Two full-length cDNAs encoding the glyoxysomal enzyme isocitrate lyase(ICL) were isolated from a λZAP cDNA library prepared frommegagametophyte mRNAs extracted from seeds imbibed at 30 °C for 8days. The cDNAs, designated Ptbs ICL 8 and Ptbs ICL 12, have openreading frames of 1740 and 1719 bp, with deduced amino acid sequences of580 and 573 residues, respectively. The predicted amino acid sequencesof Ptbs ICL 8 and Ptbs ICL 12 exhibit a 79% identity with each other,and have a greater than 75% identity with ICLs from variousangiosperm species. The C-termini of Ptbs ICL 8 and Ptbs ICL 12terminate with the tripeptide Ser-Arg-Met and Ala-Arg-Met, respectively,both being conserved variants of the type 1 peroxisomal targetingsignal. RNA blot and slot analysis revealed that Ptbs ICL 8 and PtbsICL 12 mRNAs were present at low levels in the megagametophyte of themature and stratified seeds, and that the level of both transcriptsincreased markedly upon seed germination. Protein blot analysisindicated that the steady-state level of ICL was low in the mature andstratified seed, then increased rapidly upon seed germination, peakingat around 8-10 days after imbibition (DAI). Changes in the level ofICL activity in cell-free extracts was similar to the steady-stateprotein content with the exception that ICL activity was not detected inmegagametophyte extracts of mature or stratified seeds. From 10-12 DAIwhen the megagametophyte tissue senesced, ICL activity decreased rapidlyto near undetectable levels. In contrast, steady-state levels of ICLprotein and mRNA remained relatively constant during megagametophytesenescence. In vivo synthesis of ICL protein was measured to shedlight on these differences. ICL immunoselected from[35S]-methionine labelled proteins indicated that ICL wassynthesized at very low levels during megagametophyte senescence.Together, the results show that loblolly pine ICL gene expression iscomplex. While temporal regulation appears to be primarilytranscriptional, it also involves a number of post-transcriptionalprocesses including at least one translational and/or post-translationalmechanism.
Plant Molecular Biology – Springer Journals
Published: Sep 29, 2004
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