TI - Glucokinase activities and glc signalling assays using different expression lines . AB - The growth phenotypes of the HKL1 transgenic and mutant lines that have been described could be due to an influence of HKL1 protein on HXK1 protein catalytic activity , on HXK1 signalling functions , and on the function of an unknown protein . To test for the possible influence of HKL1 protein on glucokinase activity , rate measurements were carried out using leaf extracts from the different lines . There was no significant difference for enzyme activities between the transgenic lines and their respective control lines ( Fig 5A ) . As reported previously , HXK enzyme activity in gin2-1 is about one-half of that in Ler and HKL1-HA did not have any glc PHOSphorylation activity . The possible inhibition of HXK1 by HKL1 was also tested after transiently expressing HXK1-HA and HKL1-HA in maize protoplasts . However , HKL1 protein did not affect the measured glucokinase activity (Fig 5B) . Since HKL1 lacks glucokinase activity , but has a largely conserved glc binding domain , it is possible that , instead , the protein affects glc signalling activities . A widely used screen to identify mutants in glc signalling is based on the ability of some mutants to develop normally on otherwise inhibitory concentrations of exogenous glc . Therefore , seedling growth of the different lines was assessed in the presence of varying glc concentrations ( Fig 6A , B ; see Supplementary Fig S3 at JXB online ) . At relatively high glc levels , Col-0 and Ler seedlings underwent developmental arrest , with much reduced root and shoot growth , and did not accumulate chlorophyll . The hkl1-1 seedlings were hypersensitive to developmental arrest , showing substantial repression even on 4% glc . By contrast , the HKL1-HA seedlings were glc-insensitive relative to the Ler control line . When grown on 6% glc , >90% of the HKL1-HA seedlings have green cotyledons versus 0% of the Ler seedlings . The responses of HKL1-FLAG seedlings were comparable with those of gin2-1 seedlings . As an osmotic control , all lines were shown to have a similar phenotype on MS plates with 6% mannitol (Fig 6C) . Also , mannitol did not repress cotyledon greening in any of the lines ( Fig 6D ) . The observed glc -dependent phenotype suggested that HKL1 could be a negative regulator of glc signalling . To test whether HKL1 might have a role in glc signalling , protoplast transient expression assays were carried out using pRBCS-LUC and pPPDK-LUC as established reporters of HXK1 signalling . Leaf protoplasts of Col-0 and hkl1-1 plants were used in independent assays . Relative RBCS-driven LUC activities expressed in protoplasts of either genotype was reduced by 25% with 2 mM glc ( Fig 7A ) . In both cases , co-transfection with HXK1 plus treatment with 2 mM glc reduced the reporter activity by about 55% . By contrast , transfected HKL1 did not affect the relative expressed RBCS-driven LUC activity with glc alone or with HXK1 plus glc , using protoplasts from either wild-type or mutant leaves . Similar results were obtained using pPPDK-LUC ( data not shown ) . Notably , in all cases the expression of pUBQ10-GUS was not affected by co-transfection of HXK1 , HKL1 and by addition of 2 mM glc . To complement the transient expression assays , an alternate assay of glc signalling was carried out using seedlings grown in liquid culture and treated with or without 2% glc for 8 h . The selected GLYK and T6P genes are thought to be regulated by HXK1 -dependent glc signalling , and ASN by a glycolysis -dependent glc signalling pathway . Supporting this interpretation , transcripts of ASN , GLYK and T6P were all repressed by glc treatment of Col-0 and Ler seedlings , while GLYK and T6P mRNA abundance were not affected by treatment of gin2-1 seedlings (Fig 7B) . The response of these transcripts was not differentially affected in any of the tested mutant or transgenic lines , relative to the corresponding control lines . These data indicate that HKL1 probably does not affect the commonly recognized transcriptional targets of glc signalling , whether by a HXK1 -dependent or a glycolysis -dependent pathway .