Sequence Analysis of a “True” Chalcone Synthase (chs_H1) Oligofamily from hop (Humulus lupulus L.) and PAP1 Activation of chs_H1 in Heterologous Systems

Abstract

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.

Identification and characterization of microRNAs in Humulus lupulus using high-throughput sequencing and their response to Citrus bark cracking viroid (CBCVd) infection

“…Hop is an economically important crop for the brewing industry and over the last few years has gained increasing attention due to its potential applications to pharmaceutical industries [31, 32]. In spite of that, hop miRNAs and their association with various biological processes are widely unexploited, although in the past few years several conserved miRNAs and species-specific miRNAs have been identified in various plant species with the aid of high-throughput sequencing methods.…”

“…The lupulin glands of female inflorescences (also called cones or strobiles) consist of biosynthetic cells, which synthesize specific and complex metabolomes known as terpenophenolics (hop bitter acids and prenylflavonoids) and terpenoids (essential oil components), which serve as an important raw component in beer for their unique bitterness, flavour and preservative activity [31]. In addition, hop has been attributed with health benefits, including as a relaxative and sleep inducer, an anti-inflammatory agent, an estrogenic effect, antioxidant activity and cancer chemo-preventive properties [32].…”

Combinatorial analysis of lupulin gland transcription factors from R2R3Myb, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus LupulusL.)

“…Moreover, recombinant CHS_H1 can utilize isovaleryl and isobutyryl CoA substrates, albeit at a low rate, and, therefore, could also be involved in the biosynthesis of hop bitter acids [15]. CHS_H1 is encoded by an oligofamily of genes having very specific expression in hop cones [16]. Other chalcone synthase-like enzymes associated with lupulin glands have been described.…”

Expression of SANT/HTH Myb mRNA, a plant morphogenesis-regulating transcription factor, changes due to viroid infection

“…benthamiana plants were transformed with Agrobacterium tumefaciens LBA4404 bearing the plant expression pLV07 construct (Matoušek et al, 2006) (clone 3907) containing the SlMyb gene driven by 35S promoter. SlMyb was cloned from tomato cv.…”

“…SlMyb was cloned from tomato cv. Heinz 1706 into vector pLV68 (Matoušek et al, 2006) using primers 1 and 2 listed in Table S1; subsequently, the expression cassette was re-cloned to pLV07 using restriction enzymes PacI and AscI (Fig. 5).…”