REGULATION OF 2 LOBLOLLY-PINE (PINUS-TAEDA L) ISOCITRATE LYASE GENES IN MEGAGAMETOPHYTES OF MATURE AND STRATIFIED SEEDS AND DURING POSTGERMINATIVE GROWTH

Two full-length cDNAs encoding the glyoxysomal enzyme isocitrate lyase (ICL) were isolated from a lambda ZAP cDNA library prepared from mega gametophyte mRNAs extracted from seeds imbibed at 30 degrees C for 8 d ays. The cDNAs, designated Ptbs ICL 8 and Ptbs ICL 12, have open readi ng frames of 1740 and 1719 bp, with deduced amino acid sequences of 58 0 and 573 residues, respectively. The predicted amino acid sequences o f Ptbs ICL 8 and Ptbs ICL 12 exhibit a 79% identity with each other, a nd have a greater than 75% identity with ICLs from various angiosperm species. The C-termini of Ptbs ICL 8 and Ptbs ICL 12 terminate with th e tripeptide Ser-Arg-Met and Ala-Arg-Met, respectively, both being con served variants of the type 1 peroxisomal targeting signal. RNA blot a nd slot analysis revealed that Ptbs ICL 8 and Ptbs ICL 12 mRNAs were p resent at low levels in the megagametophyte of the mature and stratifi ed seeds, and that the level of both transcripts increased markedly up on seed germination. Protein blot analysis indicated that the steady-s tate level of ICL was low in the mature and stratified seed, then incr eased rapidly upon seed germination, peaking at around 8-10 days after imbibition (DAI). Changes in the level of ICL activity in cell-free e xtracts was similar to the steady-state protein content with the excep tion that ICL activity was not detected in megagametophyte extracts of mature or stratified seeds. From 10-12 DAI when the megagametophyte t issue senesced, ICL activity decreased rapidly to near undetectable le vels. In contrast, steady-state levels of ICL protein and mRNA remaine d relatively constant during megagametophyte senescence. In vivo synth esis of ICL protein was measured to shed light on these differences. I CL immunoselected from [S-35]-methionine labelled proteins indicated t hat ICL was synthesized at very low levels during megagametophyte sene scence. Together, the results show that loblolly pine ICL gene express ion is complex. While temporal regulation appears to be primarily tran scriptional, it also involves a number of post-transcriptional process es including at least one translational and/or post-translational mech anism.