Differential expression of cytosolic and plastid pyruvate kinase isozymes in tobacco

Abstract

The expression of cytosolic and plastid pyruvate kinase (PK; E.C. 2.7.1.40) in various tissues of tobacco (Nicotiana tabacum cv. Petit Havana SR1) was investigated. To facilitate this study, a cDNA clone for cytosolic pyruvate kinase (PKc) was isolated from a tobacco seed cDNA expression library. This cDNA has an open reading frame capable of encoding a 508‐amino acid polypeptide with a predicted molecular mass of 55.1 kDa. The deduced amino acid sequence has 76% identity with the sequence of potato PKc. Southern blot analysis shows that N. tabacum contains two copies of the PKc gene, each derived from the single gene found in its progenitors, N. tomentosiformis and N. sylvestris. Northern blots detected a 1.9‐kb band in flower, seed, root, stem and leaf tissues of N. tabacum. Immunoblotting using anti‐[castor oil seed (COS) PKc]‐immunoglobulin G (IgG) detected 58‐ and 56‐kDa polypeptides in all tobacco tissues examined. In developing seeds, mRNA levels were low except for a peak at 8 days post anthesis (dpa), whereas the highest level of the PKc polypeptides was detected 10–16 dpa. The persistence of PKc well after the peak in mRNA levels suggests that PKc is stable in developing seeds. Our previous studies have shown that leucoplast PK (PKp) mRNA could be detected throughout tobacco seed development as well as in somatic tissues. In developing seeds, steady state PKc mRNA levels showed a broad peak of accumulation from 8–20 dpa, reaching maximum levels at 16 dpa. In the present study, polypeptides of 63 and 60 kDa were detected on immunoblots probed with anti‐(COS PKp)IgG in developing tobacco seeds 10–20 dpa. The observed pattern of PKp protein accumulation closely correlated with the profile of steady state mRNA levels suggesting that this isoenzyme has a much greater rate of turnover than that of PKc. In contrast to PKc, there was no detectable accumulation of PKp protein at other times during seed development or in somatic tissues even though significant levels of PKp mRNA could be detected. The differences in the protein accumulation and mRNA expression patterns of PKc and PKp suggest that the tissue‐specific and developmental expression of these isoenzymes in tobacco may be controlled by independent transcriptional and post‐transcriptional mechanisms.