Hexokinase as a sugar sensor in higher plants.

The mechanisms by which higher plants recognize and respond to sugars are largely unknown. Here, we present evidence that the first enzyme in the hexose assimilation pathway, hexokinase (HXK), acts as a sensor for plant sugar responses. Transgenic Arabidopsis plants expressing antisense hexokinase (AtHXK) genes are sugar hyposensitive, whereas plants overexpressing AtHXK are sugar hypersensitive. The transgenic plants exhibited a wide spectrum of altered sugar responses in seedling development and in gene activation and repression. Furthermore, overexpressing the yeast sugar sensor YHXK2 caused a dominant negative effect by elevating HXK catalytic activity but reducing sugar sensitivity in transgenic plants. The result suggests that HXK is a dual-function enzyme with a distinct regulatory function not interchangeable between plants and yeast.     

Isolation and characterization of hexokinase genes PsHXK1 and PsHXK2 from tree peony (Paeonia suffruticosa Andrews)

Hexokinase (HXK) plays important roles in hexose phosphorylation and sugar signaling. HXK regulates the glucose-induced accumulation of anthocyanin in many species. Little is known about the biological function of the HXK gene family in Paeonia suffruticosa. cDNA sequences of two hexokinase genes PsHXK1 and PsHXK2 were isolated using RACE-PCR and RT-PCR from P. suffruticosa. PsHXK1 encodes 498 amino acids with a 1497-bp open reading frame (ORF), and PsHXK2 contains 493 amino acids with a 1482-bp ORF. Sequence and phylogenetic analyses suggest that PsHXK1 and PsHXK2 belong to type-B HXK and may function as glucose sensors. PsHXK1 and PsHXK2 mRNA were detected in all tested tissues. PsHXK1 is highly expressed in petals and stamens, while PsHXK2 is highly expressed in stamens. At the former stages of flower opening, PsHXK1 and PsHXK2 show higher expression levels in on-tree flowers compared with cut flowers. Overexpressing PsHXK1 and PsHXK2 in Arabidopsis enhances glucose sensitivity, inhibits plant growth in response to glucose, and induces anthocyanin accumulation in response to the high level of glucose. Overall, our results primarily reveal the biological function of PsHXK1 and PsHXK2, especially their involvement in glucose-induced anthocyanin accumulation.

     

Actin-based cellular framework for glucose signaling by Arabidopsis hexokinase1

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.Key words: Arabidopsis, F-actin, glucose signaling, hexokinase, hTalin, mitochondria, polysomes, protoplasts, transient expression assay, fluorescence microscopy     

A dual role of tobacco hexokinase 1 in primary metabolism and sugar sensing

Hexokinase (HXK) is present in all virtually living organisms and is central to carbohydrate metabolism catalysing the ATP‐dependent phosphorylation of hexoses. In plants, HXKs are supposed to act as sugar sensors and/or to interact with other enzymes directly supplying metabolic pathways such as glycolysis, the nucleotide phosphate monosaccharide (NDP‐glucose) pathway and the pentose phosphate pathway. We identified nine members of the tobacco HXK gene family and observed that among RNAi lines of these nine NtHXKs, only RNAi lines of NtHXK1 showed an altered phenotype, namely stunted growth and leaf chlorosis. NtHXK1 was also the isoform with highest relative expression levels among all NtHXKs. GFP‐tagging and immunolocalization indicated that NtHXK1 is associated with mitochondrial membranes. Overexpression of NtHXK1 resulted in elevated glucose phosphorylation activity in leaf extracts or chloroplasts. Moreover, NtHXK1 was able to complement the glucose‐insensitive Arabidopsis mutant gin2‐1 suggesting that NtHXK1 can take over glucose sensing functions. RNAi lines of NtHXK1 showed severely damaged leaf and chloroplast structure, coinciding with an excess accumulation of starch. We conclude that NtHXK1 is not only essential for maintaining glycolytic activity during respiration but also for regulating starch turnover, especially during the night.     

HEXOKINASE1 forms a nuclear complex with the PRC2 subunits CURLY LEAF and SWINGER to regulate glucose signaling

The glucose sensor HEXOKINASE1 (HXK1) integrates myriad external and internal signals to regulate gene expression and development in Arabidopsis thaliana. However, how HXK1 mediates glucose signaling in the nucleus remains unclear. Here, using immunoprecipitation-coupled mass spectrometry, we show that two catalytic subunits of Polycomb Repressive Complex 2, SWINGER (SWN) and CURLY LEAF (CLF), directly interact with catalytically active HXK1 and its inactive forms (HXK1^G104D and HXK1^S177A) via their evolutionarily conserved SANT domains. HXK1, CLF, and SWN target common glucose-responsive genes to regulate glucose signaling, as revealed by RNA sequencing. The glucose-insensitive phenotypes of the Arabidopsis swn-1 and clf-50 mutants were similar to that of hxk1, and genetic analysis revealed that CLF, SWN, and HXK1 function in the same genetic pathway. Intriguingly, HXK1 is required for CLF- and SWN-mediated histone H3 lysine 27 (H3K27me3) deposition and glucose-mediated gene repression. Moreover, CLF and SWN affect the recruitment of HXK1 to its target chromatin. These findings support a model in which HXK1 and epigenetic modifiers form a nuclear complex to cooperatively mediate glucose signaling, thereby affecting the histone modification and expression of glucose-regulated genes in plants.     

Involvement of Hexokinase Hxk1 in Glucose Catabolite Repression of LIP2 Encoding Extracellular Lipase in the Yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica.