Rumi Tominaga-Wada* and Takuji Wada
Graduate School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, 739-8528, Japan
Received Date: September 15, 2017; Accepted Date: September 20, 2017; Published Date: September 27, 2017
Citation: Tominaga-Wada R, Wada T (2017) Triptychon (TRY) Protein Accumulation in the Roots of Mutant Auxin-resistant 1 (axr1) Arabidopsis thaliana. J Plant Biochem Physiol 5:192. doi: 10.4172/2329-9029.1000192
Copyright: © 2017 Tominaga-Wada R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Plant root hairs play an essential role in water and nutrient uptake. CAPRICE (CPC) family transcription factors act to induce root hairs in Arabidopsis thaliana. Previously, using a proteasome inhibitor assay, we proposed that the CPC family protein TRIPTYCHON (TRY) was degraded through the ubiquitin/26S proteasome pathway. The Auxin resistant 1 (AXR1) gene encodes the ubiquitin-activating enzyme E1. In this study, to further investigate the mechanism of TRY degradation, we introduced a CPCp:TRY-GFP construct into axr1-3 A. thaliana mutants. The CPCp:TRY-GFP transgenic plant showed weak TRY-GFP fluorescence while the CPCp:TRY-GFP in axr1-3 transgenic plant showed strong TRY-GFP fluorescence. These results support the suggestion that TRY is degraded by the ubiquitin/26S proteasome mechanism.
Arabidopsis; AXR1; Degradation; Root hair; TRY
Plant root hair is an important organ for water and nutrient absorption from the soil. In, Arabidopsis thaliana (L.) Heynh., CAPRICE (CPC) family genes, which encode the R3-type MYB transcription factor, serve as positive regulators of root hair formation [1,2]. Previously, we reported that the CPC family protein Triptychon (TRY) might be degraded through the ubiquitin/26S proteasomemediated pathway . We showed that TRY was unstable and had a longer C-terminal region (about 20 amino acids) than other CPC family proteins including CPC enhancer of TRY and CPC1 (ETC1) and CPCLIKE MYB3 (CPL3) [3-5]. Deletion of the extended C-terminal region of TRY enhanced its stability . Treatment with MG132 or MG115, proteasome inhibitors led to the accumulation of TRY, indicating that TRY proteolysis is mediated by the proteasome-dependent pathway . However, the precise mechanism of TRY degradation is still unclear.
About 5% of the A. thaliana proteome seem to be directly involved in the ubiquitin/26S proteasome system . This system influences almost every aspect of plant growth and development, including hormone signaling, morphogenesis, and environmental and pathogen responses . In the ubiquitin/26S proteasome system, ubiquitin is attached to a target protein and polyubiquitinated target proteins are degraded by the 26S proteasome. Ubiquitination is accomplished through the sequential action of E1, E2, and E3 enzymes . Ubiquitin is first activated by an E1 ubiquitin-activating enzyme and then transferred to the E2 ubiquitin-conjugating enzyme; helped by E3 ligase, the ubiquitin is finally conjugated to the target protein .
The Auxin resistant 1 (AXR1) gene encodes a protein related to ubiquitin-activating enzyme E1 in A. thaliana . To assess if TRY degradation is mediated by the AXR1 involved in the ubiquitin/26S proteasome system, we produced TRY-GFP in axr1 transgenic plants and examined its fluorescence in these mutant plants.
Plant materials and growth condition
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type background. The CPCp:TRY-GFP transgenic line used in the present study has been previously described . The axr1-3 mutant was obtained from the ABRC, as described in Leyser et al. . The CPCp:TRY-GFP construct was introduced into the axr1-3 mutant by conventional crosses. Seeds were sown on 1.5% agar plates using a previously described method .
Images of GFP-fusion in five-day-old CPCp:TRY-GFP and CPCp:TRY-GFP in axr1-3 transgenic plant roots were obtained with a Zeiss LSM-510 Meta confocal laser scanning microscope.
To further examine if the rapid degradation of TRY-GFP protein in A. thaliana root epidermis is mediated by the ubiquitin/26S proteasome system, we introduced CPCp:TRY-GFP into axr1-3 mutants (Figure 1). In accordance with our previous report on wild type plants , only weak TRY-GFP fluorescence was observed in the CPCp:TRY-GFP transgenic line (Figure 1A). By contrast, relatively strong TRY-GFP fluorescence was observed in CPCp:TRY-GFP in the axr1-3 transgenic line (Figure 1B). In addition, TRY-GFP proteins were detected in the nuclei of root epidermal cells, as in ETC1-GFP or CPL3-GFP transgenic lines, and these do not degrade GFP fusion proteins in root epidermal cells (Figure 1B) . Thus, these results suggest that TRY degradation is related to AXR1 activity.
Previously, we demonstrated that TRY was degraded due to the properties of its extended C-terminal region . In addition, and based solely on proteasome inhibitor assay results using MG132 and MG115, we concluded that TRY was degraded through the ubiquitin/26S proteasome mechanism . Our current data confirmed and reinforced this conclusion. Because AXR1 is expected to participate in the sequential actions defining ubiquitination, as it is part of E1, the non-degradation of TRY in axr1-3 mutants (Figure 1B) was predictable. This result also supports TRY degradation by the ubiquitin/26S proteasome mechanism. Further investigations will reveal the overall mechanism of root hair formation controlled by the CPC family.
We thank Y. Nukumizu and M. Iwata for their technical support, and T. Kurata, T. Ishida, and R. Sano for their useful suggestions. This work was supported by JSPS KAKENHI (Grant numbers 15K14656 and 16K07644).