Physiological analysis of transgenic Arabidopsis thaliana plants expressing one nitrilase isoform in sense or antisense direction

摘要

IAA biosynthesis in Arabidopsis thaliana can proceed via different pathways, which involve indole-3-acetonitrile (IAN) as an intermediate. The enzyme nitrilase, which converts IAN to IAA, might be a key enzyme in this biosynthetic pathway. To elucidate the role of nitrilase during plant development, transgenic Arabidopsis plants, carrying one of the four nitrilase isoforms so far identified in Arabidopsis in either sense or antisense direction, were studied. The plants were analysed for their phenotype, auxin content, nitrilase transcript and protein amounts, and nitrilase activity. Analysis of free IAA content in seedlings did not reveal any prominent differences between the different lines, whereas the total IAA content was slightly increased inthe line overexpressing NIT2 (sNIT2). In two of the antisense lines (aNIT1 and aNIT2) the total auxin content was about 75% lower than in the wild type, while IAN was increased in these two antisense lines by about 2-fold. In sNIT2 plants the IAN content was slightly decreased compared with the wild type. When IAN was added to the medium, a strong increase in IAA content was found in sNIT2 seedlings (about 22-fold), which was much lower in wild type and antisense plants. In mature plants levels of freeIAA were decreased by about 25% and 50% in the antisense lines aNIT2 and aNIT1, respectively, whereas the IAA content in the other lines did not differ much. sNIT2/aNIT2 plants were further characterized. Both nitrilase 1 and nitrilase 2 accepted the same substrates, and overexpression of nitrilase did not lead to a visible phenotype because in vivo IAN might be the limiting factor in these plants. Application of IAN led to an increase in total nitrilase protein as shown by Western blotting. Using transgenic lines with promoter-GUS fusions for each NIT gene, it was shown that NIT1 is highly expressed in seedlings and NIT2 is strongly induced (21-fold) after IAN application.