EXPRESSION OF ANTISENSE NODULIN-35 RNA IN VIGNA-ACONITIFOLIA TRANSGENIC ROOT-NODULES RETARDS PEROXISOME DEVELOPMENT AND AFFECTS NITROGEN AVAILABILITY TO THE PLANT

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3. ) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA l ibrary. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% h omology to that of soybean. The VN-35 cDNA was inserted in the antisen se orientation downstream of the CaMV-35S promoter, and transgenic hai ry roots were formed on Vigna plants using Agrobacterium rhizogenes. I nfection with Bradyrhizobium (cowpea) gave rise to root nodules on tra nsgenic hairy roots supported by the wild-type shoot. Expression of an tisense VN-35 RNA was detected in transgenic nodules on individual roo ts using polymerase chain reaction (PCR). The nodules expressing antis ense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vecto r containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysi s and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expr essing antisense VN-35 RNA. These data suggest that a reduction in ure ide biosynthesis limits the availability of symbiotically reduced nitr ogen to the plant. The nodules of tropical legumes appear to be specia lized in nitrogen assimilation and are developmentally controlled to p roduce and transport ureides.