Molecular Mechanism Of Arabidopsis NSF In Regulating Leaf Serration Development

Leaf is the main organ of plant photosynthesis,respiration and transpiration.Leaves can be simple or compound organs,depending on the leaf blade,and leaf margin could be serrated or entire.The extensive leaf lobation in serrated or lobed leaves enable greater leaf elongation in response to capture light energy,as well as improve the resistance to external stresses.It is of great significance to study the morphology of plant leaf margins to improve the utilization rate of plant light energy,the ability of plants to adapt to the environment and the ornamental value.Leaf margins are regulated by genes,hormones and environmental signals.Although the regulatory pathways of leaf margin morphology have been initially reported,the related regulatory genes still need to be further explored.In this study,we used a reverse genetic approach to characterize the function of AtNSF(Arabidopsis thaliana soluble N-ethylmaleimide sensitive factor).Our results showed the defect of AtNSF displayed more leaf serrations in leaf margin.AtNSF is also required for the primary root growth,root gravitropism and lateral root development.AtNSF is localized in the endomembrane system of Golgi and endosomes,which is required for the trafficking of auxin efflux carriers PIN1(PIN-FORMED 1)and PIN2 and regulates the auxin maxima in the leaf margin.Besides,CUC2(CUP-SHAPED COTYLEDON 2)transcription factor,the growth repressor,is negatively regulated by AtNSF,which finally establishes the maintenance of PIN1-and CUC2-mediated feed back loop in leaf margin development.By using several strategies of cell biology,molecular biology and genetics approach,we explain the function of AtNSF in regulating leaf serration development.The main results are as follows:(1)We isolated and identified a AtNSF defective mutant,atnsf-1,which exhibited more serrations in the leaf margin.Secondary teeth emerged between two outgrown teeth.Besides,the primary root growth was inhibited in atnsf-1,especially in the presence of BFA,a widely used vesicle transport inhibitor.Moreover,the gravity response and the lateral root development were abnormal when AtNSF defective,suggesting AtNSF regulates both leaf and root development.(2)The expression pattern of AtNSF is predominantly expressed in the vascular tissues.More interestingly,AtNSF expression was restricted to the leaf tip and tips of teeth,and week signal was also present in the vascular tissue.This expression pattern implicates an important role for AtNSF in the regulation of leaf serration.The localization of AtNSF protein is localized to Golgi network,cell plate,and endosomes and may mediate membrane fusion between different organelles.(3)To investigate the function of AtNSF in membrane trafficking in plants,we monitored intracellular accumulation of the endocytic tracer FM4-64 and the secretion protein marker sec GFP.We observed more severe FM4-64 internalization and more sec GFP agglomerations present at the cell periphery in atnsf-1,indicating that AtNSF might regulate multiple vesicle trafficking pathway in Arabidopsis.(4)We observed additional DR5_rev::GFP maxima between two outgrowth teeth in atnsf-1.When seedlings were treated with NPA,the polar auxin transport inhibitor,both wild type and atnsf-1 displayed smooth leaf margins,suggesting that AtNSF regulates leaf serration in a polar auxin transport-dependent manner.(5)PIN1 is required for auxin flux and the dynamic PIN1 cycling occurs between the endosome and the plasma membrane.We further investigate the auxin efflux transporter PIN1cycling in atnsf-1.More pronounced PIN1-GFP was present in atnsf-1 and more PIN1 labeled agglomerations after BFA treatment.After wash out,PIN1 internalization was still higher in atnsf-1 than in the wild type.These results demonstrated that knock-down of AtNSF disrupts PIN1 cycling between endosomes and plasma membrane.Together,AtNSF is required for PINs cycling,which may affect auxin distribution in plants.We generated the atnsf-1 pin1-8 double mutant by crossing,and evaluated its phenotype.In contrast to atnsf-1,which has more teeth than the wild type,the atnsf-1 pin1-8 double mutant had fewer teeth than the wild type and a similar number to pin1-8,suggesting AtNSF may functions upstream in PIN1-mediated polar auxin transport and further regulates the leaf serration.(6)The qPCR analysis,as well as CUC2_pro::GUS and CUC2-VENUS in atnsf-1background showed elevated CUC2 levels,indicating that AtNSF inhibits the expression of CUC2.We further investigated the genetic relationship between AtNSF and CUC2 by crossing.In contrast to atnsf-1,which had more and larger teeth than wild type,the atnsf-1 cuc2-3 double mutant had less and smaller teeth,similar to those in cuc2-3.These results show that CUC2 is required for AtNSF-mediated regulation of leaf serration and AtNSF may functions upstream of CUC2 and inhibits its expression.Finally,we added the function of AtNSF in the regulation of PIN1 and CUC2-mediated feedback loop.On the one hand,AtNSF directly influences polar auxin transport by controlling PIN1 vesicle trafficking,and finally affecting the maxima of auxin in the leaf margin.On the other hand,AtNSF inhibits CUC2 gene expression,which alters the balance of the PIN1-CUC2feedback loop.In conclusion,AtNSF is required for the PIN1-CUC2 feedback loop in the regulation of leaf serration.