RESUMO
Auxin/indole-3-acetid acid (Aux/IAA) proteins are short-lived transcriptional regulators that mediate their response through interaction with auxin response factors (ARF). Although 29 Aux/IAA proteins have been identified in Arabidopsis thaliana, their individual functions are still poorly understood and are largely defined by observed growth defects in gain-of-function mutant alleles. Here we present the isolation and characterization of a novel Aux/IAA protein in potato (Solanum tuberosum) that is named StIAA2. Down regulation of StIAA2 results in distinctive phenotypes that include, increased plant height, petiole hyponasty and extreme curvature of growing leaf primordia in the shoot apex. Gene expression analysis of transgenic plants with reduced StIAA2 transcript levels resulted in the identification of a number of genes with altered expression profiles including another member of the Aux/IAA gene family (StIAA). The phenotypes that were observed in the StIAA2 suppression clones can be associated with both common as well as unique functional roles among Aux/IAA family members indicating the importance of analyzing Aux/IAA expression in different plant species.
Assuntos
Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/biossíntese , Proteínas de Plantas/biossíntese , Solanum tuberosum/crescimento & desenvolvimento , Alelos , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas/genética , Ácidos Indolacéticos/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Reguladores de Crescimento de Plantas/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Solanum tuberosum/genéticaRESUMO
Using cDNA-AFLP RNA fingerprinting throughout potato tuber development, we have isolated a transcript-derived fragment (TDF511) with strong homology to plant steroid dehydrogenases. During in vitro tuberization, the abundance profile of the TDF shows close correlation to the process of tuber formation. However, when tuberization is inhibited by the addition of gibberellins (GAs) to the growth medium, the appearance of TDF511 in the fingerprint is delayed, then steadily increases in intensity during later stages of development. TDF511 was used to isolate the corresponding cDNA (CB12). The DNA and deduced amino-acid sequences of the cDNA show high homology to a fruit-ripening gene from tomato, a series of steroid dehydrogenases, and the maize Ts2 gene. A section of the cDNA was cloned in antisense orientation behind a 35S CaMV promoter and transformed into potato. Transgenic plants expressing the antisense gene showed significantly earlier emergence, an increase in height, and longer tuber shape. In vitro tuberization experiments reveal extended stolon lengths in comparison to the controls. The analysis of endogenous GA levels showed that the transgenic antisense plants have elevated levels of biologically active GAs and their respective precursors. We propose that this gene plays a role in the metabolism of plant-growth substances important for tuber life cycle and plant development.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , RNA Mensageiro/biossíntese , RNA de Plantas/biossíntese , Solanum tuberosum/genética , Sequência de Aminoácidos , Elementos Antissenso (Genética) , Clonagem Molecular , Impressões Digitais de DNA , DNA Complementar , Giberelinas/metabolismo , Dados de Sequência Molecular , Oxirredutases/biossíntese , Fenótipo , Filogenia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Polimorfismo de Fragmento de Restrição , Homologia de Sequência de Aminoácidos , Solanum tuberosum/enzimologia , Solanum tuberosum/crescimento & desenvolvimento , Esteroides/biossínteseRESUMO
Using the cDNA-AFLP method, we have isolated a transcript-derived fragment (TDF) which shows a differential expression pattern during tuber organogenesis of Solanum tuberosum L. The TDF was used to isolate a cDNA clone carrying a 1.5 kb insert and potentially coding for a 32.5 kDa peptide which, by homology, represents a potato homologue of an alpha-snap gene and has been designated Stsnap. Northern analysis showed that the Stsnap gene is expressed in actively dividing tissues throughout the potato plant. Analysis of genomic DNA from potato revealed that the Stsnap gene is likely to be a single-copy gene. The expression of antisense Stsnap cDNA under the control of the CaMV 35S promoter results in plants with an altered morphology such as curled leaves. Several of these transgenic lines also display cellular and developmental abnormalities with distinct changes in assimilate transport including accumulation of starch and soluble sugars in source leaves. We argue that these findings are consistent with the hypothetical function of the StSNAP gene product in vesicle targeting and fusion during plant development.
Assuntos
Proteínas de Transporte/genética , DNA Antissenso/genética , Proteínas de Membrana/genética , Solanum tuberosum/genética , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Distribuição TecidualRESUMO
Using a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLP)-derived technique for RNA fingerprinting (cDNA-AFLP), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP-glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuber-specific transcript-derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two lox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA-AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes.