The “putative” role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.)

Abstract

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

Dissection of Dynamic Transcriptome Landscape of Leaf, Bract, and Lupulin Gland in Hop (Humulus lupulus L.)

“…In a way similar to BA and terpenoids biosynthesis, 4CL generated from phenylpropanoid biosynthesis pathway condensed with three molecules of malonyl-CoA with the aid of enzyme CHS_H1 to yield the aromatic core of PF [25]. RNA-seq analysis revealed that TFs such as HlbHLH2, HlWDR1, HlWRKY1, HlMYB1, HlMYB2, HlMYB3, HlMYB7, and HlMYB8 [25,26,28] essential for regulating PF biosynthesis were present in LF, BR, and LG transcriptomes, which was in agreement with previous studies that LF contain low but detectable level of PF [9]. Nevertheless, 4.7% (2.076) of the assembled transcripts, 5.2% (1975) LF transcripts, 4.8% (637) BR transcripts, 4.9% (375) LG transcripts were annotated to encode for TFs, which could be classified into 93 TF families, including MYB, bZIP, WRKY, C2H2, and bHLH families.…”

“…The elevated levels of PAL and CHS_H1 in BR and LG compared to LF suggested that redirection of carbon flux toward flavonoid biosynthesis defines the size of the PF, flavonols, and anthocyanin in BR and LG tissues in hop. The branch point metabolite naringenin chalcone, which colimits the flux between PF and flavanols/anthocyanin biosynthetic products is regulated by the downstream enzyme activity and TFs [9,25,26]. The higher transcript levels of OMT, HlPT1 and core TFs of MBW and WW complexes in LG compared to BR and LF suggested thatthe major metabolic flux shift of primary metabolism was towards PF biosynthesis in LG tissues.…”

“…In flavonoid biosynthesis pathway the shikimate pathway serving as a starting point and sequential action of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaroyl CoA-Ligase (4CL) produce 4-coumaroyl-CoA substrate, which defines a metabolic key branch-point for the synthesis of several major classes of flavonoids, including xanthohumol (PF), flavonol and anthocyanins biosynthesis in hop [9]. The xanthohumol biosynthesis pathway is mediated by trichome-specific chalcone synthase (CHS_H1) [22], HlPT1 [23] and O-methyltransferase 1 (OMT1) [9] enzymes with fine-tuned participation of different types of TFs, belonging to bHLH, MYB, WDR, and WRKY families [24][25][26]. The activation of CHS_H1 promoter is tightly regulated by heterotrimeric ternary MBW (HlMyb3/HlbHLH2/HlWDR1 or HlMYB2/HlbHLH2/HlWDR1) or binary (HlbHLH2/HlWDR1) transcription activation complex [26] ( Figure 2).…”

“…The xanthohumol biosynthesis pathway is mediated by trichome-specific chalcone synthase (CHS_H1) [22], HlPT1 [23] and O-methyltransferase 1 (OMT1) [9] enzymes with fine-tuned participation of different types of TFs, belonging to bHLH, MYB, WDR, and WRKY families [24][25][26]. The activation of CHS_H1 promoter is tightly regulated by heterotrimeric ternary MBW (HlMyb3/HlbHLH2/HlWDR1 or HlMYB2/HlbHLH2/HlWDR1) or binary (HlbHLH2/HlWDR1) transcription activation complex [26] ( Figure 2). Recently, the transgenic analysis suggested the potential role of other binary transcription activation complex (HlWRKY1/HlWDR1) in positive regulation of MBW ternary complex and OMT1 based on significantly increased levels of PF and BA content in the hop [27].…”

Genome-wide transcriptomic analysis reveals insights into the response to Citrus bark cracking viroid (CBCVd) in hop (Humulus lupulus L.)

“…(www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 September 2018 doi:10.20944/preprints201809.0553.v1In our study, majority of genes involved in primary and secondary metabolism pathways were fluctuated in response to the CBCVd-infection. At the link between primary and secondary metabolism, phenylalanine ammonia-lyase (PAL) serve as first enzyme which is involved in biosynthesis of secondary metabolites such as flavonoids, anthocyanins, prenylflavonoids, and bitter acids in hop[79].Flavonoids constitute a large group of phenolic secondary metabolites and have been shown to have diverse biological functions such as symbiotic nitrogen fixation, pollination and floral pigmentation defense against different biotic and abiotic stresses etc. [80].…”