RESUMO
KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.
Assuntos
Carboxiliases/genética , Genes de Plantas , Família Multigênica , Ornithogalum/enzimologia , Ornithogalum/genética , Transcriptoma/genética , Açúcares de Uridina Difosfato/metabolismo , Uridina Difosfato Xilose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Compostos de Amônio/farmacologia , Biocatálise/efeitos dos fármacos , Soluções Tampão , Cálcio/farmacologia , Carboxiliases/química , Carboxiliases/metabolismo , Cromatografia Líquida de Alta Pressão , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Ornithogalum/efeitos dos fármacos , Espectroscopia de Prótons por Ressonância Magnética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Temperatura , Transcriptoma/efeitos dos fármacos , Açúcares de Uridina Difosfato/química , Uridina Difosfato Xilose/químicaRESUMO
Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of ß-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.
Assuntos
Flores/citologia , Lipídeos/química , Microtúbulos/metabolismo , Ornithogalum/citologia , Epiderme Vegetal/citologia , Flores/efeitos dos fármacos , Flores/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Ornithogalum/efeitos dos fármacos , Ornithogalum/ultraestrutura , Paclitaxel/farmacologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/ultraestrutura , Polissacarídeos/metabolismoRESUMO
AIMS: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. METHODS AND RESULTS: Several calibration steps were required to distinctly gate the GFP-labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. CONCLUSIONS: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of time- and cost-saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.
