Developmental regulation of arginase in loblolly pine (Pinus taeda L.) seeds during germination and early seedling growth

A 1366 base-pair arginase cDNA was isolated from a loblolly pine ( Pinus taeda L.) cDNA expression library constructed from whole seedling tissue collected 9 to 10 days after imbibition at 30°C (DAI₃₀). The cDNA contained an open reading frame predicting a 341 amino acid peptide with a molecular mass of 37.2 kilodaltons (kDa). When expressed in Escherichia coli, the loblolly pine arginase protein hydrolyzed arginine to ornithine and urea. The protein produced in E. coli was purified and the was partially purified from 9–12 DAI₃₀ shoot poles (cotyledons plus shoot apices) and has a molecular mass of 36.8 kDa. Based on a holoprotein size of 140 kDa, arginase is predicted to form a homotetramer.; Arginase protein and mRNA were found predominantly in the shoot poles (cotyledons plus shoot apices) of the seedling but was also detected in the root pole (hypocotyl and radicle) and the megagametophyte. Following germination, arginase protein and mRNA accumulated in the shoot pole. Arginase appeared to be regulated primarily at the RNA level; however, evidence of post-translational regulation was also observed.; An in vitro culture system was utilized to grow loblolly pine zygotic embryos with and without their associated megagametophytes. Removal of the megagametophyte caused a decrease in seedling growth and shoot pole protein accumulation, and caused a decrease in arginase enzyme activity, protein levels and mRNA abundance over the ten day culture period. The seedling was able to initiate arginase gene expression in the absence of the megagametophyte tissue, but the presence of the megagametophyte maintained and increased arginase transcript abundance during the latter stages of early seedling growth. Removal of the megagametophyte from seedlings grown in its presence caused a rapid decrease in arginase transcript abundance. This decrease could be minimized by the replacement of the megagametophyte with exogenous arginine. Application of arginine to seedlings grown in the absence of the megagametophyte was shown to stimulate arginase enzyme activity and induce arginase gene expression.