Characterization of Arabidopsis Tubby-like proteins and redundant function of AtTLP3 and AtTLP9 in plant response to ABA and osmotic stress
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Tubby and Tubby-like proteins (TLPs) play essential roles in the development and function of mammal neuronal cells. In addition to the conserved carboxyl (C)-terminal Tubby domain, which is required for their plasma membrane (PM) tethering, plant TLPs also possess an amino (N)-terminal F-box domain to interact with specific Arabidopsis Skp1-like (ASK) proteins as functional SCF-type E3 ligases. Here, we report the molecular characterization of Arabidopsis TLPs (AtTLPs). β-Glucuronidase staining showed overlapped but distinct expression patterns of AtTLPs in Arabidopsis. Yeast two-hybrid assays further revealed that AtTLP1, AtTLP3, AtTLP6, AtTLP7, AtTLP9, AtTLP10 and AtTLP11 all interacted with specific ASKs, but AtTLP2, AtTLP5 and AtTLP8 did not. Subcellular localization observations in both Arabidopsis protoplasts and tobacco pollen tubes indicated that all GFP-AtTLP fusion proteins, except GFP-AtTLP8 which lacks the conserved phosphatidylinositol 4,5-bisphosphate binding sites, were targeted to the PM. Detailed studies on AtTLP3 demonstrated that AtTLP3 is a PM-tethered PIP2 binding protein which functions redundantly with AtTLP9 in abscisic acid (ABA)- and osmotic stress-mediated seed germination. Our results suggest that AtTLPs possibly work in multiple physiological and developmental processes in Arabidopsis, and AtTLP3 is also involved in ABA signaling pathway like AtTLP9 during seed germination and early seedling growth.
KeywordsTubby Arabidopsis AtTLP ABA Stress
We thank the Arabidopsis Biological Resource Center and the Nottingham Arabidopsis Stock Centre and GABI-Kat (Kleinboelting et al. 2012) for providing us the T-DNA insertion mutants; BiFC vectors were kindly provided by Dr. Guang Li (Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China). Vectors containing the ASK cDNAs were kindly provided by Dr. Nadine Schumann and Dr. Marcel Quint (Independent Junior Research Group and Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, Halle, Germany). This work has been supported by the following grants: the National Natural Science Foundation of China 31000288, 31171169, 31100212, 31371228, 31370670; the National Basic Research Program of China 2010CB126600; the National Mega Project of GMO Crops 2013ZX08001003-007, 2013ZX08004002-006; and the Strategic Priority Research Program of the Chinese Academy of Sciences XDA08030108.
Conflict of interest
The authors declare that they have no conflict of interest.
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