Abstract
The symbiotic nitrogen fixation process requires phosphorus. In order to test whether acid phosphatase is involved in the phosphorus use efficiency for N2 fixation an in situ RT-PCR technology was applied to common bean nodules. The recombinant line 115 of Phaseolus vulgaris, obtained from a cross between BAT477 and DOR364, was inoculated with Rhizobium tropici CIAT 899 and grown in hydroponic cultures for 5 weeks under P deficiency. The in situ RT-PCR was carried out with 50 µm nodule sections using specific primers to amplify a 250 bp sequence in the acid phosphatase mRNA. The amplified gene product was visualized with fluorescence microscopy using the ELF-97 kit which allowed detection of the alkaline phosphatase conjugated anti-digoxigenin Fab fragments. The signal corresponding to acid phosphatase could clearly be observed except in samples without RT (control). Transcripts were distributed specifically in: i) non infected cells of the infected zone; ii) the vascular trace localised in the cortex surrounding the infected zone; iii) the inner cortex (i.e. cell-layers localised between the vascular trace and the infected zone); iv) the external cortex (i.e. layers beyond the endodermis). By contrast, no signal was detected in the mid-cortex and the infected cells. It is concluded that the in situ RT-PCR methodology can be applied to localise plant transcripts in nodules. The expression of acid phosphatase in the inner cortex suggests that this enzyme may be involved in the osmoregulation of nodule permeability to O2 and consequently in the adaptation of the legume- rhizobia symbiosis to low P supply