Arabidopsis FIM4 And FIM5 Are Involved In The Regulation Of Auxin-mediated Root Growth And Development

Auxin is a kind of plant hormone with small molecular weights and simple structure.It is involved in various biological roles in plants,including apical dominance,directional response,flower differentiation,vein formation and so on.Auxin is synthesized in meristematic and embryonic tissues and juvenile parts of the plant,and then is transported through the phloem tissue or by a cell-to-cell mode.The polar transport of auxin generates a local auxin concentration gradient,which regulates a broad array of physiological and developmental processes in plants.It is well known that actin cytoskeletons participate in the regulation of many physiological processes during plant growth and development,including cell morphogenesis,material transport,and signal transduction.The actin dynamics of the microfilament skeleton are regulated by a series of actin binding proteins(ABPs).Fimbrins are conserved actin bundling/cross-linking proteins widely existing in eukaryote organisms.There are five family members in Arabidopsis genome,named AtFIM1-AtFIM5.AtFIM4 and AtFIM5 have been shown to regulate both pollen germination and pollen tube polarity growth cooperately.Based on our previous work,we found that Arabidopsis thaliana seedlings showed a series of auxin related phenotypes after the simultaneous deletion of AtFIM4 and AtFIM5.Therefore,in this study,we explored the role of AtFIM4 and AtFIM5 in regulating the transport and distribution of auxin in root by means of cell biology,molecular biology and genetics.The main findings are as follows:(1)The length of primary root became longer and the number of lateral root of the double mutant is much more.Compared with the wild type,the length of the primary root of atfim4 and atfim5 single mutant did not change significantly,but the root length of the double mutant became longer,and the size of the meristem of the primary root became larger.The number of meristem cells also increased correspondingly,but there was no difference in the length of elongation zone.(2)The sensitivity of double mutant to exogenous hormones is different.Wild type and double mutant were treated with different concentrations of IAA and TIB A.The results showed that the sensitivity of double mutant to exogenous hormone was different from that of wild type.The sensitivity of double mutant to low concentration of IAA became weaker,but it was enhanced to high concentration of IAA.While the double mutant always showed strong sensitivity when it was treated with TIBA.After tissue transparency,it was found that the changes in the length of meristem zone of double mutant was not different from that of wild type,but the changes in the length of elongation zone showed great sensitivity between the wild type and the double mutant.(3)The actin bundles in the cells of the root elongation zone of the double mutant are less obvious.The microfilament conformation in the cells of the root tip elongation zone was observed by using the fluorescent marker 35s::Lifeact-EGFP.It was found that the actin bundles in the double mutant cells decreased significantly.After treated with low concentration or high concentration of IAA,it was found that auxin could promote actin bundles.(4)The expression of AtFIM4 and AtFIM5 in Arabidopsis roots are not exactly the same.The results of GUS assay of FIM4pro::GUS and FIM5pro::GUS transgenic lines showed that AtFIM4 was mainly expressed around the QC of root meristem,vascular tissue and epidermal cells,while AtFIM5 was expressed around the QC of root meristem.In the meantime,the subcellular localization of FIM4pro::GFP-FIM4 and FIM5pro::GFP-FIM5 complementary lines was observed,the localization was consistent with that of GUS assay.(5)The expression of DR5rev::3×Venus-N7 in wild type and double mutant was different.Confocal scanning revealed that the fluorescence intensity and the area of the QC in the double mutant was lower than that in the wild type,indicating that the deletion of AtFIM4 and AtFIM5 affected the distribution of auxin in atfim4/atfim5 double mutant.(6)The endocytosis in the root cells of double mutant is impaired.The endocytosis of wild type and double mutant root cells was detected by fluorescent dye FM4-64,it was found that endocytosis of FM4-64 by the root cells in the double mutant was reduced compared to the wild type.(7)The expression of the auxin export carriers PIN protein in double mutant was decreased.We crossed the double mutant with PIN1-GFP,PIN2-GFP,PIN3-GFP,PIN4-GFP and PIN7-GFP.The homozygous double mutant expressing PIN7-GFP have been isolated.It showed that the expression of PIN7 in double mutant was decreased.These results clarified that AtFIM4 and AtFIM5 are involved in auxin-mediated root growth and development in Arabidopsis thaliana.The above findings expand our understanding of the function of plant fimbrins.