Expression of Atropa belladonna Putrescine N-Methyltransferase Gene in Root Pericycle

Abstract

The cDNAs encoding putrescine N-methyltransferase (PMT), which catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine at the first committed step in the biosynthetic pathways of tropane alkaloids, were isolated from Atropa belladonna and Hyoscyamus niger. These PMTs, however, lacked the N-terminal tandem repeat arrays previously found in Nicotiana PMTs. AbPMT1 RNA was much more abundant in the root of A. belladonna than was AbPMT2 RNA. The 5'-flanking region of the AbPMT1 gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to A. belladonna. Histochemical analysis showed that GUS is expressed specifically in root pericycle cells and that the 0.3-kb 5'-upstream region was sufficient for pericycle-specific expression. Treatment of A. belladonna roots with methyl jasmonate did not up-regulate the expression of GUS or endogenous AbPMT genes. The regulation of tropane alkaloid biosynthesis is discussed and compared with that of nicotine biosynthesis.

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…RNA samples were derived from three pooled biological replicates. Notably, both the normalized cDNA pool for PE sequencing and the individual tissue samples for SE sequencing contained samples extracted from roots, the primary site of tropane alkaloid biosynthesis Suzuki et al, 1999aSuzuki et al, , 1999b.…”

“…Tropane alkaloid biosynthesis occurs in the roots of Solanaceae species with the transcripts of known biosynthetic genes preferentially or specifically accumulating in root tissues Suzuki et al, 1999aSuzuki et al, , 1999b. The A. belladonna transcriptome contains three cDNA libraries derived from root tissues: secondary root, primary tap root, and sterile seedlings (Table 2).…”

“…Several of the enzymes and genes involved in tropane alkaloid biosynthesis in the Solanaceae have been identified and characterized, and in all cases, both transcripts and proteins accumulate preferentially or exclusively in roots, the primary site of tropane alkaloid synthesis Kanegae et al, 1994;Nakajima and Hashimoto, 1999;Suzuki et al, 1999a). Several steps in tropane alkaloid biosynthesis remain to be identified, including the conversion of 4-methylaminobutanal to tropinone, the formation of phenyllactic acid and its condensation with tropine to form littorine, and the identification of the putative alcohol dehydrogenase that converts hyoscyamine aldehyde to hyoscyamine.…”

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

“…The 5'-flanking region of the Abpmt1 gene was fused to the β-glucuronidase (GUS) reporter gene and transferred to A. belladonna. Histochemical analysis showed that GUS is expressed specifically in root pericycle cells and the 0.3 kb 5'-upstream region was sufficient for pericycle-specific expression (Suzuki et al 1999).…”

Characterization of the spermidine synthase‐related gene family in Arabidopsis thaliana

“…Val236 might be important for precise positioning of the substrate for the reaction. It is unlikely that SPDS3 possesses PMT activity because 7 amino acids conserved among all available PMT sequences [22] are not conserved in SPDS3 and the amino acids at the corresponding positions in SPDS3 (Lys124, Asp125, Glu126, Asp154, Arg159, His164, and Val167) are nearly identical to those of known plant spermidine synthases.…”