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Plant Physiol. (1998) 117: 851-858 Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat1
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.
Plants play a very important role in our lives and the environment
has a significant role in plant growth and development. In different
parts of the globe, especially between 20° and 30° latitudes north
and south of the equator, the temperature decreases to 4°C to 8°C
in the winter and increases to 40°C to 44°C in the summer.
Therefore, several plant species, including annual crop plants, are
exposed to both chill and heat stress during their lifetime. When
plants are exposed to low- or high-temperature stress, Chl biosynthesis
is affected (van Hasselt and Strikwerda, 1976 There are a few reports that demonstrate that low or high temperature
reduces Chl biosynthesis. Illumination of etiolated seedlings of maize
at low temperature resulted in reduced Chl accumulation, and impairment
of Chl biosynthesis was paralleled by an aberrant development of the
thylakoid membranes (van Hasselt and Strikwerda, 1976 Although the above data are available, there has not been any
systematic and comparative study on the effect of heat and chill stress
on the detailed Chl biosynthetic pathway. Therefore, in the present
study the effects of chill (7°C) and heat (42°C) stress on several
enzymes of the Chl biosynthetic pathway were studied in the
dicotyledonous horticultural plant cucumber and, when required, were
compared with the monocotyledonous crop plant wheat.
Plant Material
Plant Growth Conditions The seeds were treated with 0.1% HgCl2 solution for 5 min and then washed with tap water three times and spread over a single layer of moist germination paper. Cucumber and wheat seedlings were grown in the dark at 25°C for 4 and 6 d, respectively, and were transferred to low (7°C) and high (42°C) temperatures for various lengths of time. One set of seedlings remained at 25°C under identical conditions for a comparison (control).ALA Content Five pairs of cotyledons were preincubated in 60 mM LA in 50 mM phosphate buffer, pH 6.0, for 4 h in the dark at 7°C, 25°C, and 42°C. After the preincubation, cotyledons were exposed to white fluorescent light (30 µmol m 2 s1) for 3 and 6 h while controls were left in the dark. Each time five pairs of
cotyledons were weighed and hand homogenized in a prechilled mortar and
pestle in 5 mL of 1 M sodium acetate buffer (pH 4.6). The
homogenate was centrifuged at 10,000 rpm (12,000g) for 10 min and supernatant was taken for assay. The assay mixture consisted of
0.1 mL of supernatant, 0.4 mL of distilled water, and 25 µL of
acetylacetone. The assay medium was mixed properly and heated in a
boiling water bath for 10 min. Then the extract was cooled at room
temperature, and an equal volume of modified Ehrlich's reagent was
added and vortexed for 2 min. After 10 min of incubation, absorbance of
the extract was measured at 555 nm and ALA content was determined from
the standard curve of ALA (Harel and Klein, 1972 ).
ALAD Two pairs of cotyledons, harvested from etiolated cucumber seedlings grown at different temperature regimes, were weighed and hand homogenized in 5 mL of 0.1 M Tris and 0.01 M mercaptoethanol solution, pH 7.6, in a mortar and pestle at 4°C. Homogenate was centrifuged at 10,000 rpm in a rotor (12,000g, model SM24, Sorvall) in a centrifuge (model RC5C, Sorvall) at 4°C. The supernatant was taken for ALAD assay. The crude enzyme extracts were assayed for ALAD activity according to the method of Shemin (1962) with a slight modification. The enzyme activity was
determined by measuring the amount of PBG formed in 1 mL of the
reaction mixture. The incubation mixture consisted of 60 mM
Tris, 0.2 mM ALA, 1 mM EDTA, 15 mM
MgCl2, 0.5% BSA (w/v), and 0.33 M
Suc, pH 7.5, and the enzyme preparation. After 10 min of preincubation,
the reaction was started by adding the substrate ALA and incubation was
carried out for 1 h at 28°C. The amount of PBG formed was
calculated using the absorption coefficient (6.2 × 104 M1
cm1) (Hukmani and Tripathy, 1994).
PBGD Two pairs of cucumber cotyledons, harvested from etiolated cucumber seedlings grown at different temperature regimes, were weighed and hand homgenized in 5 mL of 0.1 M Tris buffer (pH 7.6) at 4°C. Homogenate was centrifuged at 10,000 rpm (12,000g) for 10 min. Supernatant was taken for PBGD activity assay. The crude enzyme extracts were assayed for PBGD activity. The enzymatic activity was measured by determining the amount of uroporphyrin formed in 1 mL of reaction mixture consisting of 0.1 M Tris-HCl, 2.5 mM EDTA, 15 mM MgCl2, and 0.1% BSA (w/v), pH 7.5. The reaction was carried out for 1 h at 37°C. An aliquot of 0.85 mL of this reaction mixture was taken, to which 0.25 mL of 5 N HCl was added to stop the reaction. The porphyrinogens that were formed were oxidized in porphyrins by adding 0.1 mL of 0.1% benzoquinone to methanol. After 20 min at 4°C samples were centrifuged. A 0.1-mL aliquot was taken, to which 0.9 mL of 1 N HCl was added. Absorbance was measured at 405 nm (absorption coefficient = 5.48 × 105 M1 cm 1)
(Hukmani and Tripathy, 1994).
Preparation of Urogen III, Coprogen III, and Protogen IX Sodium amalgam was prepared by adding 18.31 g of mercury to 0.55 g of freshly cut and heated sodium metal under nitrogen gas (Jacobs and Jacobs, 1982). Reduction of porphyrins was carried out
according to the method of Poulson and Polglosse (1974) with certain
modifications. Porphyrinogens were freshly prepared by reducing
porphyrins, namely uroporphyrin III, coproporphyrin III, or Proto IX.
Porphyrins were dissolved in 0.1 N KOH consisting of 20%
ethanol. Oxygen present in the solution was removed by flushing the
solution with nitrogen gas. To this solution 5 g of 3% sodium
amalgam was added and flushing the solution with the stream of nitrogen
was continued. The reduction was carried out for 2 to 3 min; the
solution becomes colorless within this time period and the reduction
was continued for 2 to 3 min more to ensure complete reduction of the
porphyrin. Immediately, the contents were filtered. The pH of the
porphyrinogen solution was adjusted to 7.5 with dilute acetic acid.
Proto IX Synthesis from Urogen III, Coprogen III, and Protogen IX Dark-grown cucumber seedlings were transferred to different temperature regimes, and intact plastids were isolated from cotyledons over a 50% percoll gradient. The plastids were lysed in lysis buffer consisting of 10 mM Tris-HCl and 2.5 mM Na2EDTA (pH 7.7). Lysed plastids were centrifuged at 5000 rpm for 3 min in a rotor (3000g, model SS-34, Hitachi) in a centrifuge (Hitachi) at 4°C. The supernatant was used for the enzyme assay. The reaction mixture (0.3 mL) consisted of 100 mM Tris (pH 7.5), 15 mM MgCl2, 5 mM DTT, 0.1% BSA (w/v), 0.1 mL of enzyme preparation, and 0.03 mL of urogen, coprogen, or protogen. Incubation was carried out for 1 h at 28°C (Jacobs and Jacobs, 1982). The reaction was terminated by adding 1.5 mL of 90% acetone.
Pigments were quantified by spectrofluorometry (Rebeiz et al., 1975;
Hukmani and Tripathy, 1992).
Mg-Chelatase Etioplasts were isolated at 4°C using a modification of the previous method (Tripathy and Rebeiz, 1986). Five grams of cucumber cotyledons harvested from etiolated cucumber seedlings grown at different temperature regimes was hand homogenized in a prechilled mortar and pestle with 13 to 15 gentle strokes under a safe green light
in 15 mL of chilled isolation buffer consisting of 0.5 M Suc, 20 mM Hepes, 1 mM
MgCl2, 1 mM
Na2EDTA, and 0.2% BSA (w/v) at a
room-temperature pH of 7.7. The homogenate was passed through four
layers of cheesecloth and centrifuged at 1200 rpm (200g) for
2 min. The supernatant was decanted and further centrifuged at 4000 rpm
(2000g) for 7 min. All of the centrifugation was done in an
SS-34 rotor at 4°C using a Hitachi centrifuge. The pelleted plastids
were gently suspended with the help of a paint brush in suspension
buffer containing 0.5 M Suc, 0.2 M Tris-HCl, 20 mM MgCl2, 2.5 mM
Na2EDTA, 20 mM ATP, 20 mM
NAD, and 8 mM Met at a room-temperature pH of 7.7. Incubation of the 0.3-mL reaction mixture consisting of 0.1 mL of
chloroplast suspension, 0.1 mL of suspension buffer, 5 µL of Mg-Proto
in 80% acetone, 5 µL of Proto IX, and 95 µL of distilled water was
carried out at 28°C for 1 h, and 1.7 mL of ice-cold 90%
ammonical acetone was added to stop the reaction. Samples were
centrifuged at 5000g for 2 min to remove the pellet
and an equal amount of hexane was added to the supernatant. The top
hexane layer was removed and the bottom layer was again extracted
with one-third of the volume of hexane. The top hexane layer was
discarded and the bottom HEAR was taken for an estimation of MPE by
spectrofluorometry (Hukmani and Tripathy, 1992).
MPE Cyclase Plastids isolated from control, chill-, and heat-stressed cucumber seedlings were suspended in suspension buffer consisting of 0.5 M Suc, 0.2 M Tris-HCl, 20 mM MgCl2, 2.5 mM EDTA, and 20 mM ATP (pH 7.7). The reaction mixture (0.3 mL) consisted of 0.1 mL of chloroplast suspension, 0.1 mL of suspension buffer, 0.5 mM S-adenosyl Met, 5 µL of Mg-Proto in 80% acetone, and 95 µL of distilled water. The pH of the reaction mixture was adjusted to 7.7 with 1 N NaOH. The incubation was carried out at room temperature (25°C) for 1 h in the dark and 1.7 mL of ice-cold 90% ammonical acetone was added to stop the reaction. The HEAR was prepared from the acetone extract and synthesis of Pchlide was estimated by spectrofluorometry.Net Synthesis of Pchlide Three-day-old etiolated cucumber seedlings grown at 25°C were transferred to 7, 42, and 25°C for 48 h in dark. To empty the endogenous Pchlide pool, seedlings were exposed to cool-white fluorescent light (30 µmol m2
s1) for 10 min at their respective
temperatures, and 10 pairs of cotyledons were excised immediately after
the light treatment, weighed, and homogenized in 90% ammonical
acetone. After light exposure, seedlings were kept in the dark at their
respective temperatures and cotyledons were harvested after 3, 6, and
12 h. On each data point three replicates of 10 pairs of
cotyledons were hand homogenized in 20 mL of ice-cold 90% ammonical
acetone at 4°C, and homogenate was centrifuged at 10,000 rpm
(12,000g) for 10 min. Supernatant was taken for the HEAR
preparation and accumulation of Pchlide was measured
spectrofluorometrically. Dry weight was measured after keeping 100 mg
of cotyledon tissue for 72 h in an oven maintained at 80°C, and
data points were corrected for loss of moisture both at low and high
temperatures. The net synthesis of Pchlide was calculated by deducting
the Pchlide value recorded after 10 min of light exposure from those
recorded at 3-, 6-, and 12-h dark intervals.
POR Etiolated cucumber seedlings (3 d old) grown at 25°C were transferred to 7, 42, and 25°C for 48 h in dark, and 10 pairs of cotyledons were harvested, weighed, and homogenized in 90% ammonical acetone in the dark. Subsequently, seedlings were exposed to cool-white fluorescent light (30 µmol m2
s1) for 10 min and cotyledons were harvested,
weighed, and homogenized in 90% ammonical acetone. Acetone tissue
homogenates were centrifuged at 10,000 rpm in an SS-34 rotor
(12,000g) for 10 min at 4°C, and from supernatant HEAR was
prepared. Pchlide contents of cucumber cotyledons before and after
light exposure were estimated from the HEAR fraction by
spectrofluorometry (Rebeiz et al., 1975; Hukmani and Tripathy, 1992).
Three replicates were taken for each data point. The percent
phototransformation of Pchlide to Chlide in different temperatures was
calculated as: ([(Pchlide content before phototransformation)  (Pchlide content after phototransformation)]/Pchlide content before
phototransformation) × 100.
Enzymatic Activities in Wheat Seedlings Etiolated wheat seedlings (6 d old) grown at 25°C were transferred to 7, 42, and 25°C for 24 h in the dark and enzymatic activities were monitored. Longer exposure (48 h) to high temperature (42°C) almost killed the wheat tissue. All of the protocols followed for wheat seedlings were identical to that of cucumber.Spectrofluorometry Fluorometric estimation of pigments was done using a photon-counting fluorometer (model 8000C, SLM, Rochester, NY). Channel A (sample) and channel C (reference) were adjusted to 20,000 counts s1 using a tetraphenylene butadiene block as a
standard and excited at 348 nm, and fluorescence emission was monitored
at 422 nm. The HEAR samples were excited at 400, 420, and 440 nm and
emission spectra were recorded in a ratio mode from 580 to 700 nm.
Excitation and emission-slit widths were 4 nm. Emission spectra were
corrected for photomultiplier response. Using appropriate equations the concentration of plant tetrapyrroles was quantified and values expressed as nanomoles per milligram of protein of per hour (Rebeiz et
al., 1975; Hukmani and Tripathy, 1992).
Chl and Protein Estimation Chl and protein ware estimated according to the methods of Porra et al. (1989) and Lowry et al. (1951), respectively.
Chemicals All porphyrin intermediates were purchased from Porphyrin Products (Logan, UT). DTT, ALA, ATP, NAD, Benzoquinone, LA, and Hepes were purchased from Sigma. All other chemicals were purchased from Merck (Darmstadt, Germany), Sd Fine Chemicals (Mumbai, India), BDH (Poole, UK), and Qualigens (Mumbai, India).
Chl Content Four-day-old etiolated cucumber seedlings grown at 25°C were transferred to 7°C (chill stressed), 25°C (control), and 42°C (heat stressed) and exposed to cool-white fluorescent light (30 µmol m2 s1) for 6, 12, 24, and 48 h. The maximum amount of Chl synthesis was observed in
control seedlings at 48 h of light exposure. Chl synthesis was
inhibited by 60 and 90% in heat- and chill-stressed seedlings,
respectively. An initial lag period of up to 12 h was observed in
chill-stressed cucumber (Fig. 1A).
Chl Biosynthetic Intermediates To probe if inhibition of Chl biosynthesis in chill- and heat-stressed seedlings was due to reduced synthesis of tetrapyrrolic intermediates, etiolated cucumber seedlings grown at 25°C were kept for 48 h in chill- or heat-stressed conditions in the dark, and accumulation of Proto IX, MPE, and Pchlide was measured at low- or high-temperature regimes. Only the net synthesis of Pchlide could be monitored, since the amounts of MPE and Proto IX accumulated in cucumber cotyledons were negligible. In the control, the maximum amount of Pchlide was synthesized during the first 3 h (Fig. 1B). Net synthesis of Pchlide was near the maximum after 12 h of dark incubation. On the contrary, chill-stressed seedlings did not synthesize any Pchlide during the first 3 h and accumulated only small amounts of Pchlide after 12 h of dark incubation. Compared with control seedlings, Pchlide synthesis was inhibited by 90 and 70% in chill- and heat-stressed seedlings, respectively.ALA Content To study the mechanism of inhibition of Chl and Pchlide biosynthesis at different temperatures, the biosynthesis of ALA, the precursor of Chl, was monitored in chill- and heat-stressed conditions. ALA synthesis in the presence of LA was almost linear up to 6 h of illumination in control and heat-stressed seedlings. For the first 3 h ALA synthesis was completely inhibited in chill-stressed cucumber seedlings. As compared with the controls, the net synthesis of ALA was severely reduced by 78 and 70% in chill- and heat-stressed seedlings, respectively (Fig. 1C).ALAD The ALAD that synthesizes PBG from two molecules of ALA was determined in etiolated cucumber seedlings kept at 7, 25, and 42°C for 48 h in the dark. As compared with the control, the enzyme activity decreased by 24 and 45% in chill- and heat-stressed seedlings, respectively (Fig. 2A).
PBGD The next step in the Chl biosynthetic pathway is the conversion of PBG to urogen, which is catalyzed by PBGD. The enzyme activity was estimated by measuring the amount of porphyrin synthesis from PBG in cucumber seedlings kept at 7, 25, and 42°C for the last 48 h in the dark. Compared with the control, the enzyme activity was reduced by 13 and 28% in chill- and heat-stressed seedlings, respectively, suggesting that the PBGD activity was affected more in heat than in chill stress (Fig. 2B).Proto-IX Synthesis from Urogen, Coprogen, and Protogen The subsequent steps in the Proto IX biosynthetic pathway are the conversion of urogen III to coprogen III mediated by UDC, the synthesis of protogen IX from coprogen III by coprogen oxidase, and finally the conversion of protogen to Proto IX by Protox. As compared with the controls, in chill-stressed seedlings Proto IX synthesis from urogen was severely reduced by 65%, whereas in heat-stressed seedlings the activity increased by 155% (Fig. 3A). Proto-IX synthesis from coprogen III was also reduced by 34% in chill-stressed seedlings. However, in heat-stressed seedlings, Proto IX synthesis from coprogen III was not affected (Fig. 3B). Protox activity was reduced by 60% in chill-stressed seedlings, whereas there was no significant change in enzyme activity in heat-stressed seedlings (Fig. 3C).
Mg-Chelatase Mg-chelatase is the first enzyme in the Mg branch of the Chl biosynthetic pathway that inserts Mg into Proto IX to form Mg-Proto IX (Gorchein, 1972; Pardo et al., 1980; Jensen et al., 1996). In this
experiment Proto IX was used as a substrate and Mg-chelatase activity
was measured as the amount of Mg-Proto IX and its monoester synthesized
in the plastids isolated from cucumber seedlings. As compared with the
controls, the enzyme activity decreased by 60% in chill-stressed and
by 65% in heat-stressed seedlings (Fig. 4A).
S-Adenosyl Met Methyl Transferase Plus MPE-Cyclase To analyze the effect of temperature stress on Mg-Proto:S-adenosyl Met methyl transferase and MPE cyclase (Hinchigeri et al., 1981; Bollivar and Beale, 1996), plastids isolated
from cucumber seedlings grown at different temperatures were incubated
with Mg-Proto IX and S-adenosyl L-Met and
synthesis of Pchlide was monitored. Pchlide synthesis was reduced by 60 and 36% in chill- and heat-stressed seedlings, respectively (Fig. 4B).
POR POR mediates the light-catalyzed reduction of Pchlide to Chlide, which is a key regulatory step in the biosynthesis of Chl in oxygenic photosynthetic organisms (Griffiths, 1978; von Wettstein et al., 1995).
Four-day-old-etiolated cucumber seedlings grown at 25°C were
transferred to 7°C or 42°C, and were kept in the dark for 48 h. They were illuminated for 10 min in light to phototransform accumulated Pchlide to Chlide. The phototransformation of Pchlide to Chlide was 75% in control and chill-stressed seedlings and 95% in
heat-stressed seedlings (Fig. 4C).
Effect of Temperature Stress on Certain Enzymes Involved in the Chl Biosynthetic Pathway in Wheat To compare the effects of chill and heat stress on Chl biosynthesis in dicot and monocot seedlings, wheat seedlings were used and the activities of PBGD and POR were monitored.PBG Deaminase Activity in Wheat Six-day-old-etiolated wheat seedlings grown at 25°C were transferred to 7, 25, and 42°C for 24 h in the dark. PBGD activity in chill- and heat-stressed seedlings declined by 12 and 41%, respectively (data not shown). This demonstrated that both wheat and cucumber seedlings showed a similar pattern of reduction of PBGD activity in response to temperature stress.Pchlide Synthesis in Wheat Seedlings Grown at Different Temperatures The net synthesis of Pchlide in control wheat seedlings increased after 4 to 12 h of dark incubation. However, there was a lag in Pchlide synthesis for 4 h in chill- and heat-stressed seedlings after which the rate of Pchlide synthesis increased. As compared with control seedlings, in chill-stressed seedlings Pchlide accumulation was reduced by 80%, whereas in heat-stressed seedlings reduction was 65% (Fig. 5). Chill-stressed wheat seedlings had a higher accumulation of Pchlide after 12 h of dark incubation than that of cucumber. This suggested that Chl biosynthesis in wheat as compared with cucumber was relatively more resistant to chill stress.
POR in Wheat The percent phototransformation of Pchlide to Chlide was taken as an index of POR activity in wheat seedlings grown at different temperatures in the dark. Six-day-old etiolated wheat seedlings grown at 25°C were transferred to 7°C, 25°C, or 42°C and were kept in the dark for 24 h. They were illuminated for 10 min in light to phototransform accumulated Pchlide to Chlide. The phototransformation of Pchlide to Chlide was 70% in chill-stressed and control seedlings and 90% in heat-stressed wheat seedlings (Fig. 5, inset). POR activity had a similar response in heat-stressed cucumber seedlings.
Illumination of cucumber seedlings in chill- and heat-stress conditions resulted in inhibition of Chl biosynthesis by 90 and 60%, respectively (Fig. 1A), demonstrating that inhibition of Chl biosynthesis is higher in chill-stress than in heat-stress conditions. Reduced synthesis of ALA, the committed precursor of Chl, in chill- and heat-stressed seedlings (Fig. 1C) demonstrates that inhibition of Chl biosynthesis is partly due to impairment of ALA biosynthesis. ALA biosynthesis in cucumber was inhibited to a similar extent both in chill (78%)- and heat (70%)-stress conditions (Fig. 1C). However, as stated above, the inhibition of Chl biosynthesis under identical conditions in chill- and heat-stressed seedlings was 90 and 60%, respectively. To account for the discrepancy between the inhibition of ALA and Chl biosynthesis in chill- and heat-stress conditions, Pchlide synthesis was monitored. Compared with control seedlings, Pchlide synthesis was inhibited by 90 and 70% in chill- and heat-stressed seedlings, respectively (Fig. 1B), demonstrating that inhibition of Chl biosynthesis was mostly due to impairment of ALA and Pchlide biosynthesis.
* Corresponding author; e-mail bct{at}jnuniv.ernet.in; fax 91-11-6165886. Received January 7, 1998;
accepted March 11, 1998.
Abbreviations: ALA, 5-aminolevulinic acid. ALAD, 5-aminolevulinic acid dehydratase. Chl, chlorophyll. Chlide, chlorophyllide. coprogen, coproporphyrinogen. HEAR, hexane extracted acetone residue solvent mixture. LA, levulinic acid. MPE, Mg-protoporphyrin monoester. PBG, porphobilinogen. PBGD, porphobilinogen deaminase. Pchlide, protochlorophyllide. protogen, protoporphyrinogen. POR, Pchlide oxidoreductase. Proto IX, protoporphyrin IX. Protox, protogen oxidase. UDC, urogen decarboxylase. urogen, uroporphyrinogen.
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  D. P. Dixon, A. Lapthorn, P. Madesis, E. A. Mudd, A. Day, and R. Edwards Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases J. Biol. Chem., July 18, 2008; 283(29): 20268 - 20276. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
), chill-stressed (7°C, 
), and
heat-stressed (42°C, 
) cucumber seedlings. Experimental details
are as in ``
