An anther-specific gene PhGRP is regulated by PhMYC2 and causes male sterility when overexpressed in petunia anthers

Abstract

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.

Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development

“…As one of the most important reproductive organs, the development of anther is a complex process, and any disturbing of it could significantly influence the pollen’s fertility, which would bring great losses in breeding. The previous results implied that petunia anther development is a complex process, and some anther-/pollen-specific genes/promoters were obtained [ 7 , 12 , 28 , 29 ]. However, due to the different concerns of experiment designs and technical limitation, the knowledge related to petunia anther development still needs to be refined.…”

“…A TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgene, Shenzhen, China) was used to synthesize the first-strand cDNA on the basis of the manufacturer’s instructions. The qRT-PCR was carried out using the SYBR Premix Ex Taq™ IIkit (Takara, Tokyo, Japan) in the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the protocol described previously [ 28 ]. The gene expression level was calculated by the 2 −ΔΔ C t method, with the beta-actin gene of petunia as the reference gene [ 28 ].…”

“…The qRT-PCR was carried out using the SYBR Premix Ex Taq™ IIkit (Takara, Tokyo, Japan) in the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the protocol described previously [ 28 ]. The gene expression level was calculated by the 2 −ΔΔ C t method, with the beta-actin gene of petunia as the reference gene [ 28 ]. Each experiment was repeated with three independent replications, and data are shown as the mean values ± SE (standard error).…”

Biotechnological improvement of ornamental plants

“…Considering the last five years, scientific literature include transgenic petunia events obtained to study floral pigmentation (BOASE et al, 2015;CHU et al, 2015;AI et al, 2016;SHAIPULAH et al, 2015), biotic (WANG et al, 2013a;GARGUL et al, 2015;SUN et al, 2016) and abiotic stress (ESTRADA-MELO et al, 2015), selfincompatibility (LI et al, 2014;KUBO et al, 2016), adventitious root formation (LISCHWESKI et al, 2015), floral development (MOREL et al, 2017;O'DONOGHUE et al, 2017;YUE et al, 2017), plant architecture (LIANG et al, 2014;GARGUL et al, 2015), flower senescence (WANG et al, 2013a;WANG et al, 2013b;YIN et al, 2015), floral volatiles (CNA'ANI et al, 2015;SHAIPULAH et al, 2015;ADEBESIN et al, 2017;OLIVA et al, 2017), herbivore defence (KESSLER et al, 2013;SASSE et al, 2016), and others.…”

Selection of appropriate reference genes for quantitative real-time PCR in Clerodendrum trichotomum

“…and reverse amplification primers were 0.4 μl, the 2× SYBR Primer Ex Taq TM was 5 μl, and the ddH 2 O was 106 3 μl. The reaction procedure was using our usual methods (Yang et al, 2018;Yue et al, 2017). All 107 qRT-PCR reactions were repeated three times biologically and three times technically, and three times 108 template-free negative control.Ultimately, the reliability of the target reference genes were verified by using 109 ClNHX1 and ClLAC genes, the reaction volume and procedure were the same as above.…”