Cloning and expressional characterization of soybean GmLls1 gene

Maize Lls1 (lethal leaf-spot 1) gene and its Arabidopsis orthologue AtAcd1 have been sug-gested to encode pheide a oxygenase (PaO), a key enzyme catalyzing chlorophyll breakdown. To further elucidate the molecular mechanism that regulates chlorophyll catabolism during soybean leaf senes-cence, a soybean Lls1 homolog was cloned and designated as GmLls1 (GenBank Accession No. DQ154009). Database searches using the deduced protein sequence revealed that it was highly ho-mologous to Lls1 genes or Lls1 orthologues in Arabidopsis, maize, cowpea and tomato. Structural analysis of the predicted GmLLS1 protein revealed typical Rieske [2Fe-2S] and mononuclear iron-bind- ing domains as well as the C-terminus CxxC motif that were conserved in and featured PaO homo-logues. RT-PCR results showed that the transcription of GmLls1 was up-regulated in all the three tested senescence systems: the natural leaf senescence process, the dark-induced primary leaf senescence and senescing cotyledons. We have previously de-scribed the involvement of an LRR receptor-like kinase (RLK), RLPK2 in regulation of soybean leaf senescence. Here we report that the expression of GmLls1 gene was dramatically down-regulated by the RNAi-mediated suppression of rlpk2 and, as ex-pected, greatly up regulated by the CaMV 35S pro-moter derived overexpression of this RLK gene. These results suggested that the expression of GmLls1 was controlled by the RLPK2-mediated se-nescence signaling pathway. The observation that the detached rlpk2-RNAi transgenic leaves exhibitedlight-dependent necrotic lesions, which featured

Maize Lls1 （lethal leaf-spot 1） gene and its Arabidopsis orthologue AtAcdl have been suggested to encode pheide a oxygenase （PaO）, a key enzyme catalyzing chlorophyll breakdown. To further elucidate the molecular mechanism that regulates chlorophyll catabolism during soybean leaf senescence, a soybean Lls1 homolog was cloned and designated as GmLls1 （GenBank Accession No. DQ154009）. Database searches using the deduced protein sequence revealed that it was highly homologous to Lls1 genes or Lls1 orthologues in Arabidopsis, maize, cowpea and tomato. Structural analysis of the predicted GmLLS1 protein revealed typical Rieske [2Fe-2S] and mononuclear iron-binding domains as well as the C-terminus CxxC motif that were conserved in and featured PaO homoIogues. RT-PCR results showed that the transcription of GmLls1 was up-regulated in all the three tested senescence systems： the natural leaf senescence process, the dark-induced primary leaf senescence and senescing cotyledons. We have previously described the involvement of an LRR receptor-like kinase （RLK）, RLPK2 in regulation of soybean leaf senescence. Here we report that the expression of GmLIsl gene was dramatically down-regulated by the RNAi-mediated suppression of rlpk2 and, as expected, greatly up regulated by the CaMV 35S promoter derived overexpression of this RLK gene. These results suggested that the expression of GmLIsl was controlled by the RLPK2-mediated senescence signaling pathway. The observation that the detached rlpk2-RNAi transgenic leaves exhibited light-dependent necrotic lesions, which featured thells1 mutation, further supported this hypothesis.