Functional Analysis Of Endocytic Sorting Motifs In Auxin Efflux Transporter PIN2 In Arabidopsis

Polar auxin transport(PAT)is a unique regulation mechanism for plant developmental growth and environmental response.PAT is largely determined by plasma membrane(PM)-localized auxin efflux transporter PIN(PIN-FORMED).The transport activity and polar localization of PIN predominantly determine directional auxin flow and auxin gradient.Recent studies have indicated that polar localization of Arabidopsis PIN proteins is regulated by clathrim-mediated endocytosis(CME),but detailed regulatory mechanisms are poorly understood.Previous studies have shown that endocytosis of PM proteins is regulated by themselves endocytic sorting motifs including Yxx? and LL motifs in mammalian cells.However,whether similar regulatory mechanisms for PM proteins exist in plant cells remains to be elucidated.To explore functional roles of endocytic sorting motifs in PIN2 polar localization and root gravitropic growth,combined with previous work in this lab,this study used bioinformatics,genetics,cell biology,and biochemical and physiological methods to reveal regulatory roles of PIN2 endocytic sorting motifs in root gravitropism.The detailed results are as follows:(1)Bioinformatic analysis showed that PM-localized PIN proteins(PIN1,2,3,4,and 7)in Arabidopsis contain 14 Yxx? motifs and 5 LL motifs.Using molecular cloning and site-directed mutagenesis technology,19 expression constructs for mutations in endocytic sorting motifs(mPIN2-GFP)were successfully generated.(2)Using the Agrobacterium-mediated flower dipping,these 19 constructs were introduced into the pin2 mutants,and subsequently obtained all of homozygous lines in T3 generation.Genetic analysis showed that 16 constructs(mPIN2-GFP-4 to-19)effectively rescued root agravitropic phenotype of pin2 mutants,indicating that these 16 endocytic sorting motifs do not function in regulation of PIN2-mediated root gravitropism.By contrast,3 constructs(mPIN2-GFP-1,-2,and-3)could not restore the phenotype of pin2 mutants,indicating that these 3 endocytic sorting motifs act in regulation of PIN2-mediated root gravitropism.(3)Root gravistimulation response showed that root growth in the mPIN2-GFP-1 and-2 seedlings was insensitive to gravistimulation and displayed a similar phenotype of pin2 mutants;whereas root growth in mPIN2-GFP-3 exhibited a partial response to gravitropism,further confirming that 3 endocytic sorting motifs play a critical regulatory role in PIN2-mediated root gravitropism.(4)Cytological analysis revealed that the polarity of mPIN2-GFP-1 and-2 was reduced,indicating that the former two motifs are likely involved in PIN2 efflux activity and the latter motif may function in PIN2 polar localization.Analysis of auxin distribution in the root tips showed that excessive auxin was accumulated in root tips of mPIN2-GFP-2 and-3 seedlings,similar to that in the pin2 mutants.Root gravistimulation experiments showed that on the contrary to PIN2-GFP,2-h gravistimulation did not induce asymmetric distribution of mPIN2-GFP at both top and bottom sides of roots.Based on the above results from genetic and cell biology analyses,this study can conclude:endocytic sorting motifs regulate PIN2 polarity or polar efflux activity and thereby modulate PIN2-mediated root gravitropism,which will provide new insights into molecular mechanisms underlying PIN polarity and PAT in plants.