Identification and biochemical characterization of the fructokinase gene family in Arabidopsis thaliana

Abstract

BackgroundFructose is an abundant sugar in plants as it is a breakdown product of both major sucrose-cleaving enzymes. To enter metabolism, fructose is phosphorylated by a fructokinase (FRK). Known FRKs are members of a diverse family of carbohydrate/purine kinases known as the phosphofructokinase B (pfkB) family. The complete complement of active fructokinases has not been reported for any plant species.ResultsProtein sequence analysis of the 22 Arabidopsis thaliana pfkB members identified eight highly related predicted proteins, including one with previously demonstrated FRK activity. For one, At1g50390, the predicted open reading frame is half the size of active FRKs, and only incompletely spliced RNAs were identified, which led to a premature stop codon, both indicating that this gene does not produce active FRK. The remaining seven proteins were expressed in E. coli and phosphorylated fructose specifically in vitro leading us to propose a unifying nomenclature (FRK1–7). Substrate inhibition was observed for fructose in all FRKs except FRK1. Fructose binding was on the same order of magnitude for FRK1–6, between 260 and 480 μM. FRK7 was an outlier with a fructose Km of 12 μM. ATP binding was similar for all FRKs and ranged between 52 and 280 μM. YFP-tagged AtFRKs were cytosolic, except plastidic FRK3. T-DNA alleles with non-detectable wild-type RNAs in five of the seven active FRK genes produced no overt phenotype. We extended our sequence comparisons to include putative FRKs encoded in other plant sequenced genomes. We observed that different subgroups expanded subsequent to speciation.Conclusions Arabidopsis thaliana as well as all other plant species analyzed contain multiple copies of genes encoding FRK activity. Sequence comparisons among multiple species identified a minimal set of three distinct FRKs present on all species investigated including a plastid-localized form. The selective expansion of specific isozymes results in differences in FRK gene number among species. AtFRKs exhibit substrate inhibition, typical of their mammalian counterparts with the single AtFRK1 lacking this property, suggesting it may have a distinct in vivo role. Results presented here provide a starting point for the engineering of specific FRKs to affect biomass production.

An Expanded Role for WRINKLED1 Metabolic Control Based on Combined Phylogenetic and Biochemical Analyses

“…This finding can be rationalized with 373 respect to a recent complete biochemical and genetic characterization of the FRK gene family in A. 374 thaliana (Stein et al, 2016;Riggs et al, 2017). While no severe seed phenotype was found for the single-375 KO mutation of FRK3, a FRK1-FRK3 double-KO mutation resulted in a severe wrinkled seed phenotype 376 with strong reduction in seed oil content (Stein et al, 2016).…”

Identification, Expression, and Functional Analysis of the Fructokinase Gene Family in Cassava

“…Fructose must first be phosphorylated to fructose-6-phosphate by fructokinases (FRKs) or hexokinases (HXKs) before undergoing further metabolism [ 9 ]. The affinity of FRKs for fructose is much higher than HXKs [ 10 , 11 ], which suggests that fructose in plants might be mainly phosphorylated by FRKs. Plant FRKs belong to the pfkB family of carbohydrate kinases, containing a di-gly (GG) motif at the N-terminal region and GAGD motif at the C-terminal region [ 12 ].…”

“…Therefore, fructokinase may play a role in starch accumulation in the sink organs of crops. Plant FRKs are found in two locations: plastids and the cytoplasm [ 9 , 11 ]. The existence of plastidic FRK may be important for starch synthesis, since starch is accumulated in plastids.…”

TWIN SISTER OF FT (TSF) Interacts with FRUCTOKINASE6 and Inhibits Its Kinase Activity in Arabidopsis

“…Another interesting observation is that although TSF is homologous to FT, FT does not inhibit the activity of FRK6 or FRK7. All Arabidopsis FRKs except FRK1 exhibit substrate inhibition ( Riggs et al, 2017 ), and FRKs are thought to play a role in regulating starch synthesis via sucrose synthase in the sink tissue of plants ( Odanaka et al, 2002 ). Therefore, it would be interesting to further investigate a possible role for TSF in sink tissue.…”

“…According to our BiFC assay results, TSF likely interacts with FRK6 in the nucleus ( Figure 3E ), which is inconsistent with the results of a previous report ( Stein et al, 2017 ). The majority of FRKs in tomato and Arabidopsis localize to the cytosol, except for tomato FRK3 (LeFRK3) and Arabidopsis FRK6, which localize to the plastid ( Damari-Weissler et al, 2006 ; Riggs et al, 2017 ). Perhaps FRK6 interacts with TSF only in the nucleus, although FRK6 may localize to both the cytosol and nucleus.…”