compounds such as amino acids. In developing fruits, about 70 of the total amino acid content
found in the pericarp belongs to the glutamate family [12]. g-Aminobutyric acid GABA was the
predominant N-form almost 60 of the total amino acid molar content at the earlier growing
stages of the tomato fruit and glutamine ca. 30 in mature green fruits [12].
There are few reports about the enzymes in- volved in amino acid biosynthesis in the fruit.
Glutamine synthetase GS, which catalyzes the synthesis of glutamine from glutamate, ATP and
ammonium, was detected in green and red toma- toes [13] and in avocado fruit [14]. NADH-gluta-
mate
synthase GOGAT;
EC 1.4.1.14
and ferredoxin-GOGAT EC 1.4.7.1, both enzymes
catalyzing glutamate synthesis, were observed at low activities in the pericarp of green tomato fruits
[15] and in intact fruit chloroplasts [16]. Glutamate dehydrogenase GDH, which catalyzes the amina-
tion of 2-oxoglutarate synthetic reaction and the deamination of glutamate catabolic reaction,
showed an increase in the enzyme protein content during ripening of avocado fruits [14]. Other amino
acid metabolizing enzymes or transcripts for them expressed at different levels during tomato fruit
ripening were reported: aspartate aminotrans- ferase, which was exclusively found in red fruits
[17]; a putative glutamate decarboxylase GAD; EC 4.1.1.15 [18] and arginine decarboxylase EC
4.1.1.19, whose transcripts appeared to peak at the breaker stage [19], and a histidine decarboxy-
lase EC 4.1.1.22 mRNA, which accumulated dur- ing early fruit ripening and then declined [20].
The present study was carried out to contribute to the understanding of the metabolism of amino
acids during the ripening of tomato fruits. For this purpose the molar contents of free amino acids
were quantified in three different tomato cultivars Platense, Cherry and Vollendung at green, yellow
and red ripening stages of the fruits. Concomi- tantly, the activities of nitrogen-metabolizing en-
zyme and proteins of GS, GOGAT and GDH were investigated.
2. Materials and methods
2
.
1
. Plant material Tomato plants cv Platense, Cherry and Vol-
lendung were grown in a controlled environment cabinet under a light intensity at the top of a
fruit-containing plant of 700 mmol s
− 1
m
− 2
. The temperature ranged from 23°C during the light
period 14 h to 18°C in the dark and the relative humidity was 70. Plants were grown in soil,
continuously maintained under optimal irrigation and supplied daily with a standard nutrient
medium [21]. Fully expanded 4th leaves of 30-day- old plants were used for GS activity and protein
assays. Fruits were allowed to ripen naturally on the plant, and when mature, that is when fruit’s
growing stops, they were harvested and grouped as ‘green, yellow and red’. Mature green fruits were
classified as those still green on the outside, with no development of pink or red color inside and named
‘green’. The breaker stage yellow included fruits with the outside no more than 50 red, but with
development of pink or red color in the interior. Fruits named ‘red’ were completely orange or red,
but firm. Pericarp tissue of harvested fruits were obtained by removing the locule tissues and seeds
and stored at − 80°C until analysis.
2
.
2
. Determination of amino acid content Pericarp tissue was extracted with chloro-
form:methanol [12,22]. The amino acid composi- tion in the methanolic phase was determined by
derivatization with ninhydrin or o-phthaldialde- hyde and using an amino acid analyzer ALPHA
PLUS LKB 4151 or HPLC system Pharmacia- LKB [22].
2
.
3
. Enzyme extraction and assays Pericarp tissues were ground with a mortar and
pestle at 4°C in extraction buffer 10 mM ascor- bate, 2.5 mM DTT, 1 mM Na
2
EDTA, 10 mM MgCl
2
, 10 glycerol, 50 mM HEPES – KOH, pH 8.0
plus 20
ww insoluble
polyvinyl- pyrrolidone. The pericarp homogenate was filtered
through Miracloth and centrifuged at 20 000 × g for 20 min. Enzyme activities were assayed follow-
ing gel filtration through Sephadex G-25 equili- brated with the extraction buffer. For assaying
transaminases, 40 mM pyridoxal-5-phosphate was added to the extraction buffer. GDH activity was
determined in the aminating direction as described earlier [23]. Ferredoxin- and NADH-GOGAT
were assayed as reported by Gallardo and cowork- ers [15]. GS was extracted from tissues by homog-
enizing tomato leaf or fruit pericarp with a mortar
and pestle at 4°C with the addition of extraction buffer 100 mM imidazole – HCl, 0.3 vv 2-
mercaptoethanol, 50 glycerol, and centrifuged 15 min at 21 000 × g. Protein concentrations were
determined in the supernatant as described [24]. The activity of GS was measured for the trans-
ferase reaction by staining a non-denaturing poly- acrylamide gel 8 [25] loaded with protein
extracts 17 mg in a Tris – glycine buffer system and run at 120 V for 2 h. After electrophoresis,
the gel was incubated for 1 h at 37°C with GS reaction mixture 80 mM NH
2
OH – HCl, 35 mM Na – arsenate, 34 mM glutamine, 1 mM ADP,
100 mM imidazole – HCl, pH 7.1 and subse- quently the color was developed by the addition
of FeCl
3
– HCl – thrichloroacetic acid [25]. GAD was monitored by GABA formation from gluta-
mate by derivatization with o-phthaldialdehyde and HPLC [22]. The assay medium was essen-
tially as previously reported [26]. NAD-malate dehydrogenase EC 1.1.1.37, NADP-malic en-
zyme EC 1.1.1.40, aspartate aminotransferase and alanine aminotransferase EC 2.6.1.2 were
measured by standard procedures [27]. One unit of enzyme activity IU is defined as the amount
of enzyme catalyzing the transformation of 1 mmol of substrate per hour.
2
.
4
. Protein extraction for SDS-PAGE and immunoblotting
Protein extracts were obtained by homogeniz- ing the leaf or pericarp with a mortar and pestle
in 50 mM Tris – HCl pH 7.5 and 1 mM EDTA, filtered through Miracloth, incubated with 2
SDS and 0.5 mM phenylmethylsulfonyl fluoride at 25°C and centrifuged at 20 000 × g for 5 min.
The
supernatant was
precipitated with
thrichloroacetic acid final concentration 20 for 20 min in an ice bath, the pellet was washed
twice with 80 acetone, resuspended in 2 SDS and 0.5 b-mercaptoethanol and subjected to
SDS-PAGE. After electrophoresis proteins were either stained with a silver kit BioRad or elec-
troblotted
to nitrocellulose
membranes. Im-
munodetection was carried out according to the manufacturer’s procedure ECL Amersham, us-
ing antisera raised in rabbits against Zea mays chloroplastic GS, Ferredoxin-GOGAT [28] or Vi-
tis 6inifera NADH-GDH [29].
3. Results
