Cloning and expressional characterization of soybean GmLls1 gene

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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 the 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 p reviously 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 suggest that the expression of GmLls1 was controlled by the RLPK2-mediated se-nescence signaling pathway. The observation that the detached rlpk2-RNAi transgenic leaves exhibited light-dependent necrotic lesions, which featured the
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