The Regulatory Mechanisms Of SlZF3 On Flowering Time And FolB2 On Plant Root Development

Tomato(Solanum lycopersicum)is one of the most important vegetables worldwide,it also plays an irreplaceable role in the vegetable industry of China.In fundamental research,both tomato and Arabidopsis serve as important model species.It can deepen our knowledge on plant development,using tomato and Arabidopsis to study the regulatory mechanisms underlying flowering time and root architecture,which also provides important theoretical base for crop genetic improvement.In a previous study,a C2H2 type zinc finger protein,Sl ZF3,was found to enhance salt tolerance when overexpressed in tomato or Arabidopsis.Sl ZF3 possesses an ERF-motif in its C item,which exhibits transcriptional repression activity.Besides,the overexpression lines of tomato and Arabidopsis display morphological changes,such as dwarfism,smaller angle between the leaf and stem,flowering delay.In this thesis,we investigated the regulatory mechanism of Sl ZF3 on flowering time of tomato and Arabidopsis.Besides,a short root mutant named folb2 was found in heterologous overexpression of Sl ZF3 Arabidopsis lines.Map-based cloning analysis identified folb2 is a natural mutant.Positional cloning revealed that a 61 bp deletion is in the junction region of the 5`UTR and promotor of At Folb2,which results in reduced transcription of At Folb2.Consequently,this leads to the root phenotype of folb2 mutant.At Folb2 encodes a dihydroneopterin aldolase(DHNA),which acts in the upstream of folate biosynthesis.In this thesis,we also investigate how At Folb2 affects the primary and adventious root development in plants.The main findings are as follows.1.Overexpression of Sl ZF3 can significantly delay the flowering time of tomato and Arabidopsis plants,while RNA interference of this gene accelerated the flowering time in tomato.Similarly,overexpression of ZAT12,the orthologue of Sl ZF3 in Arabidopsis delayed the flowering time significantly,while its RNAi lines displayed earlier flowering.2.Yeast two hybrid and Luciferase reporter experiments demonstrated that Sl ZF3 can interact with At CO.Co-Immunoprecipitation assay showed that Sl ZF3 interacted with Sl CO1,the At CO orthologue in tomato,and this interaction was independent ofthe EAR motif in Sl ZF3.The interaction between Sl ZF3 and At CO was confirmed genetically.3.Dual luciferase assay showed that Sl ZF3 can significantly inhibit the promotor activity of At FT,while At CO can enhance the promotor activity.ChromatinImmunoprecipitation followed by quantitative PCR assay demonstrated that Sl ZF3 or ZAT12 can bind the promotor of At FT in the region nearby the 5`UTR.EMSA results showed that Sl ZF3 or ZAT12 can bind only the combinatorial cis-elements of 5`UTR Py-rich stretch and TCA of At FT promoter.The above two elements are adjacent to the CORE2 element of At FT promoter that At CO binds to,while they are 76 bp away from the CORE2 of SFT promoter.4.Taken together,Sl ZF3 negatively regulated flowering time by interacting with CONSTANS at the protein level,and also by binding to the combinatorial ciselements of 5`UTR Py-rich stretch and TCA in FLT promotor.5.In the transgenic Arabidopsis lines of Sl ZF3,a short root mutant designated as folb2 was occasionally found.folb2 mutant had a short primary root,with 1-2 adventitious roots at the shoot and root junction.Propidium iodide(PI)and iodine potassium iodide(I2-IK)staining results showed that the root apical meristem of the primary root of folb2 was damaged or even disappeared.Positional cloning revealed that a 61 bp deletion was found in the 5`UTR and promotor of At Folb2,resulting in a remarkably reduced transcription of At Folb2.Functional complementary(FC)and RNAi lines were generated,it was found that the root phenotype was recovered in the FC lines,while RNAi lines mimicked the folb2 mutant.RNAi lines were also generated for Sl FOB2,the orthologue of At Folb2 in tomato.The tomato RNAi lines showed a similar root phenotype to that of the folb2 mutant.6.The tetrahydrofolate(THF),its precursor DHF and derivatives(5-M-THF and 5-FTHF)were measured using liquid chromatography–mass spectrometry(LC-MS).It showed that the contents of THF,DHF and 5-M-THF decreased significantly in folb2 mutant and the RNAi lines of At Folb2,while their levels in the FC line recovered toa level comparable to Col-0.Exogenous 5-F-THF could restore the phenotype of folb2 mutant to wild type.Similarly,all the four compounds decreased significantly in the tomato RNAi lines of Sl Fol B.7.The effects of the At Folb2 mutation on the distribution of auxin and the auxin responsive pathway were evaluated.RNA-seq data showed the transcription level of genes involved in auxin transport and response decreased significantly in the folb2 mutant and RNAi lines,while the expression level of LBD41,a downstream target gene in auxin response,was up-regulated significantly.The RNA-seq data were further confirmed by reverse-transcription quantitative PCR.The IAA contents in leaf and root tissues of Col-0,folb2 and FC line were measured by LC-MS.It was shown that the IAA contents in folb2 was reduced compared with Col-0,while no difference was detected between folb2 and the FC line.Therefore,reduction of IAA would not be the key reason for the root phenotype of folb2 mutant.Reporter lines of DR5:m GFP,PINs:GFP,WOX5:GFP and DII:VENUS:YFP were utilized to investigate the auxin transport and distribution.It was shown that folates could facilitate the auxin downward polarity transport to the RAM and affect the auxin distribution in vascular and around tissues in the mature region of root.8.Genetic experiments indicated that LBD41 acts downstream of At Folb2.The root phenotype was recovered when LBD41 was knocked down in folb2 mutant by RNA interference.Taken together,our results revealed that folate affects auxin transport and distribution,and it was essential for the function of root apical meristem.LBD41 was a key target in the regulation of folate on primary and adventious root development.