Soybean UreD and UreF Looking for a Locus

UreD, UreF and UreG were annotated by the S. pombe genome project (http://www.sanger.ac.uk/Projects/S_pombe/). We exploited UreD and UreF as (i) heterologous probes to recover the soybean cDNA homologs, and (ii) a source of PCR primers to confirm their presence in genomic DNA clones (generously provided by Dr N. Honey, Massey Univ., NZ) complementing urease mutants of S. pombe (Kinghorn, Fluri 1984). We are now attempting to correct S. pombe mutants, identified as ureD, ureF and ureG, with the soybean orthologs.

RT/PCR analysis of an eu2 mutant allele revealed no alteration in its UreD or UreF ORF. If the sequence of a second mutant allele, and mRNA quantification, both indicate that UreD and UreF proteins are functional in eu2/eu2 we are obliged to consider that Eu2 encodes a new accessory protein. In vitro activation assays (above) indicate that Eu2 interacts with Eu3 (the UreG protein) (Polacco et al. 1999), and eu2/eu2 appears to be able to take up and translocate Ni (Holland, Polacco 1992). No UreE ORF has been identified in any eukaryotic genome to date.

RT/PCR analysis of mutant AJ6, tentatively assigned to new locus Eu5 (Table 2), revealed that 4 of 14 cDNA clones with UreD homology lacked a 15 AA region which defines an exon in Arabidopsis (Figure 2). These represent mRNA transcripts from the developing cotyledon, which is urease-positive (Table 2). Ten of ten transcripts from wild type were normal. We are now examining transcripts from leaf tissue, which is urease-negative in AJ6.

Arab 1

tomato soy tgkvvvekvggrst metgkv|vekvî vvvekvggrs tdvvwiy|itygggivsgdsis_

vapsBtdvvwiyaitygggivsgdsi tdEvwIyaKygggivsgdHis

Arab tomato soy

Figure 2. Alignment of the first 120 AA (out of -295) of UreD of Arabidopsis (Arab), tomato and soybean (soy). The 15-AA run, missing in 4 of 14 AJ6 transcripts, represents the excision of an entire exon according to the splice sites of Arabidopsis (between introns 3 and 4).

Figure 2. Alignment of the first 120 AA (out of -295) of UreD of Arabidopsis (Arab), tomato and soybean (soy). The 15-AA run, missing in 4 of 14 AJ6 transcripts, represents the excision of an entire exon according to the splice sites of Arabidopsis (between introns 3 and 4).

If we posit that splicing of the UreD transcript is impaired in AJ6, the site of the AJ6 lesion still remains to be determined. Since the sequence of normally spliced variants shows no alteration it is possible that an intron junction sequence is impaired. Regardless of the role that alternative splicing plays in the AJ6 UreD transcript, alternative splicing may be a general mechanism among dicots to maintain UreD protein at a low level. In Klebsiella, this is achieved by a sub-optimal ribosome-binding site and an unusual start codon (Park et al. 1994). We have observed a high frequency of mis-spliced UreD transcripts in tomato and Arabidopsis, often leading to the introduction of a stop codon immediately after exon IV in the latter.

In conclusion, plant urease activation differs from bacterial: (i) Plants have to activate more than one urease, usually produced at quite different levels, (ii) Plant UreG has a His-rich N-extension, like bacterial HypB and unlike all bacterial UreGs to date, (iii) Plants don't seem to have Ni-binding UreE. (iv) Plants have alternative splicing of UreD, possibly to maintain it at low levels, (v) Plants may have another accessory protein, Eu2.

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