Biochemical properties and subcellular localization of six members of the HXK family in maize and its metabolic contribution to embryo germination

Abstract

BackgroundSeed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4–9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process.ResultsFrom the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4–9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4–7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4–6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4–6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7–8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos.ConclusionsConsidering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1605-x) contains supplementary material, which is available to authorized users.

An Integrative Approach to Analyze Seed Germination in Brassica napus

“…Cluster 4 encompassed genes coding for mitochondrion and respiratory chain components, genes related to ribosomes and translation, Golgi apparatus, endoplasmic reticulum, and vesicle mediated transport. We also found genes encoding for key enzymes involved in fatty acid beta-oxidation and glyoxylate cycle (MLS, ICL, Comai et al, 2007), tricarboxylic acid cycle (ACO3, CSY3, Pracharoenwattana et al, 2005; Hooks et al, 2014), carbohydrate biosynthesis, and glycolysis (HXK1, Aguilera-Alvarado et al, 2019). In the case of BnaHKX1 , we found that the peaking time point in C033 and C032 was delayed compared to C129 ( Supplementary Figure S3 ).…”

Moonlighting Proteins: The Case of the Hexokinases

“…It is known that mammal HXKI-III glucose phosphorylating activity are product-regulated: physiological concentrations of G6P can allosterically inhibit its activity in a non-competitive way with respect to glucose, but competitive to ATP ( Van Schaftingen, 2013 ). In maize, mitochondrial attached HXKs are less inhibited by G6P and ADP than the cytosolic HXKs ( Aguilera-Alvarado et al, 2019 ), suggesting that their subcellular localization is a key component to control its enzyme activity.…”

“…A distinct feature of some hexokinases that might contribute to its moonlighting activity is their subcellular location: some of them, like S. cerevisiae HXK2 ( Fernández-García et al, ) and maize HXK7 and HXK8 ( Aguilera-Alvarado et al, 2019 ) are located at the cytoplasm; others, in glycosomes, like T. brucei HXK2 ( Blattner et al, 1995 ; Michaels et al, 2006 ); many in the mitochondrial outer membrane or MOM, like human HXKII ( Arora and Pedersen, 1988 ; Dubey et al, 2016 ), A. thaliana HXK1 ( Cho et al, 2006a ) and apple HXK1 ( Sun et al, 2018 ); or in the chloroplast, like P. patens HXK1 ( Olsson et al, 2003 ) and Oryza sativa HXK4 ( Cho et al, 2006a ). And even there are HXKs that are translocated from one subcellular compartment to the nucleus, like AtHXK1 ( Cho et al, 2006b ) and ScHXK2 ( Vega et al, 2016 ).…”

Genome-Wide Association Study of Maize Aboveground Dry Matter Accumulation at Seedling Stage

“…Out of these, 18 genes ( GRMZM2G105571 , Zm00001d027469 , Zm00001d043622 , GRMZM2G353553 , GRMZM2G167856 , GRMZM2G171373 , GRMZM2G092120 , CKX10 , GRMZM2G035217 , GRMZM2G128644 , GRMZM2G314064 , GRMZM2G407825 , GRMZM6G865522 , GRMZM2G045732 , pco092737 , Zm00001d002111 , GRMZM2G042412 , and GRMZM2G061537 ) were associated with a minimum of two traits. In addition, some of them [ GRMZM2G105571 ( Li et al, 2018 ), GRMZM2G167856 ( Li et al, 2016 ), GRMZM2G171373 ( Aguilera-Alvarado et al, 2019 ), CKX10 ( Smehilova et al, 2009 ), GRMZM2G128644 ( Song et al, 2016 ), GRMZM2G314064 ( Jiang et al, 2012 ), GRMZM2G407825 ( Yan et al, 2017 ), etc.] had been reported by previous studies of maize seed and leaf, which was consistent with our study.…”