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Purification of multiple functional leaf-actin isoforms from Phaseolus vulgaris L.

ABSTRACT: Plant actins show diversity in their gene sequences, protein isovariants and tissue distribution in eukaryotes. Besides general difficulties with the isolation of proteins from plant material (i.e. the presence of a cell wall and high proteolytic activity), the actin concentration in any vegetative plant tissue is much lower than in cytoplasmic animal tissues. In this study, we adapted a deoxyribonuclease I-Sepharose affinity purification scheme and we were able to enrich and isolate multiple functional plant actin isovariants from common bean leaves (Phaseolus vulgaris). Urea (4 M) elution proved that the DNase I column was able to bind at least eight actin isoforms with pI values ranging from 5.5 to 5.9, as observed by two-dimensional Western blots. Three of the most acidic actin isoforms, with pI values of approximately 5.6-5.7, were eluted partially with 0.75 M urea. The purified actin was also able to bind leaf and rabbit muscle profilin, phalloidin and DNase I. Moreover, the protein could polymerize into filaments that contained the main isoforms eluted from the column. The average actin recovery using this procedure was approximately 4-8 microg from 20 g of fresh tissue, of which at least 80% was able to form filaments. This is the first report of the purification of multiple plant-actin isoforms that are functional by the criteria of both binding to other ligands and polymerization.

SUBMITTER: Diaz-Camino C

PROVIDER: S-EPMC1220591 | biostudies-other | 1999 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundCommon bean (Phaseolus vulgaris L.) is the most important grain legume for human diet worldwide and the angular leaf spot (ALS) is one of the most devastating diseases of this crop, leading to yield losses as high as 80%. In an attempt to breed resistant cultivars, it is important to first understand the inheritance mode of resistance and to develop tools that could be used in assisted breeding. Therefore, the aim of this study was to identify quantitative trait loci (QTL) controlling resistance to ALS under natural infection conditions in the field and under inoculated conditions in the greenhouse.ResultsQTL analyses were made using phenotypic data from 346 recombinant inbreed lines from the IAC-UNAxCAL 143 cross, gathered in three experiments, two of which were conducted in the field in different seasons and one in the greenhouse. Joint composite interval mapping analysis of QTLxenvironment interaction was performed. In all, seven QTLs were mapped on five linkage groups. Most of them, with the exception of two, were significant in all experiments. Among these, ALS10.1DG,UC presented major effects (R2 between 16%-22%). This QTL was found linked to the GATS11b marker of linkage group B10, which was consistently amplified across a set of common bean lines and was associated with the resistance. Four new QTLs were identified. Between them the ALS5.2 showed an important effect (9.4%) under inoculated conditions in the greenhouse. ALS4.2 was another major QTL, under natural infection in the field, explaining 10.8% of the variability for resistance reaction. The other QTLs showed minor effects on resistance.ConclusionsThe results indicated a quantitative inheritance pattern of ALS resistance in the common bean line CAL 143. QTL x environment interactions were observed. Moreover, the major QTL identified on linkage group B10 could be important for bean breeding, as it was stable in all the environments. Thereby, the GATS11b marker is a potential tool for marker assisted selection for ALS resistance.

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Purification of multiple functional leaf-actin isoforms from Phaseolus vulgaris L.

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