Regulatory Roles Of An Importin Protein And Flavin Content In Desease Resistance And Flowering In Arabidopsis

The growth and development of plant faced the threat which was brought by the changes of the external environment.Plants have evolved a variety of mechanisms in response to the stress from biological and non-biological.Expression of a large number of defense response genes is one of the key steps to defense these threats.The expression of transcription factor-mediated gene played a role play in this process.When the protein is not less than 40KD,they must be combined with carrier which carry them passing through the nuclear pore complex into/out the nucleus.Therefore,The nuclear transport of transcription factor has become an important part of signal transduction to active plant defense responses.Flowering is the most important processes for its whole life of angiosperms.The flowering of Arabidopsis thaliana is regulated by multiple induced pathways,including photoperiod pathway,vernalization,gibberellins and autonomous pathway,etc..Previous studies showed that the heterologous expression of turtles riboflavin binding protein?RfBP?of the Arabidopsis unregulated FD and API in stem apex and showed early flowering.Screening of disease resistance related mutants of Arabidopsis importin geneIn eukaryotes,nucleo-cytoplasmic transport can carry on the precise regulation of signal transduction,and it also is an important way to control plant defense signal transduction.There are many proteins involved in the process of plant resistance to exogenous pathogens,especially transcription factor.Transcription factor must enter the nucleus to perform its functions after synthesis in ribosome.Therefore,nuclear transport of transcription factors become the key process in plant defense response.The current study shows that importin proteins and nucleoporin proteins,the key group of nucleo-cytoplasmic transport,participated in the process of systemic acquired resistance?SAR?and defense response mediated by resistance gene in the Arabidopsis thaliana,suggested the important role of the importin proteins and nucleoporin proteins in plant disease resistance.In this lab,we obtained all of the 99 mutant which T-DNA inserted into the 21 IMP?importin?from Arabidopsis Biological Resource Center.Subsequently,we performed the resistance screening and verification of PCR homozygote work for all mutant seeds,and we obtained 78 homozygotes.Then,we used the Pseudomonas syringae pv.tomato virulent strain DC3000 and Pectobacterium carotovora subsp.carotovora to determine disease resistance of those mutants.The statistic results show that there are 7 kinds of mutants were susceptible to two kinds of pathogenic bacteria,3 mutants were resistant,respectively belong to 7 importin genes Considering previous researches and our experimental results,we found that a gene encoding an importin?protein which specifically exist in embryophytes,the gene was named as PLANTKAP.Function analysis of importin protein PLANTKAP during defense response in Arabidopsiswe obtained 78 homozygous mutant and tested these homozygous mutant resistance by two kinds of pathogenic bacteria;finally we obtained 7 susceptible and 3 resistant mutant plants.we selected two mutants,plantkap-1 and plantkap-2,which mutation in the PLANTKAP gene for study.In the test of hormone treatment of wild-type Arabidopsis,we found that the PLANTKAP gene increased about 30-fold compared to wide-type after treatment with SA.Then we treated mutants by the pathogen Pst DC3000 and detected the expression of PR1 and PR2,the marker genes of salicylic acid pathway.qRT-PCR results showed that the expression of PR1 and PR2 in the mutant was 6?10 times lower than wild-type.And then we observed the subcellular localization of PLANTKAP in onion epidermal cells and Arabidopsis leaf by transient expression,we founded that PLANTKAP was mainly localized in the nucleus.NPR1 and TGA2 is the key transcription factor in salicylic acid signaling pathway.The expression of NPR1 and TGA2 had no significant difference between the mutant and wild-type.We observed the subcellular localization of the two transcription factors after transient expression in the mutant.The result showed that the cellular localization of NPR1 was changed in the mutant,but TGA2 wsa not affected.The yeast two hybrid assay revealed that there was no interaction between PLANTKAP and NPR1 directly.Signal mechanism of turtle riboflavin-binding protein regulates Arabidopsis flowering timeThe turtle riboflavin-binding protein?RfBP?can be combined with free flavin,and reduce the content of free flavin in organism.Recently we showed that de novo expression of RfBP in transgenic Arabidopsis increased H₂O₂ concentrations leaf cells,enhanced the expression of floral regulatory gene FD and floral meristem identity gene API at the shoot apex,and induced early flowering.While the foliar expression of 13 of 19 METC genes were decreased by 2?3 times in RfBP-expressing?RfBP+?plants compared to wide-type.Inside RfBP+ leaf cells,cytosolic H₂O₂ concentrations were increased possibly through electron leakage because similar responses were also induced by a known inducer of electron leakage from METC.Early flowering no longer occurred when the repression on METC genes was eliminated by RfBP gene silencing,which restored RfBP+ to wild type in levels of FD and API expression,H₂O₂,and flavins.Flowering was delayed 5?6 d by the external riboflavin application,which brought gene expression and flavins back to the steady-state levels but only caused 55%reduction of H₂O₂ concentrations in RfBP+ plants.Exogenous H₂O₂ treatment resulted in Arabidopsis early flowering,but flowering time returned to normal after adding the H₂O₂ scavenger.Different sources of H₂O₂,in plant tissues,could promote the Arabidopsis early flowering,but only RfBP+ could inhibit the expression of METC.