An anther-specific GRP gene, regulated by PhMYC2 , causes a significant reduction of male fertility when overexpressed in petunia, and its promoter is efficient in genetic engineering of male-sterile lines. Glycine-rich proteins (GRPs) play important roles in plant anther development; however, the underlying mechanisms and related regulatory networks are poorly understood. In this study, a novel glycine-rich family gene designated as PhGRP was isolated from Petunia hybrida 'Fantasy Red'. The qRT-PCR analysis showed that it expressed specifically in anthers, and its expression peaked earlier than those well-known tapetum-specific genes, such as TA29, and several genes with the classic cis-regulatory element 'anther-box' in petunia during its anther development. The male fertility was significantly reduced in PhGRP overexpression lines, due to the abnormal formation of pollen wall. The PhGRP promoter (pPhGRP) could drive the GUS genes expressing specifically in the anthers of the transgenic Arabidopsis plants, indicating that the anther-specific characteristic of this promoter was conserved. In addition, when pPhGRP was used to drive the expression of BARNASE, complete male-sterile petunia lines were created without changes in vegetative organs and floral parts other than anthers. Finally, when pPhGRP was used as the bait to screen a yeast-one-hybrid (Y1H) library, a transcription factor (PhMYC2) belonging to the bHLH family was successfully selected, and the binding between pPhGRP and PhMYC2 was validated both by Y1H and dual-luciferase reporter assay. Overall, these results suggest that PhGRP, which is a male fertility-related gene that expresses specifically in anthers, is regulated by PhMYC2 and whose promoter can be used as an effective tool in the creation of male-sterile lines.
Jasmonate ZIM-domain (JAZ) family proteins are the key repressors in the jasmonate signaling pathway and play crucial roles in plant development, defenses, and responses to stresses. However, our knowledge about the JAZ protein family in petunia is limited. This research respectively identified 12 and 16 JAZ proteins in two Petunia progenitors, Petunia axillaris and Petunia inflata. Phylogenetic analysis showed that the 28 proteins could be divided into four groups (Groups A–D) and further classified into six subgroups (A1, A2, B1, B3, C, and D1); members in the same subgroup shared some similarities in motif composition and sequence structure. The Ka/Ks ratios of seven paralogous pairs were less than one, suggesting the petunia JAZ family might have principally undergone purifying selection. Quantitative real-time PCR (qRT-PCR) analysis revealed that PaJAZ genes presented differential expression patterns during the development of flower bud and anther in petunia, and the expression of PaJAZ5, 9, 12 genes was generally up-regulated after MeJA treatment. Subcellular localization assays demonstrated that proteins PaJAZ5, 9, 12 were localized in nucleus. Yeast two hybrid (Y2H) elucidated most PaJAZ proteins (PaJAZ1-7, 9, 12) might interact with transcription factor MYC2. This study provides insights for further investigation of functional analysis in petunia JAZ family proteins.
Tissue-specific promoters can drive genes specifically expressed in the target organs and have been widely used in plant molecular breeding. In this study, a 1.2-kb promoter region of an anther-specific gene PhLRR from Petunia hybrida BFantasy^was isolated and fused to the β-glucuronidase (GUS) gene. The pPhLRR::GUS vector was heterogeneously transformed into tobacco in which the GUS staining was only detected in the early development stage of anthers and no GUS expression in any other three floral whirls or vegetative organs was observed. It is very different from other well-studied anther-specific promoters which drive genes specifically expressed in the later development stage of anthers or only in the pollens. Furthermore, the pPhLRR::Barnase was introduced into petunia and induced complete male sterility without influencing the ornamental characteristics or the female fertility in transformed plants.These results indicate that PhLRR promoter is a new kind of petunia anther-specific promoter and could be taken as a valuable tool in ornamental plant breeding.
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