases de Leitura Aberta/fisiologia , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Plântula/metabolismo , Zea mays/crescimento & desenvolvimentoRESUMO
Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.
Assuntos
Proteínas de Plantas/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas/química , Zea mays/enzimologia , Animais , Reações Antígeno-Anticorpo , DNA Complementar/química , Ativação Enzimática/efeitos dos fármacos , Germinação/efeitos dos fármacos , Humanos , Soros Imunes/metabolismo , Insulina/farmacologia , Peso Molecular , Fosforilação/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Testes de Precipitina , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/imunologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/imunologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Homologia de Sequência de Aminoácidos , Sirolimo/farmacologia , Treonina/metabolismo , Zea mays/imunologiaRESUMO
Acidic ribosomal proteins (ARPs) are highly conserved phosphoproteins in eukaryotic organisms. They participate in translation regulation by interacting with eEF-2 elongation factors in the peptide elongation process. During maize germination, protein synthesis is tightly regulated by different mechanisms that are not yet clearly understood. The objective of this research is to characterize the expression patterns of the two maize ARPs (P1 and P2) and their phosphorylated status in germinating maize embryonic axes. Expression of P1 and P2 mRNA transcripts was analyzed by Northern blots with specific cDNA probes. Results indicated that both transcripts are among the mRNA stored pool of the quiescent axes and each displays a distinctive expression pattern during germination. P1 and P2 synthesis initiates very early in germination, as demonstrated by [(35)S]methionine pulse-labeling experiments. This synthesis was not insulin/IGF-stimulated as the synthesis of the bulk of ribosomal proteins that was responsive to this stimulus. P1 and P2 proteins were purified from ribosomes of maize embryonic axes and their physicochemical characteristics determined. A cytoplasmic pool of dephosphorylated P1 and P2 proteins was found in axes of quiescent and germinated stages that freely assembled into the ribosomes. IEF analysis of ARPs revealed one P1 (P1-1) and two P2 (P2-1 and P2-2) forms in the ribosomes of 24 h germinated axes. Kinetic studies of ARP phosphorylation during germination revealed a specific order of phospho-ARP appearance, suggesting that this process is under regulation within this period. It is concluded that P1 and P2 phosphorylation rather than ARP expression or assembly into ribosomes is the main step that regulates ARP function in axes during maize germination.