Phytochemtstry, Vol 28, No 4, pp 1031-1035, 1989 Prtnted anGreat Brltaln 003 I -9422/89 $3 00 + 0 00 0 1989 PergamonPressplc EVIDENCE FOR THE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA DE NOVO SYNTHESIS OF POLY(A) POLYMERASE IN GERMINATED WHEAT EMBRYOS SUJATA LAKHANI,* ROMA KAPOOR, NEERAJ VERMA and R C. SAcHARt Biochemistry and Molecular Btology Laboratory, Department of Botany, Umverstty of Delht, Delht 110007, India (Recetued zn revised form 30 August 1988) Key Word Index-Wheat embryos; poly(A)+RNA, poly(A) polymerase, de nouo synthesis, in uiuo labelhng zyxwvutsrqponmlkjihgfed Abstract-A linear rise m the activity of poly(A) polymerase occurs followmg germination of excised wheat embryos. A roughly three-fold increase in poly(A) polymerase activity was paralleled by a rise (2.4-fold) m the relative abundance of poly(A)+RNA isolated from 72 hr germinated embryos over that at six hr after tmbibition. Administration of cyclohextmide (20 pg/ml) to germinated embryos stgmficantly mhtbtted (80%) poly(A) polymerase activity with a dramatic decline (77%) in the levels oftotal poly(A)+RNA. This indicated that the relative abundance of poly(A)+RNA ts regulated by the modulation of poly(A) polymerase acttvtty The enhancement of poly(A) polymerase activity followmg embryo germinatton is primarily achieved through de nouo synthesis of the enzyme. This has been conclusively shown by rn uzuo labellmg of the newly synthesized total proteins with “SO:- m wheat embryos and ultimately recovering the label m the purtfied preparatton of poly(A) polymerase. Fracttonation of purified poly(A) polymerase on native polyacrylamtde gels revealed a single protein band, thereby estabhshmg the electrophoretic homogenetty of the enzyme preparatton. Autoradiography of this gel showed a single radioactive band which corresponded with the protein band of the purified poly(A) polymerase. Further characterization of the purified labelled poly(A) polymerase was obtained by acid hydrolysis of the enzyme protein followed by the chromatographic separation of the 35S Iabelled amino acids. Autoradiography of the chromatogram revealed the presence ofthe label in the cysteine residues of poly(A) polymerase These studies clearly indicated that de nmo synthesis of poly(A) polymerase indeed occurs during growth of wheat embryos. INTRODUCTION Poly(A) polymerase plays an important role in the post- transcriptional polyadenylation of mRNA and hnRNA m germinating seeds [l] The enzyme catalyses the covalent addition of AMP residues from its substrate ATP to the 3’ terminus of primer RNA [Z]. Among plants, poly(A) polymerase activity has been reported m tobacco [3], maize [4], wheat [S] and pea [6]. Two dtstmct isozymes of poly(A) polymerase have been reported from wheat seedling chloroplasts and nuclear fractions. These iso- zymes differ in then primer RNA requirement [7]. Phytohormonal regulatton of poly(A) polymerase by gibberelhc acid (GA,) has been reported from this labora- tory m wheat aleurone layers [S], germinating excised wheat embryos [9] and pea epicotyls [6]. Gibberelhc actd also increases the relative abundance of poly(A)+RNA in barley and wheat aleurone layers [IO, 111, and m germinated seeds of maize [12], castor bean [13] and hazel [14]. It has been suggested that the GA,-induced poly(A) polymerase activity is responstble for the increase in the levels of poly(A)+RNA in wheat aleurone layers and excised embryos [8, 93. Inhibitor data (CHI) has indt- cated that the GA,-stimulated poly(A) polymerase activ- ity is dependent on de nova protein syntheses. However, * Present address ICGEB, NII Campus, New Delhi-67, Indta. t Author to whom correspondence should be addressed blocking of transcription by cordycepm in GA,-treated aleurone layers and embryos of wheat failed to inhibit poly(A) polymerase activity. This suggested that fresh transcription is not mandatory for the hormone-triggered poly(A) polymerase activity both in wheat aleurone layers [S] and excised embryos [9, 151. Modulatton of poly(A) polymerase activity could also occur by the post-translattonal structural modification of the enzyme. Rose and Jacob [16, 173 have purified the phosphorylated form of poly(A) polymerase from rat liver and hepatoma cells. The hepatoma enzyme was phos- phorylated to a greater extent in viuo than the liver enzyme. Phosphorylation of the enzyme resulted in the activation (7-fold) of poly(A) polymerase isolated from the rat liver cells. Phosphorylation dtd not alter the extent, but augmented the rate of poly(A) synthesis as a result of increased affinity of the enzyme for tts poly- nucleotide primer [ 16, 171. So far, the enhancement of poly(A) polymerase follow- mg germination of wheat embryos was shown to be dependent on de novo protein synthesis. This was based on the circumstanttal evidence obtained by inhibitor studies. We now provide a more conclustve proof for the de novo synthesis of poly(A) polymerase following germination of excised wheat embryos. The enzyme m- duction was paralleled by an increase m the total popu- lation of poly(A)+RNA. Repression of the newly syn- thesized poly(A) polymerase by a translation inhibitor (CHI) simultaneously lowered the levels of total poly(A)+ RNA. 1031