Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant

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Abstract
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 library. Sequence analysis of Vigna uricase (VN‐35) cDNA revealed 90% homology to that of soybean. The VN‐35 cDNA was inserted in the antisense orientation downstream of the CaMV—35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes. Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild‐type shoot. Expression of antisense VN‐35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN‐35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing β‐glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N‐35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN‐35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.
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