Cloning and molecular analyses of hop transcription factors

Abstract

Expression of hop chs_H1 and other hop chalcone synthase (CHS)-like enzymes was assayed in developing pollen tissue. Both, chs_H1 and valerophenone synthase (vps) are highly expressed in pollen suggesting the existence of common specific regulatory element(s). While vps is highly specific for female and male floral organs, the oligofamily of chs_H1 is expressed in both generative and somatic tissues, suggesting a more universal promoter. To identify novel transcription factors involved in the regulation of chs_H1 genes in lupulin glands, cone-specific ESTs showing homology to bZIP and bHLH were utilized to screen hop cDNA and genomic libraries. Additionally, inverse PCR was used to amplify full length cDNAs of members of two bZIP oligofamilies from hop. Transcription factors H/bZIP1A and H/bZIP2 have predicted molecular masses of 27.4 and 34.2 kDa, respectively. While H/bZIP1A is rather neutral (pI 6.42), H/bZIP2 is strongly basic (pI 8.51). A truncated variant of H/bZIP1 (H/bZIP1B) that is strongly basic but lacks the zipper domain was also cloned from hop. Additionally, the first bHLH transcription factor was cloned from hop. H/bHLH1 is a protein of 33.6 kDa with a predicted pI of 6.08. Based on semiquantitative RT PCR results, this TF is expressed specifically in hop floral and cone tissue. The combinatorial action of H/bZIP2 and other hop TFs that have been cloned, especially H/Myb1 and 3 were analyzed in P. hybrida and N. benthamiana transient expression systems. TF H/bZIP2, as well as both Myb TFs, were found as activators of chs_H1 promoter. In the combinatorial system, H/bZIP2 was found to modulate expression of H/Myb1 and H/Myb3 Trs from hop