The Regulatory Mechanism Of NUCLEAR Pore ANCHOR In Vegetative Phase Change In Arabidopsis

The life cycle of higher plants consists of an embryonic,a vegetative and a reproductive phase,and the vegetative phase can be further divided into a juvenile vegetative and an adult vegetative phase.The juvenile-to-adult transition is referred to as vegetative phase change,which is a pivotal developmental stage in plant development.The juvenile-to-adult transition is regulated by a conserved micro RNA---miR156 and its target SPL（SQUAMOSA PROMOTER BINDING PROTEIN-LIKE）genes.The miR156-SPL pathway plays a vital regulatory role in plant development and growth,biotic/abiotic stress response,and secondary metabolite biosynthesis.Therefore,investigation on the role of the miR156-SPL pathway is of theoretical and practical significance since it would lay a solid foundation for future molecular breeding of plants with high yield,high quality,and high resistance.There already have some researches on the miR156-SPL pathway in various fields in plants,but how other regulatory factors integrate into the miR156-SPL pathway to modulate vegetative phase change is still quite limited.To further understand the regulatory mechanism of vegetative phase change and identify more regulatory factors of the miR156-SPL pathway,we performed a forward genetic screen to identify mutants with abnormal transition from the juvenile to the adult phase in an EMS-mutagenized Arabidopsis Col-0 seeds.We identified three characteristic mutants with a precocious vegetative phase change phenotype,which we named pvt2,pvt3,pvt4（promoted vegetative transition2/3/4）.This research carried out a series of studies on the above mutants,and obtained the following results:1.Through map-based cloning,the pvt2 mutation was narrowed down to a region between marks YUP8H12R and F20B17 on chromosome I,pvt3 between marks T21F11 and F23A5 on chromosome I,and pvt4 between marks T9L6 and F10C21 on chromosome I.Whole genome sequencing,d CAPS analyses and allelic complementation tests indicated that there was a C-to-T substitution at the 659^th base of the coding sequence of the NUCLEAR ANCHOR PROTEIN（NUA）gene（AT1G79280）in the pvt2 mutant,this substitution led to the introduction of a stop codon at the original arginine position to cause a premature termination of NUA translation.Likewise,there was a C-to-T substitution at the 1240^th base of the coding sequence of the NUP96 gene（AT1G80680）in the pvt3 mutant,leading to the substitution of the glutamine amino acid by a stop codon,and a C-to-T substitution at the 2214^th base of the coding sequence of the NUP160 gene（AT1G33410）in the pvt4mutant,leading to the substitution of the glutamine amino aicd by a stop codon.2.Quantitative real time RT-PCR（q RT-PCR）indicated that miR156 and MIR156A were both downregulated significantly in nua,while the target genes of miR156,including SPL2,SPL5,SPL6,SPL9,SPL10,SPL13,and SPL15 were all upregulated remarkably in nua.Consistent with this result,the GUS activity in the transgenic MIR156A transcriptional reporter line p MIR156A::GUS was significantly down-regulated in the nua background than that in the Col-0 background,this result indicated that NUA might affect miR156 by regulating the transcription of MIR156A.3.GUS activity in the miR156-insensitive SPL9 translational level transgenic line p SPL9::r SPL9-GUS was significantly enhanced in the nua background than that in the Col-0 background.Western blot indicated that the SPL9 protein level in the p SPL9::3×FLAG-r SPL9 transgenic line was significantly higher in the nua background than that in the Col-0 background.These results suggest that NUA regulates SPL9independently of miR156.4.q RT-PCR analysis of the GUS m RNA in the p SPL9::r SPL9-GUS transgenic line showed no significant difference between the Col-0 and the nua background.Similarly,the SPL9 promoter luciferase report experiment showed that NUA was not involved in the transcriptional regulation of SPL9.Western blot analysis of the SPL9protein level in protoplasts transformed with the 35S::3×FLAG-r SPL9 plasmid indicated that the SPL9 protein level was significantly higher in the nua background than that in the Col-0 background,this result implied that NUA regulates SPL9 at the protein level.5.MG132 treatment experiment indicated that SPL9 was degraded through the26S proteasome pathway.The degradation rate of GST-SPL9 purified from Escherichia coli in the cell free system was significantly slower when incubated with proein extracts from nua than that from Col-0.Therefore,the nua mutation leads to a slower degradation rate of SPL9,thus maintaining a high level of SPL9 in the nua background.6.Immunoprecipitation mass spectrometry（IP-MS）revealed that the 147^th lysine in SPL9 was ubiquitinated,and the 152^th,154^th,158^th serines were phosphorylated in the Col-0 background,but not in the nua background.These results indicated that NUA might affect SPL9 degradation by influencing SPL9 amino acids modification.7.Protein interaction prediction suggested that NUA interacted with ESD4（EARLY IN SHORT DAYS 4）.Phenotypic characterization of nua,esd4,and nua esd4mutants indicated that there was no significant difference in the first leaf with abaxial trichomes and in leaf shape between different genotypes.Since the SPL9 protein level was upregulated in esd4,this suggests that ESD4 and NUA functions in the same pathway to regulate SPL9 protein level.8.Yeast two-hybrid and Bi FC confirmed that NUA interacts with SPL9 and ESD4at the protein level.ESD4 acts as a SUMO（small ubiquitin-like modifier）protease,SUMO protects the target protein from ubiquitination for degradation.In the nua mutant,SPL9 SUMO level is increased.This suggested that NUA may influence the SUMO modification level of SPL9 and its protein stability through ESD4.9.Phenotypic characterization of nua spl9,and esd4 spl9 double mutants showed that the introduction of spl9 mutation could partially restore the early phase change phenotype of nua and esd4 in that both leaf shape and the first leaf with abaxial trichomes,and nua spl9,esd spl9 double mutants are similar to those of Col-0.This result implied that NUA and ESD4 participated in the regulation of phase change by affecting SPL9 or by other unknown pathways.10.Subcellular localization result showed that NUA was localized in the nuclear,and NUA is a hydrophilic protein without a transmembrane domain that can form a stable tertiary structure,this provides a stable environment for macromolecule transport.The results of protein interaction,SPL9 subcellular localization,and nucleus-cytoplasmic separation experiment indicate that NUA functions as a nucleoporin by interacting with SPL9 to transport SPL9 from the cytoplasm to the nucleus.In the nua mutant background,SPL9 fails to enter the nucleus completely by being trapped in the cytoplasm.Based on the above results,we propose a model for the regulation of plant vegetative phase change by NUA.On the one hand,NUA regulates vegetative phase change by affecting the transcription of MIR156A,and thus the mature miR156 level.On the other hand,NUA influences the SUMO modification of SPL9 through ESD4,thereby affecting its ubiquitination modification to maintain SPL9 protein level.In the nua mutant,the SUMO modification of SPL9 increases to antagonize ubiquitination,thus inhibiting SPL9 degradation to maintain a high level of the SPL9 protein,causing a precocious vegetative phase change phenotype.