Plant Science 160 2001 209 – 218
Cloning and characterization of a cDNA encoding hexokinase from tomato
Thierry Menu
a
, Christophe Rothan
a
, Nir Dai
b
, Marina Petreikov
b
, Christelle Etienne
a
, Agnes Destrac-Irvine
a
, Arthur Schaffer
b
, David Granot
b
, Be´re´nice Ricard
a,
a
Unite´ de Physiologie Ve´ge´tale, Institut National de la Recherche Agronomique, B.P.
81
,
33883
Villena6e d
’
Ornon, France
b
Institute of Field and Garden Crops, Agricultural Research Organization, Volcani Center, Bet Dagan
50250
, Israel Received 17 April 2000; received in revised form 30 June 2000; accepted 30 June 2000
Abstract
Two different partial sequences encoding putative hexokinase HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1 were isolated from tomato Lycopersicon esculentum by RT-PCR using degenerate primers. Southern blot analysis suggested the
existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK
2
contained an open reading frame encoding a 496 amino acid protein that has 69 identity with the two Arabidopsis HXKs, 83 and 85 identity with potato StHXK
1
and tobacco NtHXK, respectively. However, this clone had 97 amino acid identity with potato StHXK
2
and, therefore, was named LeHXK
2
. LeHXK
2
cDNA was expressed in a triple mutant yeast Saccharomyces cere6isiae strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK
2
grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK
2
expressed in yeast were determined after the purification of LeHXK
2
by HPLC-ion exchange chromatography, confirming the identity of LeHXK
2
as hexokinase with higher affinity to glucose. LeHXK
2
mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK
2
was most highly expressed in flowers. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Sugar phosphorylation; Lycopersicon esculentum; cDNA www.elsevier.comlocateplantsci
1. Introduction
Hexose phosphorylating enzymes are function- ally classified as fructokinase FRK, glucokinase
GLK or hexokinase HXK, based on their sub- strate specificities. FRK and GLK phosphorylate
strictly fructose Fru and glucose Glc, respec- tively, while HXK can phosphorylate a range of
hexoses e.g. Fru, Glc and Man. Together, these enzymes catalyze the first, irreversible step of
hexose metabolism. These reactions potentially constitute an important regulatory step in plants
and in other organisms [1 – 3].
Only a few genes encoding hexose phosphory- lating enzymes have been cloned from plants in-
cluding tomato. Two tomato cDNAs encoding FRK were isolated and their ability to comple-
ment a yeast triple mutant unable to phosphory- late either Glc or Fru showed that both cDNAs
encoded genuine FRK [4,5]. Several HXK genes have been cloned so far, two from Arabidopsis
Abbre6iations
:
DAA, days after anthesis; FRK, fructokinase; Fru, fructose; Gal, galactose; Glc, Glucose; Glc 6-P, glucose 6-phosphate;
GLK, glucokinase; HXK, hexokinase; Man, mannose; PGI, phos- phoglucose isomerase; PGK, phosphoglycerate kinase; RT, reverse
transcriptasetion; Suc, sucrose; Ura, uracil. The nucleotide sequence of LeHXK
2
will appear in the GenBank Nucleotide
Sequence Database
under the
accession number
AF208543. Corresponding author. Tel.: + 33-55-6843234; fax: + 33-55-
6843245. E-mail address
:
ricardbordeaux.inra.fr B. Ricard. 0168-945201 - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 3 2 - 0
AtHXK
1
and AtHXK
2
and potato StHXK
1
and StHXK
2
, one from tobacco and one from spinach [1,6,7] and accession numbers X94302;
AF118133. The Arabidopsis HXK genes were pro- posed to be sugar sensors based on results ob-
tained with
transgenic Arabidopsis
plants overexpressing AtHXK [1]. Similar results were
obtained with transgenic tomato plants overex- pressing AtHXK suggesting that the sugar signal-
ing pathways in tomato might also be mediated by hexokinase [8]. To analyze the role of tomato
hexokinase in tomato plants it was important to isolate and characterize tomato HXKs. In this
paper, we describe the isolation of two different partial sequences of tomato HXKs that were used
to demonstrate the existence of two divergent HXK genes in tomato plants. A full-length cDNA
encoding one of the two HXKs was cloned and designated LeHXK
2
based on its higher homology to StHXK
2
. Kinetic analyses of LeHXK2 follow- ing expression in yeast cells suggest that LeHXK2
is not the GLK previously reported by Martinez- Barajas and Randall [9]. Expression analysis of
LeHXK
2
indicates that LeHXK
2
is expressed in a large spectrum of tissues and organs and at differ-
ent stages of fruit development with the lowest expression in leaves and the highest in flowers.
2. Materials and methods
