3. Results
3
.
1
. Cloning of the full-length LeHXK
2
cDNA Total RNA extracted from tomato fruit har-
vested 10 DAA was reverse transcribed. The cDNA obtained was amplified using degenerate
primers for two conserved regions of Arabidopsis and potato HXKs. A single band about 700 bp
long was obtained, purified and subcloned. Several clones were partially sequenced and hinted at the
presence of two different HXK genes Fig. 1. Of the 125 nucleotides, 17 were different in the two
HXK sequences, suggesting considerable diver- gence. However, every nucleotide change but one
was in the third position and silent, so that the deduced amino acid sequences were practically
identical. The two sequences were designated LeHXK
1
and LeHXK
2
, according to their higher similarity to potato StHXK
1
or StHXK
2
, respec- tively Fig. 1. Both the sequences were used as
probes for screening of a cDNA library prepared from young cherry tomato fruit [10]. A single type
of cDNA clone corresponding to LeHXK
2
was isolated and the clone containing the largest insert
of 1770 bp was sequenced. LeHXK
2
contained an open reading frame of 1491 bp followed by a
142-bp sequence and a poly A
+
tail. The ATG triplet beginning at nucleotide position 138 was
assigned as the likely site of translation initiation since an in-frame termination codon TAA was
located at positions 108 – 110. The ORF encoded a putative HXK protein of 496 amino acids with a
calculated molecular weight of 53754 D and a pI of 6.3. The putative LeHXK
2
protein showed sim- ilar homology with the two Arabidopsis HXK
proteins 69 identity, and higher homology with potato StHXK
1
and StHXK
2
proteins, 83 and 97 identity, respectively Fig. 2. ATP and two
phosphate binding sites, as well as a sugar recogni- tion site were located in the same highly conserved
domains in all of the plant hexokinases Fig. 2.
3
.
2
. Number of HXK genes in tomato plants While two different sequences encoding HXK
were found by RT-PCR of tomato fruit, only one full-length sequence was recovered by the screen-
ing of a fruit cDNA library. To determine the number of different HXK genes in tomato, south-
ern blot analyses were carried out on tomato genomic DNA digested with four restriction en-
zymes Fig. 3. The LeHXK
1
and LeHXK
2
partial sequences shown in Fig. 1 were radiolabeled with
32
P and hybridized with blots shown in Fig. 3. The LeHXK
2
probe hybridized intensely to a single band in the digests with EcoRI E, HindIII H
and XbaI X panel A. Other bands hybridized less intensely. These bands hybridized intensely
with the LeHXK
1
probe panel B and C. These
Fig. 1. Deduced amino acid and nucleotide sequence of two partial HXK sequences, LeHXK
1
and LeHXK
2
, obtained by RT-PCR from tomato fruit 10 DAA. 125 nucleotides of the approximately 700 nucleotide PCR product were determined by
manual sequencing as described in Section 2. Amino acids and nucleotides are numbered on the right. The amino acids corresponding to the forward RT-PCR primer are overlined. The two partial sequences were aligned with the corresponding
sequences of StHXK
1
and StHXK
2
. Asterisks indicate identical nucleotides. Only seven nucleotide changes exist between LeHXK
1
and StHXK
1
and only five between LeHXK
2
and StHXK
2
, while 17 changes exist between LeHXK
1
and StHXK
2
, and 12 changes exist between LeHXK
2
and StHXK
1
.
Fig. 2. Comparison of predicted amino acids of LeHXK
2
with potato HXK StHXK
1
, StHXK
2
and Arabidopsis HXK AtHXK
1
, AtHXK
2
. ATP, phosphate binding and substrate recognition sequences [19], as well as identical and conserved .: residues are marked. The positions of degenerate forward Fwd and reverse Rev primers, as well as the specific S2 and AS1 primers used
for RT-PCR are indicated by overlining. GeneBank accession numbers are AtHXK
1
-U28214, AtHXK
2
-U28215, StHXK
2
- AF106068, LeHXK
2
-AF208543. The sequence of StHXK
1
is the corrected sequence of accession number-X94302 Veramendi, pers. commun..
Fig. 3. Southern hybridization of genomic DNA isolated from leaves of tomato plants. The DNA 10 mg was restricted with
EcoRI E, HindIII H, XbaI X and BamHI B, as indi- cated above each lane. The same blot was hybridized succes-
sively with the original 700 bp LeHXK
2
A and LeHXK
1
C fragments. A duplicate blot was hybridized with the LeHXK
1
sequence B. The position of molecular weight markers Kbp are shown at the left of panel A.
Fig. 4. Growth of DFY632 triple mutant cells transformed with pFL61-LeHXK
2
on selective media. LeHXK
2
cDNA was subcloned into pFL61 and expressed in DFY632. Yeast
cells transformed with pFL61 were used as controls. 110 mM of either Gal, Glc or Fru was added to the selective media
− Ura for the uracil auxotrophic strain.
higher affinity to Glc. High affinity to Glc was confirmed by determining the kinetic properties of
LeHXK
2
in protein extracts from DFY632 yeast cells with either pFL61 or pFL-LeHXK
2
, follow- ing MonoQ chromatography Fig. 6A. Purified
extracts from cells transformed with pFL61 alone had no measurable activity for hexose phosphory-
lation while extracts from cells with pFL-LeHXK
2
showed high affinity for Glc and Man, K
m
of 21
Fig. 5. Substrate specificity of LeHXK
2
assayed in a native PAGE. Native PAGE and activity staining were carried out
as previously described [15], except that the staining solution included 0.6 agarose. When added, hexoses were present at
1 mM.
results suggest that in tomato, there are two dis- tinct HXK genes, which are sufficiently different
to give only slight cross-hybridization.
3
.
3
. Functional identification of LeHXK
2
cDNA To analyze whether LeHXK
2
encodes an au- thentic HXK, LeHXK
2
was cloned into a yeast expression vector pFL61 to obtain pFL-LeHXK
2
. pFL-LeHXK
2
was expressed in triple mutant yeast cells, DFY632, which are unable to phosphorylate
hexoses and therefore are unable to grow on Glc or Fru as carbon sources. As shown in Fig. 4,
DFY632 cells transformed with pFL61-LeHXK
2
grew on Glc and Fru while control cells containing only pFL61 failed to grow. These results were
retested by native gel analysis of crude extracts prepared from yeast cells with pFL61 or pFL61-
LeHXK
2
. However, activity was detected with Glc as substrate but not with Fru Fig. 5. Accord-
ingly, the doubling time on Glc was 5 h while that on Fru was 11 h suggesting that LeHXK
2
has
Fig. 6. Partial purification and characterization of hexokinase from yeast extracts expressing LeHXK
2
. A HPLC-ion ex- change MonoQ chromatography of yeast protein extracts
separated using a KCl gradient. B Hexose phosphorylation activity of LeHXK
2
with Glc, Man and Fru. Note different scales of sugar concentrations. V, nmoles of Glc 6-phosphate,
Man 6-phosphate or Fru 6-phosphate produced per mg of protein per min.
Table 2 Effect of HXK inhibitors on LeHXK
2
a
Activity Concentration
Compound mM
Glucosamine 50
83.8 9 6.2 n = 4
50 Mannoheptulose
80.4 9 7.2 n = 4
5 38.7 9 5.9
ADP n = 4
81.1 9 2.2 Glucose-6-Phosphate 5
n = 3
a
Glucose phosphorylation activity was measured using de- salted crude extracts in the presence of 1 mM Glc. The results
are expressed as the percentage of activity without inhibitor. N, number of repetitions
carried out with the desalted crude extract indi- cated that LeHXK
2
was weakly inhibited by man- noheptulose, glucosamine and Glc 6-P but was
strongly inhibited by ADP Table 2.
3
.
4
. Expression of LeHXK
2
mRNA LeHXK
2
mRNA was easily detected by RT- PCR using 2 mg of the total RNA for RT and 670
ng of the product for PCR with specific primers S2 + AS1. Fig. 7 shows that an approximately
700-bp band was amplified under these conditions in roots, leaves, flowers and developing fruit.
These conditions permit the detection of specific mRNAs but not their relative amounts. Modifica-
tions were, therefore, introduced in order to per- mit quantitative RT-PCR analyses to be carried
out. Of the 2 mg total RNA used in the oligo-dT primed RT reaction, aliquots corresponding to 200
ng of the total RNA were amplified in PCR reactions with primers specific for LeHXK
2
and for Actin. These conditions gave linear amplifica-
and 38 mM, respectively Fig. 6B and Table 1. Like most plant HXKs, LeHXK
2
had a K
m
value for Fru in the millimolar range, 7.7 mM, but a
higher V
max
than for Glc or Man. Inhibitor studies
Fig. 7. Expression analysis of LeHXK
2
in different organs. RT-PCR was carried out with specific primers for LeHXK
2
and for Actin as given in Section 2. MG, mature green; Br, breaker.
Table 1 Kinetic parameters of LeHXK
2
enzyme expressed in yeast Substrate
V
max
nmolmg K
m
mM V
max
K
m
Glucose 7.5
158 0.021
0.038 Mannose
3.7 142
Fructose 7.74
284 .04
Fig. 8. Quantitative RT-PCR of cDNA from different organs and tissues and from different stages of fruit development. RNA 2 mg was used for RT as described in Section 2. 200 ng from the same RT reaction were used as template for quantitative PCR
reactions using primers specific for LeHXK
2
and Actin LeAc. PCR products were transferred to nylon membranes and detected by hybridization with specific rabiolabeled probes. Imbibed seeds, seeds placed on moistened filter paper for 5 days at 25°C in the
dark.
tion but the products could only be detected by hybridization with radiolabeled probe. Fig. 8
shows that LeHXK
2
was most highly expressed in flowers but had also high expression in ovary
tissue at anthesis and in seeds imbibed for 5 days. As quantified with an Instant Imager Packard,
the induction of LeHXK
2
messenger in fruit at anthesis and in imbibed seeds was 2.5 – 2.7-fold
higher than the low-level expression measured in most tissues, while induction in flowers was 292-
fold.
4. Discussion
Kategori