Functional Study Of RAV Family Transcriptional Factors NGAL1-3 In Arabidopsis

Leaves and roots are important vegetative organs of higher plants,which are involved in photosynthesis,and water and mineral nutrient absorption and transport,respectively.Leaf margin is one of the important characteristics of leaf morphological diversity,and it is also an important basis for botanical classification.In biological stress responses,abundant leaf margin teeth reduce the possibility of plants being attacked by herbivorous insects.During the development of Arabidopsis leaf margin,the NAC family transcription factor CUP-SHAPED COTYLEDON 2(CUC2)plays a dominant role.Loss-of-function mutation of CUC2 leads to smooth leaf margins while gain-of-function mutation of CUC2 shows enhancement of leaf margin outgrowth and serration formation.Roots are the main way for terrestrial plants to obtain water and mineral nutrition from the environment.With the development of roots,middle cortex will form.The development of middle cortex is regulated by many genes such as SCR,SHR,SCL3,and phytohormone such as,auxin and gibberellin.Among them,b HLH family transcription factors TMO5 and LHW regulate root radial division and periclinal division as a dimer.The over-expressions of TMO5 and LHW lead to the shortening and thickening of roots with premature formation of middle cortexThis study focused on the NGATHA(NGAL)subfamily of RAV family transcription factors NGAL1,NGAL2,and NGAL3 in Arabidopsis.Starting from the triple mutant of NGAL genes,ngal1-2 ngal2-1 ngal3-1(ngaltri),it was discovered that ngaltri harbored enhanced leaf margin teeth,longer root length,and earlier formed middle cortex compared with the wide type.Based on these phenotypes,this study mainly explored the function of NGAL1 in the development of leaf margin and the main root in Arabidopsis.The results were obtained as follows:(1)NGALs may be involved in the development of flowers,leaves and roots in Arabidopsis thaliana:Transgenic reporter lines expressing the GFP gene driven by the endogenous NGAL gene promoters,p NGAL1:GFP,p NGAL2:GFP,and p NGAL3:GFP showed that NGAL1,NGAL2,and NGAL3 were all expressed in flowers,leaves,and roots.Additionally,p NGAL1:GUS showed signals in vascular tissues.(2)NGAL1 act upstream of CUC2 in promoting the formation of leaf-margin serrations:Compared with the wild type,ngaltri exhibited significantly enhanced leaf margin serrations.Genetic analysis showed that the leaf serrations of the quadruple loss-of-function mutant ngaltri cuc2-101 were smoother than that of ngaltri,while the quadruple mutant ngaltri cuc2-1D displayed a significantly more serrated leaf margin phenotype compared with either ngaltri or cuc2-1D.Moreoever,abs2-1D/+ cuc2-1D showed the similar smooth leaf edge with abs2-1D/+.(3)NGAL1 and TMO5 co-regulate the development of the main root:As for the underground part,ngaltri displayed increased root length and diameter but also more cells in meristem.In additon,middle cortex could be observed in advance.The expression of p NGAL1:NGAL1-GFP in ngaltri restored the phenotypes of ngaltri in roots and showed that NGAL1-GFP was widely located in the root tip.Over-expression of NGAL1 led to shorter roots with the decreased number of cells in meristem and the root diameter in Arabidopsis.Protein interaction studies indictated that NGAL1 could interact directly with TMO5 and LHW,respectively,in the nucleus.NGALs and TMO5 genetically interact to regulate root growth.A loss-of-function mutation of TMO5 partially reversed the root phenotype of ngaltri with decreased meristem and meristem cell number.Taken together,our fingdings uncovered the function of NGAL1 in the development of leaf margin teeth and root in Arabidopsis,and provided new genetic materials for the analysis of flower,leaf,and root development signaling pathways in plants.