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VariationsinthePhotosynthesisRateandActivity
ofPhotosyntheticEnzymesinMaizeLeafTissueof
DifferentAges
ArticleinPlantandCellPhysiology·October1984
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Plant & Cell Physiol. 25(7): 1297-1301 (1984)
JSPP © 1984
Short Communication
Variations in the Photosynthesis Rate and Activity of
Photosynthetic Enzymes in Maize Leaf Tissue
of Different Ages
Hideaki Usuda
Laboratory of Chemistry, Faculty of Medicine, Teikyo University, Ohtsuka, Hachioji, Tokyo, Japan
Highly good correlations for the extractable activities of ribulose-l,5-bisphosphate
carboxylase, pyruvate,Pj dikinase, phosphoenolpyruvate carboxylase, NADP-malate
dehydrogenase and NADP-malic enzyme and the rate of photosynthesis were found in
maize leaves of various ages. The activities of the first two enzymes were similar to, or
slightly higher than, the photosynthesis rate, whereas the activities of the other enzymes
were 2 to 6 times higher than the photosynthesis rate. These results suggest that
pyruvate,Pj dikinase and ribulose-l,5-bisphosphate carboxylase may be rate-limiting
factors in maize.
Key words: C4 photosynthesis — Maize leaf — Pyruvate,Pj dikinase — RuBP
carboxylase — Zea mays.
It is important to examine the rate-limiting step(s) in C4 photosynthesis in order to obtain a
clear understanding of the photosynthetic process in C4 plants. The co-operative functions of
the mespohyll and bundle sheath cells in these plants, however, make it difficult to analyze the
limiting factors in C4 photosynthesis. A determination of the correlation between the photosynthesis rate and the activities of several key enzymes in C4 photosynthesis is one way to investigate
rate-limiting processes. RuBP carboxylase activity is highly correlated with the photosynthesis
rate in C3 plants when CO2 is limited (e.g. Bjorkman 1966, Makino et al. 1983, Uchida et al.
1980, Wareing et al. 1968).
In C4 plants, RuBP carboxylase also has an important role in the net CO2 assimilation; but,
there have been conflicting reports on the relationship between the RuBP carboxylase activity or
content and the photosynthesis rate, or the biomass productivity, in C4 plants. Sugiyama and
Hirayama (1983) reported that there is no good correlation between the biomass and the RuBP
carboxylase content on the basis of fresh weight. They also found that there is a highly negative,
correlation between the biomass and this protein in maize plants grown with different concentrations of nitrate when the latter was expressed as a unit-soluble protein. But, Avdeeva and
Andreeva (1973) and Wong (1979) reported a good correlation between RuBP carboxylase
activity and the rate of photosynthesis in maize leaves grown with different N supplies the activities of RuBP carboxylase being similar to the photosynthesis rates.
The object of the study reported here was to determine whether correlations exist between
the extractable activities of RuBP carboxylase, PPDK, PEP carboxylase, NADP-MDH and
Abbreviations: BSA, bovine serum albumin; DTE, dithioerythritol; MDH, malate dehydrogenase; ME, malic
enzyme; PEP, phosphoenolpyruvate; PPDK, pyruvate,P| dikinase; RuBP, ribulose-1,5-bisphosphate.
1297
1298
H. Usuda
NADP-ME and the photosynthesis rate in maize leaves of various ages grown with a sufficient N
supply. For each treatment the determination of both the enzyme activities and the photosynthesis rates were carried out on the same leaf tissues to obtain a high degree of accuracy.
Seeds of Zea mays L. (variety Chuseishu-B) were obtained from Nihonsogyo, Tokyo, Japan.
Plants were grown in an artificially illuminated growth chamber with a 14-h light/10-h dark
period with day/night temperatures of 26j : 2 0 C/19 : t2°C. Each plant was cultured in a 1-liter
pot containing a mixture of vermiculite and soil and 2 g of chemical fertilizer (Kumiai-yukiirikasei-toku No 888, N : P : K = 1 :1 : 1, Seibu-kagakukogyo, Tokyo, Japan). Plants were
watered with a nutrient solution (Edwards and Walker 1983) 5 times a week. The light intensity
was approximately 500 ^E-m~2-s-1.
Leaf gas exchange was measured with a single attached leaf enclosed in an acrylic plastic leaf
chamber (6x 10x0.4 cm). The light sources were four, 400-W metal-halide lamps (DR 400,
Toshiba, Tokyo, Japan). The light beam was passed through a 10-cm layer of water to eliminate
heat. Varying levels of irradiance were obtained by changing the number of cheesecloth screens
between the leaf chamber and the light sources. Light intensities were measured with a quantum
sensor (model 185B, Li Cor Instruments, NE, U.S.A.).
The leaf temperature was kept at 30:t0.2oC by circulating temperature-controlled water in
the water jackets of the leaf chamber. CCVcontaining air (CO2 concentration = 330 /il-liter"1)
was stored in a large balloon and used for the experiments. The water vapor of the inlet air
stream was 20.0 g-m~3. The air stream was passed into the leaf chamber at a constant rate of
2.0 liter-min"1. The CO2 uptake in the chamber was measured with an IR gas analyzer (ZAU,
Fuji-denki, Tokyo, Japan).
After the gas exchange measurements, about half of each leaf that had been used for the gas
exchange measurement was frozen in liquid nitrogen under light of 1,500/tE-m"2^"1. The
frozen leaves (ca. 3-6 cm2) were immediately and rapidly homogenized in a chilled mortar with
5 mg of insoluble polyvinylpolypyrrolidone and 0.2 g of acid-washed sand plus 0.5 ml of ice-cold
medium containing 50 mil Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2.5 mM pyruvate and
0.5% (w/v) BSA. Pyruvate was added to the extraction medium to prevent cold inactivation of
PPDK (Shirahashi et al. 1978). After homogenization, 2.5 ml of the same medium was added,
and the homogenates filtered thrpugh miracloth. A sample of the filtrate was used for the Chi
determination. The homogenate was centrifuged at 10,000 Xg for 1 min, and its supernatant
was used as the crude enzyme extract.
PEP carboxylase, NADP-MDH, and PPDK were assayed as described previously (Usuda
et al. 1984). RuBP carboxylase was assayed spectrophotometrically after activation with CO2
and Mg2+. The assay mixture (1 ml) of RuBP carboxylase contained 50 mM HEPES-KOH,
pH 7.8, 10 mM KC1, 1 mM EDTA, 30 mM MgCl2, 1 mM DTE, 5 mM ATP, 0.2 mM NADH, 10 mM
NaHCC>3, 5 mM phosphocreatine, 3 units of phosphoglycerate kinase, 3 units of NAD-glyceraldehyde-3-phosphate dehydrogenase, 1 unit of phosphocreatine kinase and the leaf extract. The
reaction was initiated by adding RuBP at a final concentration of 0.5 mM. The assay mixture
(1 ml) of NADP-ME contained 50 mM HEPES-KOH, pH 8.0, 5 mM EDTA, 5 mM DTE, 0.5 mM
NADP, 5 mM malate and the leaf extract. The reaction was initiated by adding MgCl2 at a final
concentration of 22.5 mu. All enzymes were assayed at 30°C.
About half of each leaf that had been used in the gas exchange measurements was used in the
determinations of the Chi and the soluble protein contents that were based on leaf area. The leaf
was homogenized in 50 mM Tris-HCl, pH 7.5. The Chi concentration was determined according
to Arnon (1949). Soluble protein was determined by the method of Bradford (1976) using the
Bio-Rad protein assay reagent (Bio-Rad, California, U.S.A.).
Light response curves of the rate of CO2 assimilation in maize leaves of different ages are
shown in Fig. 1. At 150 /jE-m-^s"1 and below, the rates of CO2 assimilation were similar for
Photosynthesis and enzyme activities in maize
1299
leaves of different ages, b u t the maximum rates of CO2 assimilation ranged from 16.5
CO2-m~ 2 -s- 1 to 38.6 /imol CO2 > m~ 2 -s- 1 depending on leafage (see also Fig. 2). T h e respective
light intensities required for half m a x i m u m photosynthesis for young a n d recently m a t u r e d leaves,
- 22-s
.0-1
middle aged leaves and old leaves were 360, 250 and 150 /xE-m~
The correlations of several enzyme activities with the photosynthesis rate were investigated
in maize leaves of different ages that had a range of maximum photosynthesis activity. The
0
1000
500
Light Intensity
1
-V
Fig. 1
1500
20
30
CO2 Assimilation
40
10
20
30
CO2 Assimilation
2
( l r i
'
40
Fig. 2
Fig. 1 Light response curves of the rate of photosynthetic CO2 assimilation in maize leaves of different ages.
The
fourth leaf of an 18-day-old plant (O), 2nd leaf of a 19-day-old plant (O), 3rd leaf of a 33-day-old plant (A) and
2nd leaf of a 36-day-old plant (•) were used.
Fig. 2 Correlations of enzyme activities, soluble protein and Chi contents with the photosynthesis rate in maize
leaves of different ages.
Photosynthetic CO2 assimilation was measured under 1,500 ^Em~ 2 s - 1 of light. Dotted
lines represent the enzyme activities equal to the photosynthesis rate. The second leaves of 19- (O), 21- (©), 26(<D) and 36-day-old (•) plants, the 3rd leaves of 25- (A), and 33-day-old (A) plants, the 4th leaves of 18- (O),
20- (<3>), and 25-day-old (•) plants and the 6th leaves of 24- (•) and 32-day-old (•) plants were used.
1300
H. Usuda
values of the extractable activities of PPDK were similar to those of the maximum photosynthesis
rates and those of RuBP carboxylase were close to, or slightly higher than, the values of the maximum photosynthesis rates (Fig. 2). Activities of NADP-MDH, PEP carboxylase, and NADPME were 2 to 6 times higher than the maximum photosynthesis rates. These results indicate that
there are high correlations for these enzyme activities and the maximum photosynthesis rate.
A high correlation between the soluble protein content and the maximum photosynthesis rate also
was found, but there was no high correlation between the Chi content and the maximum photosynthesis rate.
The similarities of the extractable activities of RuBP carboxylase and PPDK to the maximum
photosynthesis rates and the high correlations for these two enzyme activities and the maximum
photosynthesis rate in maize leaves of different ages suggest that both enzyme activities may limit
the maximum photosynthesis rate in maize leaves. RuBP carboxylase was assayed after activation with CO2 and Mg2+. The activities of RuBP carboxylase without activation after extraction
were similar to those of the fully activated RuBP carboxylase when C4 leaves were illuminated
(Usuda, Ku and Edwards, unpublished data). Thus there is no possibility of an over-estimation
of RuBP carboxylase activities in situ. There is, however, the possibility of an under-estimation
of these two enzyme activities in the crude extract system.
The specific activities of these two enzymes therefore were calculated from the values of the
enzyme activities and the amount of soluble protein obtained, on the assumption that RuBP
carboxylase and PPDK, respectively, made up 35% and 6% of the total soluble protein. These
values were taken from the recent findings of Sugiyama et al. (1984). The respective specific
activities of RuBP carboxylase and PPDK were 2.3-4.1 U-mg-1 protein and 13.7-23.7 U-mg-1
protein. These values are very close to, or even higher than, those previously reported for maize
leaves (e.g. Reger et al. (1983) reported 2.36 U-mg"1 protein of RuBP carboxylase and Ashton
et al. (1984) reported 10 U-mg-1 protein of PPDK).
Sugiyama et al. (1984) also have reported a very good correlation between the extractable
PPDK activity and the PPDK protein content in crude extracts from maize leaves. This finding
and the specific activities of the two enzymes calculated in my study tend to exclude the possibility
of an under-estimation of the activities of the two enzymes. The presence of an inactive form
of RuBP carboxylase as a major storage protein, however, has been pointed out (see Huffaker
and Peterson 1974). The possibility of an under-estimation of the specific activity of RuBP
carboxylase due to this inactive form remains. Even so, my present data support the good correlation reported previously between the extractable RuBP carboxylase activity and the photosynthesis rate in maize leaves (Avdeeva and Andreeva 1973, Wong 1979).
RuBP carboxylase has a strong correlation with the photosynthesis rate in C3 plants (see the
introduction). Considering that in C4 photosynthesis, the net CO2 assimilated is ultimately
catalyzed by RuBP carboxylase, RuBP carboxylase activity could be one of the rate-limiting
factors in C4 photosynthesis. The lack of a good correlation between the biomass and RuBP
carboxylase content in maize plants grown with different concentrations of N (Sugiyama and
Hirayama 1983) could be due to the following: Biomass is the integration of leaves of various ages
which differ in photosynthetic ability (Fig. 2); e.g. the stem and non-photosynthetic tissues such
as the vascular systems. There was no significant correlation between the dry weight of the
ground-up tissues with the maximum photosynthesis rate in the largest expanded leaf of maize
grown under various light intensities with a sufficient N supply (Usadu, Ku and Edwards,
unpublished data).
The activities of RuBP carboxylase and PPDK obtained in the present study under optimum
assay conditions were just enough to sustain the maximum rate of photosynthesis. This is an
indication that they have an important function in rate-limiting processes in maize photosynthesis.
Photosynthesis and enzyme activities in maize
1301
Questions remain about the actual substrate concentrations for these and other enzymes during
photosynthesis in situ.
I thank T. Sugiyama for his invaluable discussions prior to publication on the contents and specific activities of
RuBP carboxylase and PPDK and G. E. Edwards for his critical review of the manuscript.
This research was partially supported by a grant (5854035) from the Japanese Ministry of Education, Science
and Culture.
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(Received May 12, 1984; Accepted July 5, 1984)