| Arabidopsis hexokinase1 (AtHXK1) is a moonlighting protein with roles in both glucose signaling and catalysis. In this study, we first cloned and characterized the six HXK related genes from Arabidopsis. Three of the six encoded proteins were shown not to phosphorylate hexoses and thus, are designated as hexokinase-like (HKL) proteins. Though they are only 50% identical to HXK1, the amino acid sequences of HKL1 and HKL2 both have well conserved glucose binding domains and other recognized structural elements. The possible basis for their lack of catalytic activity was further probed by site-directed mutagenesis and ultimately was attributed to a suite of amino acid substitutions. Gene expression studies showed that transcripts of HKL1 and HKL2 occur in most plant tissues, thus supporting the hypothesis that they have regulatory functions.The function of AtHKL1 was more closely examined using a reverse genetics approach. We identified a T-DNA knockout mutant for HKL1 and made HKL1 overexpression lines in different genetic backgrounds. Their phenotypes showed that HKL1 is a negative regulator of plant growth. Interestingly, many of the phenotypes required the presence of HXK1 protein. Both HKL1-GFP and HXK1-GFP are expressed at mitochondria and both were shown to interact with each other by coimmunoprecipitation assays. However, even though the HKL1 phenotypes included some dependence on glucose treatments, we conclude that HKL1 likely does not have a direct role in glucose signaling. Instead, we found from seedling signaling assays and a novel root hair phenotype that HKL1 mediates plant growth responses at least in part by promoting ethylene biosynthesis and/or signaling. Overall, these studies have helped to identify, characterize, and define the function of non-catalytic HXKs from Arabidopsis. |
