Studies On The Function Of CKRC1 In Auxin Biosynthesis And Its Relation To Cytokinin Action

Cytokinin (CK) influences many aspects of plant growth and development, whose function often involves its intricate interactions with other phytohormones such as auxin and ethylene. However, the molecular mechanisms underlying the role of CK and its interactions with other growth regulators are still poorly understood.Here we describe the isolation and characterization of the Arabidopsis CK induced root curling 1 (ckrcl) mutant. The ckrcl mutant exhibits defective root gravitropic response (GR) and an increased resistance to CK in primary root growth. These defects can be rescued by exogenous auxin (IAA, NAA,2,4-D) and IPA.Inverse-PCR amplification of T-DNA flanking sequence identified a T-DNA insertion in the 4th intron of an annotated gene Atlg70560. Allelic analysis and molecular complementation showed that Atlg70560 was the CKRC1 gene.CKRC1 encodes a previously identified tryptophan aminotransferase (TAA1) involved in the indole-3-pyruvic acid (IPA) pathway of indole-3-acetic acid (IAA) biosynthesis. Consistent with a defect in auxin biosynthesis, GC-MS result showed that the endogenous IAA level in ckrcl-1 mutant was significantly reduced.To investigate the details of tissue-specific expression of CKRC1/TAA1, we introduced a CKRC1 promoter (2.2 kb) GUS reporter fusion (pCKRC::GUS) into Arabidopsis WT plants. Staining of the GUS activity in independent transgenic lines revealed that the CKRC1 promoter was highly active in the shoot apical meristem, vascular tissues of cotyledons, stems, sepals, stamen filaments, and primary and secondary roots.Furthermore, we show that CK up-regulates CKRC1/TAA1 expression in roots in AHK3/ARR1/12-dependent and ethylene-independent manner using chemical inhibitors and mutants. To more precisely determine the amount of the IAA increase upon the CK treatment, we conducted the GC-MS analysis of the endogenous IAA levels. The IAA level in 7-d old seedlings significantly increased by 41% in WT, but only 23%(not statistically significant) in ckrcl-1, indicating that a significant part of CK induced auxin biosynthesis is dependent on CKRC1. [3H]IAA transport assays showed that ZT significantly reduced the rate of both acropetal (a-, from shoot to root) and basipetal (b-, from root tip to the elongation zone) PAT in WT and ckrcl-1 roots, through its AHK3 ARR1/12 signalling pathway that is independent of ethylene.Our results suggest that CK regulates root growth and development not only by down-regulating PAT, but also by stimulating local auxin biosynthesis.