| As sessile organisms,plants can only change cell size,shape or physiological function in face of various environmental changes.Root hairs are tubular-shaped outgrowths of epidermal cells essential for plants acquiring water and nutrients from the soil,and the longth of root hairs is also a potentially important agronomic trait.Despite their importance,the growth of root hairs is finite.But it is not clear how such determinate growth of root hairs is precisely achieved.We identified a novel key gene LONG ROOT HAIR(LRH)that controls root hair elongation via screen of T-DNA insertion mutants.LRH encodes a GYF domain-containing protein potentially involved in translational regulation.In this study,we investigated the function of LRH at physiological,biochemical,molecular and genetic levels,revealing the underlying molecular mechanism in the determinate growth of root hairs.The main findings are as follows:1.LRH is a repressor of root hair elongationIn the preliminary study,we obtained two mutants(lrh-1 and lrh-2)with substantially longer root hairs from the Arabidopsis T-DNA insertion mutant library.The two mutants displayed higher growth rates,longer growth duration and earlier initiation of root hairs versus wild type(WT),but showed normal root hair density.Further analysis showed that the longer root hairs of these mutants were not attributed to the mutation of the genes harbor known T-DNA insertion.We hence performed Whole Genome Sequencing(WGS)for lrh-1 and lrh-2 to detect the background mutation,and found that both mutants harbor an additional T-DNA insertion in LRH.To confirm the function of LRH,we analyzed other alleles of LRH T-DNA insertion mutants(lrh-3 and lrh-4),finding that they displayed longer root hairs,similar to lrh-1and lrh-2.Furthermore,transgenic expression of LRH could fully restore the root hair growth of lrh-1 to that of WT.These results indicate that the LRH gene encods a repressor of root hair elongation.2.LRH inhibits root hair elongation by repressing RSL4 protein accumulationROOT HAIR DEFECTIVE6-LIKE 4(RSL4)is a central transcription factor in regulation of root hair elongation.To determine how LRH represses root hair elongation,we first tested the expression of some key genes of root hair elongation in lrh-1 mutants,and found that the transcription of RSL4 was not affected in lrh-1,but the expression of several genes that are regulated or affected by RSL4 was altered,suggesting that LRH may affect RSL4 abundance or activity at protein level.We further showed that the protein biosynthesis inhibitor cycloheximide(CHX)could completely suppress the promotion of root hair growth in lrh-1 mutants,favoring the role of LRH in regulation of protein synthesis.RSL4-GFP protein accumulated earlier and expectedly higher with much longer duration in hair(H)cells of lrh-1 versus WT,indicating that LRH inhibits the accumulation of RSL4.Meanwhile,we found that lack of RSL4 obviously suppressed the promotion of root hair elongation and initiation conferred by lrh-1,and that the triple mutant lrh-1/rsl2/rsl4 was hairless as similar as the rsl2/rsl4 double mutant,suggesting that RSL4 is epistatic to LRH.In addition,LRH and RSL4 are spatially co-expressed in H cells of root elongation zone,which indicates that LRH regulates RSL4 translation probably via a cell-autonomous way.Taken together,LRH inhibits root hair elongation by repressing RSL4 protein accumulation.3.LRH interacts with eIF4Es translation initiation factors to inhibit RSL4translationGiven that LRH was predicted to contain a conserved eIF4E-binding motif,we demonstrated that LRH interacts with eIF4Es translation initiation factors(eIF4E1 is the predominant one),using Split-luciferase(LUC)complementation assay in tobacco(N.benthamiana)leaves,Yeast Two-hybrid system(Y2H),bimolecular fluorescence complementation(Bi FC)and protein pull-down assay indicate that LRH can interact with eIF4Es.Additionally,we showed that this interaction relied on the N terminal of eIF4E1.Furthermore,we demonstrated that LRH directly affects the association of eIF4E1 with the m RNA cap by m~7GTP pull-down assay,and that LRH inhibits the association of eIF4E1 with RSL4 m RNA and RSL4 translation revealed by RNA immunoprecipitation(RIP)and translating ribosome affinity purification(TRAP)assays.In addition,we demonstrated that eIF4E1 and eIF(iso)4E are epistatic to LRH in root hair regulation.These results collectively indicate that LRH represses RSL4translation and protein accumulation via inhibition of the association of eIF4Es with the 5’cap of RSL4 m RNAs.4.RSL4 directly regulates LRH expression in a feedback wayRSL4 protein was expectedly and markedly induced by phosphorus deficiency or several hormone treatments,showing a similar pattern with LRH.Moreover,we found three typical RHEs(Root hair-specific cis-elements,the RSL4 binding sites)in the LRH promoter.These observations suggest a possible regulation of RSL4 on LRH expression.Further transactivation assay showed that both RSL4 and RSL2 were capable to transactivate LRH expression in tobacco leaves.In addition,the electrophoretic mobility shift assay(EMSA)and chromatin IP(Ch IP)-q PCR proved that RSL4 can directly bind to the promoter region of LRH in vitro and in vivo.Correspondingly,we found that the LRH m RNA abundance was reduced but in a mild way in the rsl2/rsl4versus WT,and that the LRH protein accumulation in H cells of root elongation zone was substantively reduced.Taken together,these results indicate that RSL4 directly transactivates LRH expression.In summary,we identified a novel factor LRH in regulation of root hair elongation.LRH represses RSL4 translation via interaction with eIF4Es,thereby to inhibit root hair growth.RSL4 in turn directly transactivates LRH expression to form a feedback regulatory loop.This LRH-RSL4 feedback loop may shed new light on the mechanism underlying the determinate growth of root hairs,and may also provide a new theoretical support for the breeding of novel crop germplasm with stronger root system and higher nutrient use efficiency. |
