Auxin-mediated Root Growth Inhibition In Response To Aluminum Stress In Arabidopsis

As a sessile organism,plants have developed plasticity of growth in their livelong evolution to adapt ever-changing external environment and the plasticity is more representative in the root.Root system of plants are the major organ to absorb water and nutrients from the soil,and participate in the response to various stress as well.Plants can change the structure and function of the root system,which reflect the root plasticity to response to diverse adversity.Then it would be meaningful to explore the molecular mechanism of root plasticity,and to provide the theoretical basis for crop breeding in the future.Aluminum(Al)is the most abundant metal in the soil.When soil pH drops below 5.5,Al3+ is released into the soil solution and becomes one of the limiting factors for crop production in most acid soils,which is only exceeded by cdrought stress with regard to abiotic limitations to crop production.It has been estimated that as much as 50%of the world's potentially arable lands are acidic,60%of which are in the tropics and subtropics where many of the developing countries reside.Hence,Al toxicity is a worldwide agronomic problem that limits crop production and affects food security.To explore the molecular mechanism of Al stress in acid soil has attracted more and more attention.And there will be meaningful by cultivating antiacid varieties to solve the limiting crop production in acid soil.As crucial factors in regulating plant growth and development,phytohormone mediates the plasticity of plant growth in response to surrounding environment.Although phytohormone such as ethylene,auxin and abscisic acid have been reported to regulate the Al-induced root growth inhibition,the crosstalk of these hormones in regulating this process is still not yet known,and needs to further lucubration.In this dissertation,model organism Arabidopsis thaliana is used to investigate the interaction between phytohormone and aluminum by utilizing molecular biology,cell biology,genetics physiology and other techniques.As a result,auxin,cytokinin and ethylene are involved in response to aluminum stress in the root-apex transition zone(TZ)through hormonal biological synthesis,signal transduction or directly regulating by other transcription factorsWe first detected the phenotypes of YUC mutants under Al stress treatment,Al-dependent root growth inhibition was least severe in yuc9 mutant,while its extent in multiple yucQ mutant was more obvious.When yucasin,an inhibitor of YUC activity,was introduced in the growth medium,the effect of Al stress on root-growth inhibition was also strongly reduced.Besides,the strong Al stress-induced DR5rev:GFP signals produced in the root-apex TZ of a AICl3 treated-WT root was remarkably attenuated in yuc mutants or yucasin treatment.Thus,this indicated that YUC activity was involved in the auxin response in the root-apex TZ and subsequent root growth inhibition when plants were subjected to Al stress,and the functional redundancy existed among YUC genes.To address how YUCs regulate the local auxin signaling in the root-apex TZ in the presence of Al stress,the effect of exposure to Al stress on the spatial expression of the YUC genes was detected.Under non-stressed conditions,only YUC9 was expressed in the root tips,but these YUC genes were specifically induced in the root-apex TZ in response to Al.A comparison of the Al stress-induced expression of the YUC genes in the presence of either ACC or AVG showed that the induction of GFP signals by Al treatment in the root-apex TZ was enhanced by the former,but repressed by the latter,indicating that Al stress-regulated up-regulation of the YUC genes in the root-apex TZ was regulated by ethylene.Next,we demonstrated that EIN3 may directly regulated YUC9 expression to participate in the Al-induced response of YUCs in the root-apex TZ.Al stress induced local up-regulation of EIN3 and EIL1 in the root-apex TZ,while the strong Al stress-induced expression of YUC9 was markedly decreased in ethylene mutant ein3-1 eill-1,demonstrated that the response of EIN3 under Al stress was relied on YUC9.Furthermore,AlCl3 plus ACC treatment resulted in an intensification of the DR5rev:GFP signals throughout the root tip,but the level of enhancement was more modest in the yuc mutants or yucasin treatment.Al/ACC co-treatment also had a pronounced inhibitory effect on the WT root but not on the yuc mutants or yucasin treatment.Thus,we concluded that YUCs-mediated local auxin biosynthesis and thus root growth inhibition in response to Al were ethylene-dependent.Previous studies have proved that PIF4(transcription factor belong to bHLH family)could directly regulate the expression of YUC8 to mediate hypocotyl elongation under heat stress.Here,we demonstrate that YUC5 and YUC9 can also be directly regulated by PIF4.Loss of function mutant pif4-101 showed Al-tolerant phenotype,while overexpression transgenes line 35S:PIF4 was hypersensitive to Al stress.Furthermore,up-regulation of YUC8,YUC9 and DR5rev:GFP in the root-apex TZ under Al stress was obviously attenuated in pif4-101 mutant,indicated that Al-induced local auxin biosynthesis and response was regulated through PIF4,while the Al-induced expression of PIF4 was still regulated by EIN3/EIL1-mediated ethylene signaling.Besides auxin,we analyzed if cytokinin is involved in response to Al stress as well.Loss of function mutants of CK synthesis or receptor presented more resistance to Al stress than WT,while CK metabolism mutant or type-B ARR overexpression lines were more sensitive to Al stress.ARR3,ARR4 and TCSn:GFP were unsurprising up-regulated in the root-apex TZ under Al stress,implied that Al stress induced a local CK response in the root-apex TZ.To test whether other phytohormones participate in the regulation of Al stress-induced CK response in the root-apex TZ,we detected the TCSn:GFP signals under Al treatment in yuc1D mutant or NAA treatment.The results showed that increased auxin levels led to the elevatory Al stress-induced TCSn:GFP signal,while reduced auxin signaling caused the highly attenuated TCSn:GFP signals under Al stress.Moreover,auxin-enhanced root growth inhibition in response to Al stress was less obvious in the arr1 arr10 arr12 triple mutant,whereas CK-enhanced root growth inhibition under A1 stress was still observed in auxin synthesis or signaling mutants.These results indicated that CK acted downstream of auxin in the regulation of Al stress-induced root inhibition.Next,we detected the TCSn:GFP signals under Al treatment in etol-2 mutant or ACC treatment.The results showed that increased ethylene levels led to the elevatory Al stress-induced TCSn:GFP signal,while in ein3-1 eill-1 double mutant or AVG treatment caused the highly attenuated TCSn:GFP signals under Al stress.Furthermore,ethylene-enhanced root growth inhibition in response to Al stress was less obvious in the arrl arr10 arr12 triple mutant,while CK-enhanced root growth inhibition under A1 stress was still observed in ein3-1 eill-1 double mutant,demonstrating that ethylene signals acted upstream of CK in the process of root inhibition under Al stress.In addition,the Al/ACC co-treatment enhanced TCSn:GFP signals could be obviously relieved by PEO-IAA treatment,but reduced TCSn:GFP signals under Al/PEO-IAA co-treatment can't be recovered by ACC.Overall,we suggested that the ethylene-enhanced CK response in the root-apex TZ under Al stress was regulated by auxin.To confirm whether Al-induced local CK response in the root-apex TZ is the result of local CK biosynthesis,we detected the root phenotype of ipt mutants under Al stress.As a result,ipt mutants showed insensitive root-inhibition in response to Al stress,which was more pronounced in iptl ipt3 ipt5 ipt7 multiple mutant.Otherwise,Al stress-induced IPTs expression in the root-apex TZ was remarkably attenuated in the arf7 arf19 double mutant or PEO-IAA treatment,indicating that local up-regulation of IPTs induced by Al stress in the root-apex TZ was regulated by auxin.Next,we analyzed the sequence of IPT promoters,the typical auxin response element(AuxRE)was found in the promoters of IPT1,IPT5 and IPT7.Hence,a series of assays such as yeast one-hybrid assay and ChIP were conducted to confirm that ARF7 bound directly to the IPT5 and IPT7 promoters,thus mediating local CK biosynthesis in response to Al stress.In conclusion,our research demonstrated that the Al stress-induced auxin response in the root-apex TZ and thus root growth inhibition was regulated by YUC genes,while EIN3 and PIF4 could mediate this process through direct regulation of YUC.Moreover,we further found that up-regulation of IPT expression in the root-apex TZ in response to Al stress and thus root growth inhibition was direct regulated by auxin signaling.These findings not only confirmed the vital role of CK in response to Al stress,but also illuminated the synergistic action of auxin and CK in regulating Al stress-mediated root growth inhibition except for the antagonism relationship between auxin and CK in normal development conditions.