CRISPR/Cas9-mediated targeted mutation reveals a role for AN4 rather than DPL in regulating venation formation in the corolla tube of Petunia hybrida

Venation is a common anthocyanin pattern displayed in flowers that confers important ornamental traits to plants.An anthocyanin-related R2R3-MYB transcription factor,DPL,has been proposed to regulate corolla tube venation in petunia plants.Here,however,we provide evidence redefining the role of DPL in petunia.A CRISPR/Cas9-mediated mutation of DPL resulted in the absence of the vein-associated anthocyanin pattern above the abaxial surface of the flower bud,but not corolla tube venation,thus indicating that DPL did not regulate the formation of corolla tube venation.Alternately,quantitative real-time PCR analysis demonstrated that the spatiotemporal expression pattern of another R2R3-MYB gene,AN4,coincided with the formation of corolla tube venation in petunia.Furthermore,overexpression of AN4 promoted anthocyanin accumulation by increasing the expression of anthocyanin biosynthesis genes.CRISPR/Cas9-mediated mutation of AN4 led to an absence of corolla tube venation,suggesting that this gene in fact determines this key plant trait.Taken together,the results presented here redefine the prime regulator of corolla tube venation,paving the way for further studies on the molecular mechanisms underlying the various venation patterns in petunia.

Intron-retained alternatively spliced VIN3-LIKE 2 gene from Chimonanthus praecox promotes flowering in transgenic Arabidopsis

Vernalization is a process of acquiring or accelerating the flowering ability by prolonged cold exposure.VERNALIZATION INSENSITIVE3(VIN3)is induced by chilling and is extremely important for the vernalization response in Arabidopsis thaliana.However,the issue of the induction of the VIN3-LIKE genes in wintersweet(Chimonanthus praecox)has been largely neglected.In the present study,we explored the molecular regulation of the PHD type finger protein-encoding gene CpVIL2 in relation to the growth and development of wintersweet in Arabidopsis.In wintersweet,quantitative real-time PCR(qRT-PCR)analysis showed that the relative expression of CpVIL2-As2i(intron-retained alternatively spliced in the second intron)was extremely higher in the pistils than in the other tissues.And the relative CpVIL2-As2i expression in flower buds(FBs)treated at 8°C was higher than that of FBs in December,2016 under natural conditions,which was not detected in non-flowering FBs at 16°C.In Arabidopsis,the expression patterns of the CpVIL2-As2i gene were detected at first in CpVIL2-As2i pro::GUS(β-glucuronidase)lines,with predominantly higher expression in flowers and inflorescence.Meanwhile,the hormone-induced expression profiles of the CpVIL2-As2i promoter were confirmed using exogenous induction by abscisic acid(ABA)and indole acetic acid(IAA)phytohormones,where the GUS enzyme activity obviously decreased compared with that of control.In comparison with Arabidopsis/Col-0,early flowering was detected in ectopic 35S::CpVIL2-As2i lines.Overall,these results demonstrated the function of the CpVIL2-As2i gene,at the same time,provided us with new insights into the molecular mechanisms of early flowering and complex regulatory networks of vernalization in wintersweet.

Integrated transcriptome and proteome analysis provides insight into chilling-induced dormancy breaking in Chimonanthus praecox

Chilling has a critical role in the growth and development of perennial plants.The chilling requirement(CR)for dormancy breaking largely depends on the species.However,global warming is expected to negatively affect chilling accumulation and dormancy release in a wide range of perennial plants.Here,we used Chimonanthus praecox as a model to investigate the CR for dormancy breaking under natural and artificial conditions.We determined the minimum CR(570 chill units,CU)needed for chilling-induced dormancy breaking and analyzed the transcriptomes and proteomes of flowering and non-flowering flower buds(FBs,anther and ovary differentiation completed)with different CRs.The concentrations of ABA and GA3 in the FBs were also determined using HPLC.The results indicate that chilling induced an upregulation of ABA levels and significant downregulation of SHORT VEGETATIVE PHASE(SVP)and FLOWERING LOCUS T(FT)homologs at the transcript level in FBs when the accumulated CR reached 570 CU(IB570)compared to FBs in November(FB.Nov,CK)and nF16(non-flowering FBs after treatment at 16℃for−300 CU),which suggested that dormancy breaking of FBs could be regulated by the ABA-mediated SVP-FT module.Overexpression in Arabidopsis was used to confirm the function of candidate genes,and early flowering was induced in 35S::CpFT1 transgenic lines.Our data provide insight into the minimum CR(570 CU)needed for chilling-induced dormancy breaking and its underlying regulatory mechanism in C.praecox,which provides a new tool for the artificial regulation of flowering time and a rich gene resource for controlling chilling-induced blooming.