Arabidopsis ECS1 Gene Mediates Root Gravitropism Through Modulating Vesicle Trafficking

Auxin controls plant development through a complex regulatory network including biosynthesis, transport, and perception. In this study, we isolated an interesting mutant from an ethylmethanesulfonate-mutagenized Arabidopsis pool. This mutant was named as ecsl for its swelled pavement cell in the abaxial epidermis of rosette leaves. In addition, ecsl shows shorter primary roots, bent hypocotyls in dark, and reduced sensitivity in root gravitropic responses. Map-based cloning showed that the mutant gene was located in 123 kb interval on the 5th chromosome. Then, we sequenced all genes in this interval, and found a C to T substitution at 278bp of ECS1, which caused a conversion of serine to phenylalanine. ECS1 gene encodes a protein which related to disease resistance. The ECS1 protein consists of three domains, including a TIR structure, a nucleotide binding domain (NB), and a rich leucine repeat (LRR).ecsl has shortened primary root in light, which is mainly caused by shortened mature zone cells. The hypocotyls of ecsl show right bending in dark. SEM analyses revealed that these cells are right spiral. In addition, NPA treatment could partially restore the hypocotyl benting of ecsl.Previous studies confirmed that PIN3 plays an important role in regulating hypocotyl bending. We found that the expression level of PIN3 is increased in the hypocotyl column cells of ecsl.ecsl is hypersensitive to the auxin analogue NAA, and insensitive to 1-N-naphthylphthalamic acid (an auxin transport inhibitor), Furthermore, we confirmed endogenous auxin is reduced in ecsl by analyzing the DR5-GFP in ecsl background. Exogenously applied NAA and NPA can increase DR5-GFP expression in the roots of ecsl mutant, indicating that ecsl might be involved in the regulation of auxin polar transport.The roots of ecsl showed reduced sensitivity to gravity. Recent studies demonstrated that PIN2 is required for root gravitropism through regulating auxin distribution and transport. We found that the polar distribution of PIN2 protein is normal in the cortex and epidermis cells of ecsl. But the fluorescence intensity of PIN2-GFP was increased on the plasma membrane of these cells, and obvious particles were observed in these cells. qRT-PCR analysis showed that the expression of PIN2 is significantly increased in ecsl than that of wild type, implying that ECS1 regulate PIN2 protein level by modulating the transcription of PIN2 gene.FM4-64 staining revealed that the number of vesicle is significantly increased in ecsl compared to that of wild type. The ecsl seedlings were less sensitive to brefeldin A (BFA). Similarly, BFA can induce PIN2 vesicles formation in cells of ecsl mutant. These findings indicate altered vesicle trafficking in ecsl mutant seedlings.All of the results suggested that ECS1 may modulate auxin transport by regulating vesicle trafficking, and thereby play a critical role in root gravitropism.