DIFFERENTIAL EXPRESSION OF CYTOSOLIC AND PLASTID PYRUVATE-KINASE ISOZYMES IN TOBACCO

The expression of cytosolic and plastid pyruvate kinase (PK; E.C. 2.7. 1.40) in various tissues of tobacco (Nicotiana tabacum cv. Petit Havan a SRI) was investigated. To facilitate this study, a cDNA clone for cy tosolic pyruvate kinase (PKc) was isolated from a tobacco seed cDNA ex pression library. This cDNA has an open reading frame capable of encod ing a 508-amino acid polypeptide with a predicted molecular mass of 55 .1 kDa. The deduced amino acid sequence has 76% identity with the sequ ence of potato PKc. Southern blot analysis shows that N. tabacum conta ins two copies of the PKc gene, each derived from the single gene foun d in its progenitors, N. tomentosiformis and N. sylvestris. Northern b lots detected a 1.9-kb band in flower, seed, root, stem and leaf tissu es of N. tabacum. Immunoblotting using anti-[castor oil seed (COS) PKc ]-immunoglobulin G (IgG) detected 58- and 56-kDa polypeptides in all t obacco tissues examined. In developing seeds, mRNA levels were low exc ept for a peak at 8 days post anthesis (dpa), whereas the highest leve l of the PKc polypeptides was detected 10-16 dpa. The persistence of P Kc well after the peak in mRNA levels suggests that PKc is stable in d eveloping seeds. Our previous studies have shown that leucoplast PK (P Kp) mRNA could be detected throughout tobacco seed development as well as in somatic tissues. In developing seeds, steady state PKp mRNA lev els showed a broad peak of accumulation from 8-20 dpa, reaching maximu m levels at 16 dpa. In the present study, polypeptides of 63 and 60 kD a were detected on immunoblots probed with anti(COS PKp)-IgG in develo ping tobacco seeds 10-20 dpa. The observed pattern of PKp protein accu mulation closely correlated with the profile of steady state mRNA leve ls suggesting that this isoenzyme has a much greater rate of turnover than that of PKc. In contrast to PKc, there was no detectable accumula tion of PKp protein at other times during seed development or in somat ic tissues even though significant levels of PKp mRNA could be detecte d. The differences in the protein accumulation and mRNA expression pat terns of PKc and PKp suggest that the tissue-specific and developmenta l expression of these isoenzymes in tobacco may be controlled by indep endent transcriptional and post-transcriptional mechanisms.