RESUMEN
Sixteen post-harvest upregulated genes from sugar beet comprising five novel sequences were isolated by subtractive cloning. Transcription profiles covering a period of up to 49 days after harvest under controlled storage conditions and in field clamps are reported. Post-harvest induced genes are involved in wound response, pathogen defense, dehydration stress, and detoxification of reactive oxygen species. An early induction of a cationic peroxidase indicates a response to post-harvest damage. Wound response reactions may also involve genes required for cell division such as a regulator of chromatin condensation and a precursor of the growth stimulating peptide phytohormone phytosulfokine-alpha. Surprisingly, also three putative non-protein coding genes were isolated. Two of these genes show intron specific and storage temperature dependent splicing of a precursor mRNA. The temperature dependent splicing of an intron containing sugar beet mRNA is also maintained in transgenic Arabidopsis thaliana. The storage induced genes are integrated into a model that proposes the response to several post-harvest stress conditions. Temperature regulated splicing may be a mechanism to sense seasonal temperature changes.
Asunto(s)
Beta vulgaris/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/genética , Empalme Alternativo , Beta vulgaris/fisiología , Clonación Molecular , Cartilla de ADN , Genes de Plantas , Hibridación de Ácido Nucleico , Raíces de Plantas/fisiología , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN de Planta/genética , Estaciones del AñoRESUMEN
The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.
Asunto(s)
Beta vulgaris/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Beta vulgaris/anatomía & histología , Beta vulgaris/metabolismo , Clonación Molecular , Genes Reporteros , Hipocótilo/anatomía & histología , Hipocótilo/metabolismo , Luciferasas/análisis , Proteínas de Plantas/metabolismo , Raíces de Plantas/anatomía & histología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/anatomía & histología , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/anatomía & histología , Nicotiana/genéticaRESUMEN
BACKGROUND: Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation. RESULTS: Suppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet. CONCLUSIONS: This work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed.
Asunto(s)
Beta vulgaris/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes Reporteros/genética , Complejos de Proteína Captadores de Luz/genética , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genética , ADN Complementario/genética , Genes de Plantas/genética , Luz , Hibridación de Ácido Nucleico/métodos , Especificidad de Órganos/genética , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
The taproot from sugar beet (Beta vulgaris L.) undergoes a specific developmental process to function as a food storage organ. Suppression Subtractive Hybridization (SSH) was utilized for the isolation of cDNA fragments for taproot expressed genes. Isolation and molecular analysis of six cDNAs encoding the complete gene product revealed that these genes comprise homologues of a drought-inducible linker histone, a homologue of a major latex-like protein, a phosphoenolpyruvate carboxylase kinase, a putative vacuolar processing enzyme, a thaumatin-like protein and an alanine- and glutamic acid-rich protein. All genes are transcribed in taproots while transcription in leaves is low or undetectable.