Sequence analysis of a "true" chalcone synthase (chs_H1) oligofamily from hop (Humulus lupulus L.) and PAP1 activation of chs_H1 in heterologous systems.
Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for "true" chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the "native" chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome.
Matousek
Jaroslav
J
Biological Centre AS CR, Institute of Plant Molecular Biology, Branisovska 31, 37005 Ceské Budejovice, Czech Republic.
Vrba
Lukás
L
Skopek
Josef
J
Orctova
Lidmila
L
Pesina
Karel
K
Heyerick
Arne
A
Baulcombe
David
D
De Keukeleire
Denis
D
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000096:biosynthesis; D029681:Arabidopsis Proteins; D001483:Base Sequence; D004789:Enzyme Activation / Q000187:drug effects; D027582:Humulus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000494:pharmacology*; D014466:Ultraviolet Rays
D000217:Acyltransferases; D000872:Anthocyanins; D029681:Arabidopsis Proteins; D000073718:Pancreatitis-Associated Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C502612:AT1G56650 protein, Arabidopsis; C000612141:REG3A protein, human; C020141:flavanone synthetase