HlMyb3, a Putative Regulatory Factor in Hop (Humulus lupulus L.), Shows Diverse Biological Effects in Heterologous Transgenotes

Abstract

A hop-specific cDNA library from glandular tissue-enriched hop cones was screened for Myb transcription factors. cDNA encoding for R2R3 Myb, designated HlMyb3, was cloned and characterized. According to the amino acid (aa) sequence, HlMyb3 shows the highest homology to GhMyb5 from cotton and is unrelated to the previously characterized HlMyb1 from the hop. Southern blot analyses indicated that HlMyb3 is a unique gene, which was detected in various Humulus lupulus cultivars, but not in Humulus japonicus. Reverse transcription and real-time PCR revealed the highest levels of HlMyb3 mRNA in hop cones at a late stage of maturation and in colored petiole epidermis, while the lowest levels were observed in hop flowers. Two alternative open reading frames starting in the N-terminal domain of HlMyb3, encoding for proteins having 269 and 265 amino acids with apparent molecular masses of 30.3 and 29.9 kDa, respectively, were analyzed as transgenes that were overexpressed in Arabidopsis thaliana, Nicotiana benthamiana, and Petunia hybrida plants. Transformation with the longer 269 aa variant designated l-HlMyb3 led to a flowering delay and to a strong inhibition of seed germination in A. thaliana. Nearly complete flower sterility, dwarfing, and leaf curling of P. hybrida and N. benthamiana l-HlMyb3 transgenotes were noted. On the contrary, the shorter 265-aa-encoding s-HlMyb3 transgene led in A. thaliana to the stimulation of initial seed germination, to fast initiation of the lateral roots, and to quite specific branching phenotypes with many long lateral stems formed at angles near 90 degrees . Limited plant sterility but growth stimulation and rather branched phenotypes were evident for s-HlMyb3 transgenotes of P. hybrida and N. benthamiana. It was found that both HlMyb3 transgenes interfere in the accumulation and composition of flavonol glycosides and phenolic acids in transformed plants. These effects on heterologous transgenotes suggest that the HlMyb3 gene may influence hop morphogenesis, as well as metabolome composition during lupulin gland maturation.

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.)

“…The previously cloned Hl Myb1 [19] is clustering together with Hl Myb7, however, this sequence is less related to ZmP1. Hl Myb3 that was found earlier as metabolome regulator [20] is clustering together with C. sativa, V. vinifera , and G. hirsutum Mybs as described previously [20]. This TF is clustering together with Hl Myb6, while two other hop Mybs 4 and 5 are quite unrelated and are not included in the analyzed dataset of Myb sequences (see Additional file 1).…”

“…During our previous work, we cloned two authentic R2R3Myb TFs having specific expression in cones of the Czech hop variety Osvald's 72, i.e., Hl Myb1 [GenBank:AJ876882] [19] and Hl Myb3 [GenBank:AM501509] [20]. In the present work, we isolated and purified lupulin glands from Osvald's 72 according to Nagel et al [41] and constructed a lupulin gland-specific cDNA library.…”

“…At Myb75 and Hl Myb2, the strongest ternary activators of P chs _H1, showed low capability to activate P chs 4. Interestingly, a clear-cut difference was observed for the subvariants of Hl Myb3 [20]. While l-Hl Myb3 shows a maximum of activation for P chs 4 in the MB combination, the maximum of activation of P chs _H1 was observed for the shorter variant s- Hl Myb3 in the MBW combination.…”

“…Neither the combinations with hop-derived Mybs, Hl Myb2 or s-Hl Myb3 that activated P chs _H1, i.e., s- M3B2, s- M3B2W1 nor M2B2W1 resulted in blue pigmentation in the petunia leaves. The metabolically highly active s-Hl Myb3 [20], caused yellow spots on the leaves in combination with Hl bHLH2 (Figure 9). The hop TFs, Hl Myb2, s-Hl Myb3 and WDR1 were not able to co-induce anthocyanin biosynthesis in petunia leaves.…”

“…Two hop R2R3Myb TFs, i.e. Hl Myb1 and Hl Myb3, have been characterized in our previous studies [19,20] and diverse biological effects caused by Hl Myb3 subvariants have been demonstrated in heterologous transgenotes [20]. Although these Myb TFs were suggested to be involved in the lupulin metabolome production based on sequence similarity and specific expression in hop cones [19,20], their influence on the activation of chs genes has not been investigated in detail.…”