TI - cerevisiae and A .nidulans and also in Arabidopsis . . AB - Whether non-catalytic homologues of known enzymes are commonly present in other protein families is not known . The Arabidopsis glutathione transferase family does include both non-catalytic as well as catalytic forms , although their relative distribution between the groups apparently has not been strictly determined . Recently , beta-amylase4 ( BAM4 ) of Arabidopsis was shown to lack apparent catalytic activity , yet somehow to facilitate starch breakdown . BAM4 is one of perhaps four chloroplastic isoforms within Arabidopsis . Also , the plant shikimate kinase gene family includes two non-catalytic homologues which have been present in all major plant lineages for over 400 million years . In Arabidopsis , these express novel functions , one of which is required for chloroplast biogenesis . Non-catalytic enzyme homologues might occur somewhat more often among plant gene families than what is currently appreciated , since sequence divergence levels within families are often >25% . That is , in order to transfer all four digits of an EC number at an error rate below 10% , the estimated level of sequence identity needs to be >75% . It is suggested that when non-catalytic homologues of known enzymes do occur , they are likely to have important regulatory functions . For example , several catalytically inactive homologues of phosphoinositide 3-phosphatases have been linked to specific human diseases ( Robinson and Dixon , 2006 ) . As one general approach to understand protein function , the tissue expression pattern and regulation of gene expression can provide an important physiological context . The AtHKL1 transcript was previously shown to be expressed in the principal plant organs . These observations have been extended in this study by demonstrating that pHKL1-GUS activity occurs predominantly in the vascular tissues of different sink organs such as roots , stems , and anthers ( Fig 8 ) . In stem cross-sections , the vascular staining was associated with phloem tissue . While we are not aware of any HXK family members having been reported in surveys of the phloem proteome , nonetheless many phytohormones and a number of regulatory proteins have been detected in phloem sap . The HKL1 promoter activity was also found to be influenced by several phytohormones , including being repressed by ABA and induced by both ACC and cytokinin . Hormone induction of the HKL1 promoter occurred in both vascular and non-vascular tissues ( Fig 8 ) . Our analysis of the HKL1 promoter sequence for known regulatory elements indicates that the promoter does have motifs proposed to be regulated by several hormones ( data not shown ) . Phenotypes of the AtHKL1 overexpression lines provide evidence that the HKL1 protein is a negative regulator of plant growth . HKL1 overexpression in Ler ( HKL1-HA ) resulted in reduced seedling growth on sucrose plates ( Fig 2 ) , reduced hypocotyl elongation under low light conditions ( Fig 3 ) , severely reduced rosette size under LD conditions ( Fig 2 ) , and a decreased sensitivity to auxin-induced lateral root formation ( Fig 4 ) . In a recent initial report , some rice HXK family members also were considered to be possible negative regulators of seedling growth ( Yu and Chiang , 2008 ) . The status of the glc binding domain in possible regulatory HXKs needs to be evaluated experimentally . It has previously been shown that AtHXK1-G173A has a 90% decrease in glc PHOSphorylation activity . Since AtHKL1 has the same recognized glc binding domain as does this mutated protein , we speculate that glc binding affinity is reduced in AtHKL1 , but not eliminated . Thus , for a negative regulator , decreased glc binding affinity could be a feedback mechanism to limit plant growth in the presence of excessive carbohydrate availability . The HKL1 protein might function as a negative regulator of cell expansion , based on reduced hypocotyl growth of HKL1-HA seedlings and on increased hypocotyl growth of the hkl1-1 seedlings ( Fig 3 ) . Seedling hypocotyl growth by cell elongation integrates diverse signals including light , temperature , nutrients , and most plant hormones . In gin2-1 , reduced hypocotyl growth has been attributed to the possible insensitivity of seedlings to auxin signalling . However , ethylene also can repress hypocotyl elongation in seedlings grown under conditions similar to those in our experiment . Thus , it is possible that HKL1 expression promotes ethylene sensitivity instead of attenuating auxin sensitivity . Consistent with this possibility , while lateral root formation does require auxin synthesis , transport , and signalling , enhanced ethylene signalling has more recently been shown to repress lateral root formation by modulating auxin transport . Thus , the observed HKL1 repression phenotype for auxin-induced root formation ( Fig 4 ) might instead be associated with an altered ethylene response . Further experiments are needed to clarify the mechanisms involved . The mode of action of AtHKL1 is not known , but does merit further consideration . On the one hand , since both HXK1 and HKL1 are targeted to mitochondria , the two proteins have the potential to interact such that HKL1 could act as a dominant negative effector . In this case , the overexpression of HKL1 in the gin2-1 background might not result in a novel phenotype relative to its overexpression in the presence of HXK1 . Assay results for hypocotyl growth ( Fig 3 ) , for auxin induction of lateral root growth ( Fig 4 ) , and for glc tolerance ( Fig 6 ) are consistent with this possibility . Furthermore , the contrasting phenotypes observed by these assays with the hkl1-1 mutant also support this interpretation . On the other hand , the overexpression of HKL1 in WT did result in a much more diminutive plant under LD conditions than was observed in gin2-1 ( Fig 2 ) . This implies that HKL1 could have a more complicated mode of action by also independently affecting one or more targets possibly involved in mediating phytohormone responses . In summary , the present results indicate that the non-catalytic AtHKL1 protein can negatively influence plant growth , possibly by somehow influencing cross-talk between glc and other plant hormone response pathways . Elucidating the functions of non-catalytic proteins will be an ongoing challenge for contemporary biologists .