Abstract
BackgroundThe hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression.ResultsThe transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines.ConclusionsTaken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.
Highlights
The hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils
The generated construct harboring HlWRKY1 cDNA was fused to the tetramer of enhancer of cauliflower mosaic virus (CaMV) 35S promoter and HlWDR1 cDNA was fused to mannopine synthase bidirectional promoter, respectively (Fig. 2) and was transferred into the A. tumefaciens strain GV3101 by electroporation [47]
Southern blot analysis using HlWDR1 and hygromycin phosphotransferase (HPT) gene confirmed that T-DNA was stably inserted into chromosomal DNA (Fig. 2b)
Summary
The hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. The female plants of hop produce cone-like inflorescences, commonly referred to as “hop cones” or “hops” contain a large number of highly metabolically active glandular trichomes (lupulin glands) on the inner side of bracts and bracteoles, which synthesize and/or secret specific secondary metabolites such as essential oils, bitter acids (humulone or α-acid and lupulone or β-acid) and prenylated flavonoids (xanthohumol and desmethylxanthohumol) during its phased maturation [7, 8]. After a three-year of normal growth, the hop cones completely develop and ripe with highest metabolome content [13]
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