Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.)

Abstract

SummaryArginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5¢-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately ±117 kcal mol ±1 ) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/ translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the ®rst report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for ef®cient translation.

Tissue-specific expression of olive S-adenosyl methionine decarboxylase and spermidine synthase genes and polyamine metabolism during flower opening and early fruit development

“…Alternatively, SPDS might be regulated by post-transcriptional mechanisms, as during apple fruit development (Zhang et al 2003) or during nitrogen-fixing root-nodule development of Lotus japonicus (Efrose et al 2008). Translational and/or post-translational regulation of other enzymes in PA biosynthesis, such as ADC (Chang et al 2000) and ODC (Kwak and Lee 2001), has also been documented. Experiments are still needed to elucidate how the protein levels and the enzyme activity of SPDS are regulated during early fruit development.…”

Change in Free Polyamine Contents and Expression Profiles of Two Polyamine Biosynthetic Genes in Citrus Embryogenic Callus under Abiotic Stresses

“…the concrete reason for this result needs to be clarified. As a matter of fact, such phenomenon may be reasonable considering the fact that the rate-limiting enzymes in many metabolic pathways are regulated transcriptionally, translationally or post-translationally (3,13).…”

Known and novel post-transcriptional regulatory sequences are conserved across plant families

“…The entire polyamine synthesis pathway is a ''hotspot'' for translational control that incorporates CPuORFs, non-CPuORFs, non-AUG uORFs, and ribosomal frameshifting (Ivanov et al 2010). In plants, thermospermine synthase is regulated by a uORF-dependent transcription factor (Imai et al 2006), and arginine decarboxylase contains a conserved uORF (Chang et al 2000), as do several catabolic polyamine oxidases (Hayden and Jorgensen 2007;Table 3;Supplemental Fig. S2).…”