1-1-1997
Glutamate-1-semialdehyde aminotransferase
activity during tomato fruit development and
ripening
Richard Edward Finger
Iowa State University
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Recommended Citation
Finger, Richard Edward, "Glutamate-1-semialdehyde aminotransferase activity during tomato fruit development and ripening" (1997).Retrospective Theses and Dissertations. 17408.
during tomato fruit development and ripening
by
Richard Edward Finger
A thesis submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
Major: Plant Physiology
Major Professors: Richard J. Gladon and David J. Hannapel
Iowa State University Ames, Iowa
Graduate College Iowa State University
This is to certify that the Master's thesis of Richard Edward Finger
has met the thesis requirements of Iowa State University
TABLE OF CONTENTS
GENERAL INTRODUCTION
Introduction 1
Thesis Organization 2
Literature Review 2
CHARACTERIZATION OF GLUT AMA TE-1-SEMIALDEHYDE AMINOTRANSFERASE AND ACTIVITY THROUGHOUT TOMATO FRUIT DEVELOPMENT
AND RIPENING 7
Abstract Introduction
Materials and Methods Results
Discussion Literature Cited
GENERAL CONCLUSIONS LITERATURE CITED ACKNOWLEDGMENTS
7
8 9 11
12 15
Introduction
Ripening involves a series of reactions within fruit tissue that allow it to soften,
sweeten, become aromatic, and change color. These characteristic changes tell us that the fruit is horticulturally mature and may be consumed. The loss of green color in fruit is probably the most evident to the consumer because it is a visual feature, and this color change is something that we would like to understand and control, if possible. In order to control this process, we
have to know how the plant controls or signals this almost universal degreening process, the loss of chlorophyll within the fruit during development and ripening. It is logical to begin the search for a control of chlorophyll concentration in fruit with enzymes responsible for
chlorophyll biosynthesis. Reinbothe and Reinbothe ( 1996) list two major points of regulation in chlorophyll biosynthesis: the formation of protoporphyrin IX via Mg chelatase and the formation of ALA via glutamate-1-sernialdehyde aminotransferase (GSA T).
The purpose of this research was to measure the activity of GSAT, a key enzyme in chlorophyll biosynthesis, throughout development and ripening to aid in determining a control point for chlorophyll concentration within the fruit during maturation. Our results show that GSAT activity was great in immature fruits, declined to day 25 postanthesis, rose to a peak at day 40 (mature green) fruits, and then receded during ripening. GSAT activity corresponded solely to the sharp decline in total chlorophyll and chlorophyll a concentrations from day 10 to
Thesis Organization
This thesis is organized into three chapters. The first chapter is a general review that includes an overview of recent and relevant literature. The second is a journal manuscript, and the third is general conclusions that can be drawn from the research. Literature cited in the General Introduction and General Conclusions follow the last chapter.
Literature Review
The disappearance of chlorophyll during development of the fruit may have a biochemical significance to fruit ripening. In the process of elucidation of the control of chlorophyll concentration in fruit, recent studies have focused on chlorophyll degradation as a controlling factor by measuring chlorophyllase activity in both olives and citrus (Minguez-Mosquera and Gallardo-Guerrero, 1996; Trebitsh et al., 1993). However, it is logical to predict that chlorophyll biosynthetic enzymes could control chlorophyll content within the fruit. Previous studies with biosynthetic enzymes of chlorophyll in tomatoes have shown a decrease in activity early in fruit development when chlorophyll content decreases sharply (Kyriacou et al., 1996; McMahon et al., 1990).
Chlorophyll and heme share a common biosynthetic pathway in higher plants, algae, photosynthetic eubacteria, nonphotosynthetic eubacteria, and archaebacteria (Beale et al., 1975; Beale, 1976; Grimm et al., 1991; Li et al., 1989; Palmier et al., 1997). The initial reactions have been named the C-5 pathway because the 5-carbon skeleton of glutamate remains intact to the formation of 5-aminolevulinic acid (ALA; Beale and Castelfranco, 1974). Higher animals and organisms that cannot produce ALA from glutamate contain ALA synthase that catalyzes a condensation of glycine and succinyl coenzyme A to form ALA for heme production (Leeper,
1985). ALA is considered the first committed precursor for tetrapyrrole synthesis in all
uroporphyrinogen III, which is processed through various steps into protoporphyrin IX. This molecule is an important branch point in the pathway as it accepts either an Fe2+ to be processed
into heme or a Mg2+ to be converted into chlorophyll (Reinbothe and Reinbothe, 1996).
Glutamate, the precursor of chlorophyll, is prevalent within the immature tomato fruit and increases in concentration throughout development (Yu et al., 1967). Glutamyl-tRNA
synthase ligates glutamate to its corresponding tRNA molecule, the same complex that is used in protein synthesis within the chloroplast (Beale and Weinstein, 1991). The reaction requires ATP hydrolysis to AMP and a divalent metal cofactor, either Mg2+ or Mn2+ (Mayer et al.,
1994). Glutamyl-tRNA synthase binds first to the tRNA molecule (Hara-Yokoyama et al., 1986) and shows specificity to chloroplast and prokaryotic tRNA (Schon et al., 1986). It will not bind to cytosolic tRNA even within the same species (Hess et al., 1992). Yet, the enzyme sometimes misacylates with a tRNA corresponding to glutamine (Chen et al., 1990a). The tertiary structure of the tRNA is involved in binding, as point mutations in the T -loop of the
RNA altered its conformation and uncoupled chlorophyll synthesis (Stange-Thomann et al., 1994).
Cytokinins stimulate ALA biosynthesis by increasing the number of chloroplast
tRNA Giu molecules (Masuda et al., 1994) and by upregulating the activity of the next enzyme in
ALA synthesis, glutamyl-tRNA reductase (Masuda et al., 1995). This enzyme reacts with the
glutamyl-tRNA complex and uses NADPH (Chen et al., 1990b) to reduce the a-carboxylic
acid group of the C-terrninus of glutamate to an aldehyde with a concomitant release of the tRNA (Jahn et al., 1992). The reaction is inhibited by heme (Huang and Wang, 1986); however, it is difficult to imagine this step as an important point of regulation in chlorophyll synthesis. The product of this reaction is glutamate-1-semialdehyde (GSA), which is very unstable in linear form. It can chemically break down into glutamate and ALA at high
1991). A cyclized form of GSA has been proposed (Jordan et al., 1993), but GSA must be linear to bind with the next enzyme in the tetrapyrrole synthesis pathway (Pugh et al., 1992). Linear GSA used for assays of GSAT is synthesized in vitro by the ozonolysis method of Gough and coworkers (1989).
Glutamate-1-semialdehyde aminotransferase (GSAT; also glutamate-1-semialdehyde-2, 1-aminomutase) converts GSA to ALA, and it is considered to control ALA synthesis and to be the rate-limiting step in tetrapyrrole biosynthesis (Beale and Castelfranco, 1974). In barley, GSAT is encoded in the nuclear genome and is transcribed into a 46 kDa protein dimer in the cytoplasm. It has a 34 amino acid transit peptide sequence that is cleaved after chloroplast import (Grimm, 1990), and the active enzyme is associated with the soluble fraction (stroma) of leaf tissue (Kannangara and Gough, 1978; Berry-Lowe et al., 1992). GSAT is an important control point for chlorophyll synthesis as it is transcriptionally regulated by white light (Ilag et al., 1994) and blue light, but not red light (Mayer and Beale, 1991). In Chlamydomonas, 30 min of light caused a doubling of mRNA that encodes GSAT, and 2 hours of light induced a 26-fold increase in GSA T transcript (Matters and Beale, 1994 ). Circadian control of GSAT showed mRNA encoding for GSAT peaked every 24 hrs (Kruse et al., 1997).
The GSAT mechanism was once thought to be a transamination that occurred between two open-chained GSA molecules, anN-transfer that takes place in Chlorella (Mayer et al., 1993). However, Avissar and Beale (1989) showed that, like the majority of
aminotransferases, GSAT requires a vitamin B6 cofactor for optimal activity, and the cofactor is the source of the N-transfer. Hennig and coworkers ( 1997) found that the GSAT protein is an asymmetric dimer in Synechococcus with one cofactor per subunit. Through point
studies by llag and Jahn (1992) introduced putative intermediates to the reaction: dioxovaleric acid (DOV A) and diaminovaleric acid (DA VA). Brody and coworkers (1995) found that the native enzyme and enzyme with DA VA released the reduced form of the cofactor into solution. DOV A released the oxidized form of the cofactor, showing that DA VA is the most probable intermediate for the reaction.
Two genes that encode GSAT protein have been cloned from Arabidopsis and soybean (Wenzlau and Berry-Lowe, 1995; Frustaci et al., 1995). In tomato, one gene has been cloned that encodes for GSAT (Po1king et al., 1995). This gene is located within the nuclear genome and is translated in the cytosol with a 44 amino acid transit peptide sequence that is cleaved to a final 46 kDa protein in tomato. In tomato fruit, GSAT mRNA levels decreased to day 35 postanthesis and then stay relatively constant. GSAT protein concentrations decreased to day 25 postanthesis and were barely detectable thereafter throughout development and ripening (Polking et al., 1998).
There is some evidence that GSAT activity is not associated with control of chlorophyll concentration as ALA produced via GSA T may not necessarily be processed into tetrapyrroles. Some studies have shown that ALA can be metabolized through nonporphyrin pathways with a concomitant release of C02. Both [4-14C]ALA and [5-14C]ALA fed to etiolated barley leaves
evolved 14C0
2 (Duggan et al., 1982). [5-14C]ALA was also fed to ripening tomato fruit tissue
and 14C0
2 was collected (El-Rayes, Ph.D. Dissertation, 1987). These studies suggest that
under certain circumstances the plant can metabolize ALA away from chlorophyll synthesis and that this metabolic process is associated with respiration.
However, other studies suggest that increased chlorophyll within the fruit (via mutants) may also be associated with the timing of respiration. The tomato mutant dark green ( dg ),
content were similar between the two genotypes. Another tomato mutant, Green ripe ( Gr ), has a delayed degreening process in development. This mutant produces a burst of respiration 10 to 20 days beyond the wild-type (Jarret et al., 1984).
CHARACTERIZATION OF GLUTAMATE-1-SEMIALDEHYDE AMINOTRANSFERASE AND ACTIVITY THROUGHOUT
TOMATO FRUIT DEVELOPMENT AND RIPENING
A paper to be submitted to the Journal of Plant Physiology Richard E. Finger, David J. Hannapel1, and Richard J. Gladon1
Abstract
Glutamate-1-sernialdehyde aminotransferase (GSAT) is a key enzyme in chlorophyll biosynthesis and is a logical point to control chlorophyll loss during development in fruits. The objective of this research was to measure the activity of GSAT throughout development and ripening of the tomato fruit and compare this activity to chlorophyll concentration in fruits at the same time intervals. GSAT activity was also characterized in immature tomato fruits, and its activity was measured in tomato organs. An optimum pH of7.0, a maximum velocity (V maJ of 2.3 nmol/g of tissue, and a Km of 119 IJ.M were found for GSAT from fruits 15 days postanthesis. Activity was greatest in leaves of 4-week-old seedlings at 3.84 nmol/g tissue, 1.67 nmol/g tissue in fruits 15 days postanthesis, and low activity was measured in roots, stems, and overripe fruits. Throughout tomato fruit development, GSAT activity was greatest at day 10 postanthesis and declined to day 25. The activity rose to day 40 and then decreased again during ripening to 60 days postanthesis. Chlorophyll concentration in tomato fruit declined throughout development and ripening with the greatest decrease between days 10 and 25 postanthesis. Our results indicate that GSAT activity is a possible control point for
chlorophyll concentration in early tomato fruit development, but it shows no sign of controlling chlorophyll content in the fruit after day 25 postanthesis.
Introduction
In higher plants, algae, and certain bacteria and archaebacteria, the initial steps of tetrapyrrole biosynthesis are the 'C-5 pathway' that converts the 5-carbon skeleton of glutamate to 5-aminolevulinic acid (ALA) (Beale et al., 1975; Jahn et al., 1991; Reible et al., 1989; Palmier et al., 1996). The last step of the C-5 pathway is the conversion of glutamate-1-semialdehyde (GSA) to ALA, which is an important regulation step in the chlorophyll biosynthesis pathway (Reinbothe and Reinbothe, 1996), and is considered the rate-limiting step in tetrapyrrole biosynthesis (Beale and Castelfranco, 1974). The enzyme that catalyzes
this reaction is glutamate-1-semialdehyde aminotransferase (GSAT; E.C. 5.4.3.8; also glutamate-1-semialdehyde-2, 1-aminomutase), and it is located within the stroma of the
chloroplast (Kannangara and Gough, 1978; Berry-Lowe et al., 1992). This enzyme, like the
majority of aminotransferases, requires a vitamin B6 cofactor for optimum activity (Avissar and Beale, 1989).
GSA T protein crystallized from Synechococcus is an asymmetric ~ dimer that has one
cofactor binding site per subunit (Hennig et al., 1997). GSAT functions in a ping-pong bi-bi mechanism (Smith et al., 1992) that alternates the cofactor between pyridoxal-5-phosphate and pyridoxamine-5-phosphate, releasing diaminovaleric acid as the most probable intermediate (Brody et al., 1995). GSA, the substrate, must be linear to have the carbonyl and amino groups in a cis position to react with the cofactor (Pugh et al., 1992). For most assays of
GSAT, GSA is synthesized in vitro by the method of Gough et al. (1989).
One gene that encodes GSAT has been cloned from tomato (Polking et al., 1995). Messenger RNA accumulation of this gene was high in young fruits and decreased to day 35 postanthesis whereafter they stayed constant during the remainder of fruit development and ripening. Immunoblot analysis has characterized GSA T protein throughout tomato fruit development, and it showed that GSA T protein decreased in young fruits to 25 days
Enzyme activity of GSAT has not been characterized in any fruit tissue or measured throughout the development or ripening of a fruit. The objectives of this research were to characterize GSA T in tomato fruit, obtaining an optimum pH and a
K,
for the enzyme, and to measure GSAT activity during tomato fruit ontogeny. GSAT activity during development can then be compared to total chlorophyll, chlorophyll a, and chlorophyll b concentrations within the fruit at these time intervals, and this will aid in discerning if GSAT is a control point for chlorophyll loss during tomato fruit maturation.Materials and Methods
Chemicals. GSA was a gift from Dr. C. G. Kannangara (Carlsberg Laboratory, Copenhagen, Denmark). Reagent grade levulinic acid, pyridoxal-5-phosphate, dithiothreitol, 3-(4-morpholino) propane sulfonic acid (MOPS) buffer, and polyvinylpolypyrrolidone (PVPP) were purchased from Sigma Chemical Co. (St. Louis, MO).
Plant materials. Lycopersicon esculentum Mill. 'Rutgers' seeds were germinated in rockwool cubes (Grodan # A036/4010/10) in a greenhouse (24
oc
day, 20oc
night) undersupplementary irradiance of 300 11mol·s·1·m·2 from high pressure sodium lamps with a 20-hr
photoperiod. Seedlings were watered only for the first week. Thereafter, they were fertilized with a hydroponic solution with elemental concentrations of (mg-L-1): N 200, P 40, K 370, Ca
190, Mg 36, Fe 2.5, Cu 0.22, Zn 0.32, Mn 0.57, B 0.1 0, and Mo 0.005. The pH of the nutrient solution was 5.5 and had an electrical conductance (EC) of 2.2 mS·cm·1•
Two-week-old uniform seedlings were transplanted and maintained in a rockwool substrate (Grodan #
DMGG/42/40) and fertilized 7 times daily. Plants were trained to a single stem, and the stem apex was removed above the first inflorescence (McAvoy et al., 1989). Flowers were
Enzyme extraction. One gram of tissue was removed from the blossom end of the fruit (epidermis included) with a stainless-steel cork borer (1 em diameter). One gram of root, stem, and leaves of 4-week-old seedlings were dissected with a stainless-steel razor blade. All tissues were frozen in liquid N2 and ground in a mortar. PVPP (1 g) was immediately added
to prevent oxidation of phenylates. Five mL of O.lM MOPS buffer that contained 0.3 M glycerol and 0.1 mM DTT was added and blended with a Brinkman Polytron homogenizer (Westbury, NY). The extract was centrifuged at 4
oc
at 10,000 x g for 10 min to pellet cellular debris. The supernatant that contained soluble protein was passed through a Sephadex G-50 column to restrict endogenous low molecular weight contaminants to the assay (data not shown). This eluate was used for the enzyme source. The Bradford (1976) method was used to determine soluble protein content.Chlorophyll determination. Because it has a greater sensitivity to chlorophyll than other methods, the method developed by Knudson and coworkers ( 1977) was used to determine chlorophyll content within the fruit with the following modifications. One gram of fruit tissue was removed in the same manner as the enzyme assay and placed in a 40 mL glass vial. The tissue was covered completely with 5 mL of 95% ethanol, and the vial was capped. After 24 hrs in darkness, the ethanol/chlorophyll mixture was decanted into another vial. The tissue was again covered with 5 mL of 95% ethanol and kept in darkness for 24 hrs. The two
solutions were combined and chlorophyll a, chlorophyll b, and total chlorophyll concentrations were determined spectrophotometrically using the equations of Winterrnans and DeMots (1965).
Enzyme assay. GSAT activity was determined by measuring the rate of enzymatic ALA synthesis (ALA produced without enzyme was subtracted). GSAT activity was assayed by the method of Hoober et al. (1988) with the following modifications. Each reaction
was added to give final concentrations of 150 j..LM, 20 j..LM, and 10 mM, respectively. The
reaction was initiated by the addition of substrate and incubated at 30
oc.
After 30 min, whilethe reaction was increasing linearly with respect to time (data not shown), 38 j..LL of perchloric acid was added to terminate the reaction. The extracts were centrifuged at 16,000 x g for 8 min to remove protein. The supernatant was added to 308 j..LL of 1 M sodium acetate buffer (pH
4.8) and adjusted to pH 4.6 with 3M NaOH. After adding 300 j..LL acetylacetone to the mixture, the tube was shaken vigorously and incubated at 95
oc
for 20 min. The volume was adjusted to 2 mL with sterile deionized H20 and mixed with an equal volume of modified Erhlich's reagent. Absorbance was measured at 553 nm, 5 min after mixing. A molarextinction coefficient of 7.2 x 104 M·'·cm·' was used to quantify ALA (Mauzerall and Granick,
1956).
Experimental design. Fruits and plant material assayed for GSAT activity or
chlorophyll determination were chosen at random for a completely randomized block design. Each block contained means of 4 replications of fruits of the same physiological class (days after pollination). Graphs of each block gave similar results, therefore, the data were pooled. ANOVA was calculated for the data using SAS 6.06 (Cary, N.C.).
Results
GSAT activity from fruits 15 days postanthesis was greatest at pH 7.0 with a saturating substrate concentration, and 90% of its activity remained at our standard assay conditions of pH 6.8 (Fig. 1 ). GSA T activity, also from 15 day fruits, increased linearly with GSA concentration until it exceeded 120 j..LM, and the enzyme became saturated (Fig. 2). A
Lineweaver-Burke plot of IN vs. 1/[S] yielded a V max of 2.3 nmol/g fruit tissue and a~ of
leaves from 4-week-old seedlings at 3.84 nmol/g tissue. This value was 230% greater than activity measured in 15 day postanthesis fruits (Table 1). Less activity was observed in roots and stems from 4-week-old seedlings and 65 day (overripe) fruits, and all these organs exhibited GSAT activity less than 0.70 nmol/g tissue.
GSAT activity during fruit development was greatest at 10 days postanthesis with a value of 2.0 nmollg tissue. GSAT activity decreased sharply between days 10 and 25 postanthesis. After day 25, the activity increased to day 40 postanthesis to a value of 1.2
nmollg tissue. GSAT activity then decreased after day 40, reaching a low in 60 day fruit of 0.3 nmol/g tissue (Fig. 3C). The activity found at day 25 postanthesis was contrasted with the activity found at day 40 and were shown to be significantly different according to Fisher's LSD
(P=0.05), indicating a significant peak in activity during development. Total chlorophyll and
chlorophyll a concentrations within the fruit decreased throughout development and ripening with the most pronounced declines between days 10 and 25 postanthesis (Fig. 3B).
Chlorophyll b concentration stayed relatively constant throughout the ontogeny of the fruit. Soluble protein content decreased similarly to total chlorophyll with a large decrease between days 10 and 25 postanthesis and a smaller decline thereafter (Fig. 3A).
Discussion
We found that the optimum GSAT activity in tomato fruits was at pH 7.0, and this is similar to other plant systems. In Chlamydomonas, GSAT had an optimum pH of 7.2 (J ahn et al., 1991), whereas in soybean nodules, GSAT activity was optimal between pH 6.5 and 7.0 (Sangwan and O'Brian, 1992). Kannangara and Gough (1978) reported a pH optimum of 7.9 from barley, but nonenzymatic ALA, which form optimally at pH 8.5 (Hoober et al., 1988), may not have been subtracted as part of the assay protocol.
Substrate kinetics of GSAT from crude tomato fruit extracts showed that concentrations
barley eDNA expressed in E. coli was found to saturate with 60 to 80 MM GSA and a~ of
25~M was calculated (Berry-Lowe et al., 1992). Synechococcus GSAT also saturated around
60 ~M GSA, and they reported a lower ~n of 12 ~M (Smith et al., 1991). In barley, 100 ~M
GSA was shown to half-saturate the partially purified enzyme (Kannangara and Gough, 1978).
Our~~ value is similar to that of barley, which is the only measurement found that used an
enzyme source from a higher plant.
Previously, GSAT activity had never been measured throughout the development or ripening of a fruit. GSAT activity was greatest during development at the earliest stage of 10 days postanthesis and decreased sharply in activity to day 25 postanthesis (Fig. 3C). In this same developmental time frame, soluble protein, total chlorophyll, and chlorophyll a
concentrations within the fruit declined at a high rate as well. It is at this stage of development that GSAT activity declines and correlates with chlorophyll concentration. GSAT could be a logical point of control of chlorophyll concentration in the fruit in this early development, since GSA T is a major point of control in chlorophyll biosynthesis (Reinbothe and Reinbothe,
1996). A later enzyme of chlorophyll biosynthesis, 5-aminolevulinic acid dehydratase, decreased abruptly during this stage of tomato fruit development as well (Kyriacou et al.,
1996). This suggests a more complex downregulation of chlorophyll biosynthetic enzymes during this developmental stage of the tomato fruit.
From day 25 to 40 postanthesis, GSAT activity nearly doubled. Soluble protein levels within the tomato during this period declined at a slower rate than in earlier development. Total chlorophyll and chlorophyll a concentration decreased more slowly during this phase of
By day 40, the tomato fruit began ripening and chlorophyll concentration showed another small decrease within the fruit Soluble protein remained low during ripening, but GSAT activity is comparatively high at this stage of development. It is unclear why GSAT activity would be at its third highest level during development when the tomato is breaking color during ripening and is no longer green. This suggests that the increase in GSAT activity and the corresponding increase in ALA that this activity would supply is not being used solely for chlorophyll biosynthesis. One possibility is that ALA is metabolized through the
tetrapyrrole pathway into heme that may need for the climacteric burst of respiration that begins tomato ripening. This climacteric peaks in the pink tomato, just a few days after this peak of GSAT found at day 40. Another possibility is an alternate route of ALA metabolism via a nontetrapyrrole pathway that is also associated with respiration. 14C0
2 was collected when
both [4-14C]ALA and [5-14C]ALA were fed to etiolated barley (Duggan et al., 1982). This
suggests that such an alternate pathway for ALA metabolism exists.
Questions remain concerning the precise mechanism for control of chlorophyll
concentration in tomatoes. A sensible continuation would be a study of another major point of chlorophyll biosynthesis: the activity of Mg chelatase, which inserts Mg2+ into the tetrapyrrole
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Table 1. GSAT activity from roots, stems, and leaves of 4-week-old seedlings, from immature fruit (15 days postanthesis), and from overripe fruit (65 days postanthesis)z.
Enzyme ALA produced
source (nmol/g tissue)
Root 0.60 c
Stem 0.67 c
Leaf 3.84 a
Fruit (15 days) 1.67 b
Fruit (65 days) 0.56 c
Figure Captions
Fig. 1. Effect of pH on GSAT activity. Fruits 15 days postanthesis were used as the enzyme
source, and they were assayed with 150 ~M GSA. Each point is a mean of eight replications
(Fisher's LSD, P = 0.05). The regression equation is y = -0.71 x2
+
9.98x- 32.90 with an R2value of 0.68.
Fig. 2. Effect of GSA concentration on GSAT activity. Fruits 15 days postanthesis were used as the enzyme source, and they were assayed at pH 6.8. Each point is a mean of eight
replications (Fisher's LSD, P = 0.05). The regression equation is y = 0.00003x2
+
0.015x+
0.046 with an R2 value of 0.975.
Fig. 3. Soluble protein concentration (A), total chlorophyll (. ),chlorophyll a (e ), and
chlorophyll b (A.) concentrations (B), and GSAT activity (C) during tomato fruit development.
Each point is a mean of eight replications (Fisher's LSD, P = 0.05). The regression equation for soluble protein is y = 0.0103x2- 1.160x
+
44.174 (R2 = 0.985). The regression equationsfor total, a, and b chlorophylls, respectively, are y = 0.0056x2
- 0.947x
+
39.480 (R2 =0.962), y = 0.0064x2 - 0.884x
+
30.517 (R2 = 0.939), andy= -0.102x+
8.646 (R2 = 0.583).The regression equation for GSAT activity is y = -0.000061x3
+
0.0068x2- 0.254x
+
3.9442
'"d ,..-.,
1.5
Q) Q)
u
::s::s
~ '"d ... 0 ...1
;....bJ)
I
o..-...
,... LSD=
0.21(0.05) ~0
~s
~5
0.5
0
~--~--~----~--~----~--~--~----~--~6.4
6.6
6.8
7
7.2
7.4
7.6
7.8
8
8.2
pH
[image:23.568.54.477.244.527.2]2
1.5
'"d ,--..., (l) (l)
() ;::j
;::j ~ 1
I
LSD = 0.45
'"d ... 0 ...
(0.05)
;... bj) ~~
<O
~
8
0.5<5
0----~--~----~--~--~----~--~--~--~
0
25 50 75 100 125 150 175 200 225 [image:24.563.53.474.241.490.2]GSA concentration (J.!M)
40
0
35
·~
0)
30
..j-1 ;::::$
8
~
25
O.·p
a) OJ)
20
-
.._
:a
~
15
0 '-'
10
Clj5
A
I
LSD=
3.4(0.05)
0
r-~~--~~~~~~--~~~--~~~Fig. 3.
40
35
c
I
LSD=
6.6(0.05)
I
LSD=
0.21(0.05}
0
~~~--~~~--~~--~~~--~~~0 5 1 0 15 20 25 30 35 40 45 50 55 60 65
[image:25.567.47.443.74.691.2]GENERAL CONCLUSIONS
This is the first report of GSAT activity being measured in ripening organs of tomato, and throughout development and ripening of its fruit. Characterization of the enzyme from 15-day fruits yielded an optimum pH of 7 .0, a V max of 2.3 nmol/g of fruit tissue and a ~1 of 119 ~-tM, and these values were similar to those found in other plant species. Activity of GSAT
was measured in tomato organs, and GSAT activity was greatest in young leaves and was <50% that activity in fruits 15 days postanthesis. GSAT activity was low in young roots, stems, and overripe fruits (65 days postanthesis). Throughout development and ripening, GAT activity fluctuated from greatest activity from 1 0-day fruits to decrease to day 25 and then rose to a peak at day 40 before receding during ripening.
The change in GSAT activity at various stages of tomato fruit development does not seem to influence chlorophyll concentration in the fruit after day 25 postanthesis. Total
chlorophyll and chlorophyll a contentration declined during tomato fruit development, from 10 to 60 days postanthesis, with the largest decline coming early in development. Chlorophyll b remained at low, relatively constant concentrations throughout development. Our results suggest that GSAT activity does not direct chlorophyll concentration in the tomato fruit after 25 days past anthesis.
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ACKNOWLEDGMENTS
I want to start out by thanking five tremendous people from St. Louis University, who have all directed me to this position in life. In alphabetical order: Dr. Bob Bolla, the Biology department head and soybean nematologist; Dr. Steven Dina, my Biology advisor and plant ecologist who I could never thank enough but just remember everything that he taught me; Dr. Retha Edens, my lab instructor for Plant Physiology who got me interested in this subject; Mrs. Patricia Kozlowski, the lab tech in Bob Bolla's lab who taught me the wonders of plant nucleotides; and finally, the late Dr. Robert Neidlinger, my Music advisor and theory
professor, who loved music as life and taught me the same.
