Tech. Bull. Fac. Agr. Kagawa Univ., Vo1.42, No 2, 175-180,1990
Seasonal Changes in Photosynthetic Enzyme Activity
in Relation to Malate Content of Sugarcane Leaf
Toshiyuki M
ATSUIThis paper describes the seasonal changes in weight and malate content of two varieties of sugar- cane, 'N:CO 310' (N:CO) and 'Chikusha' in relation to malate dehydrogenase, phosphoenolpyruvate car- boxylase, phosphoenolpyruvate carboxykinase and malic enzyme activity. The stalk weight of 'N:CO' is apt t o be higher than that of 'Chikusha'. Since the difference could be attributed t o photosynthetic and malic acid enzymes, these enzymes were analyzed in the two varieties during the growing period. The highest malic acid content in the basal leaf of 'N:CO' was shown in August, whereas that of 'Chikusha' was in July. The phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK) in the middle and bottom leaves of 'N:CO' showed higher activities than those of 'Chikusha' in August and the malate dehydrogenase (MDH) and malic enzyme (ME) in the middle leaf of 'N:CO' show- ed higher activities than those of 'Chikusha' in August. The difference in weight of the two varieties might depend on these enzyme activities during the growing period.
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t
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~ t : .
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a
9 h%%?3;&71>3j;. 'N:CO' D+%%O MDHt
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a
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= - ; k b i % % ~ + a 6 ) L O 2 4 ; i ~ 6 .Introduction
Two varieties of sugarcane, Chikusha and N:CO 310 have been used a s the raw material for making the traditional commer cia1 sugar, " Wasanbon"
.
'Chikusha' h a s higher amino acid content and grows faster but weighs less than N:CO 310('-~! In general, sugarcanes which belong t o C-4 group of plants a r e known t o be superior to C-3 plants in photosynthetic ability. The difference in weight in the two va-176 T o s h i y u k i MATSUI : Photosynthetic Enzyme Activity in Sugarcane.
rieties is thought to depend on the differences in photosynthetic ability. The seasonal changes in photo- synthesis and TCA-cycle in relation t o malate and fresh weight were inrestigated. Phosphoenolpyruvate carboxylase (PEPC) acts on CO2 entering the stomata. The enzyme is localized in chloroplasts of the mesophyll cells and it produces oxaloacetate. The oxaloacetate is reduced to malate by malate dehy- drogenase (MDH) C-4 plants utilize malate a s a carrier of CO2 NADP-malic enzyme catalyzes the formation of CO2 from malate and it is localized in the bundle sheath chloroplasts. Phosphoenolpyruva- te car boxykinase (PEPCK) in mitocondr ia catalyses the for mation of phosphoenolpyruvate from oxalo- acetate.
Materials and Methods Muter ials
Sugarcane varieties Chikusha and N:CO were harvested from the field of Kagawa University located in Nagao, Kagawa Prefecture. The fertilization and planting operations a r e described in t h e previous paper (4). Samples were collected on the 25th of each month from May to November and the 10th of De-
cember. Three sugarcanes of each variety were harvested a t random every month. Methods
Enzyme extraction: Two g of a costa-removed leaf was cut into fine pieces and ground in a cooled mortar and pestle with lOml of 0.1 M Tris-HC1 buffer (pH7.5) containing 10 mM L-cystein and 2 g of sea sand. The resulting homogenate was filtered through 4 layers of cotton cloth and the filtrate was centri- fuged a t 10,000 x g for 10 min. The supernatant was filled up t o 10 ml with 10 mM of the same buffer (pH 7.5) and the mixture was used a s the source of crude enzyme after passing through Sephadex G-
25. Two ml of the enzyme solution was applied to a Sephadex G-25 fine column (1.5 i d . X 15 cm) which had been equilibrated with 10 mM Tris-HC1 buffer (pH 7.5). The column was eluted with the same buffer a t a flow rate of 12 ml per h. Each 2 ml fraction was collected and was monitored by quantitative ab- sor bance a t 280 nm for protein. The fractions combined from tubes 3 and 4 were used for enzyme assays and investigations of their properties.
Enzyme a s s a y (Malate dehydrogenase): The formation of malate was assayed spectrophotometrically by measuring the decrease in NADH a t 340 nm, pH 7.5 and 30
after
a 3 min preliminary incubation. The assay mixture contained 50 mM potassium phosphate buffer (pH 7.5), 10 mM NADH, 5 mM oxalo- acetate and 2 pl crude enzyme. Distilled water was used in the blank experiment instead of oxaloacetate and the other conditions were the same as described above.(Phosphoenolpyruvatecarboxylase) The formation of oxaloacetate was measured by coupIing with added malate dehydr ogenasec6! The reaction mixture contained 50 mM Tr is-HC1 buffer (pH 7 5), 20 mM NaHC03, 2 mM MgC12, 0.25 mM phosphoenolpyruvate, 0.1 mM NADH, 1 mM mercaptoethanol, 2 units malate dehydrogenase and 0.2 ml of crude enzyme. Distilled water wasused in the blank instead of phos- phoenolpyruvate. PEPC activity was measured by following the r a t e of NADH oxidation, monitored a t 340 nm and 30 "C.
Tech. Bull. Fac. Agr. Kagawa Univ., Vo1.42, No.2 (1990) 177
(Phosphoenolpyruvate carboxykinase) PEPCK activity was measured by coupling with added malate d e h y d r ~ g e n a s e ' ~ ) . The PEPCK assay was carried out in the same way a s PEPC except that 2 mM ADP was added to the reaction mixture. Soluble protein was determined according to the method of LOWRY et al.(8)using bovine serum albumin a s the standard. MDH, and PEPC and PEPCK activities were ex- pressed a s the amount of malate and oxaloacetate, respectively, liberated per min per mg of protein.
(Malic enzyme) Malate incorporation of NaHi4C03 into acid-stable products was determined a t 30 "c"! The reaction mixture contained 100 mM Trfs-HCl buffer (pH 7.5), 25 mM NaHC03, 8 mM MgC12, 20 mM pyr uvate, 2 mM mercaptoethanol, 0.15 mM NADPH, 2 fiCi ~ a ~ ' ~ C 0 3 (20 pCi/100 p1) and crude cnzyme. After 10 min incubation, the reaction was stopped by adding 0.5 ml of 1.5 N HC1 and incubation was continued for 90 min a t 30 "C. Radioactivity of the reaction mixture was determined by a liquid sc- intillation counter. The activity was expressed a s pmol CO2 incorporation per min per mg of protein.
Determination of malic acid by gas chromatography ( G . C.).
Sample preparation and G.C. condition were carried out in a simmilar manner a s in the previous pa- per '3).
Results and Discussion
Properties of malate dehydrogenase (MDH), phosphoenolpyruvate car boxyluse(PEPC), andphospho- enolpyruvate carboxykinase (PEPCK) in sugarcane leaves: The optimum pH for MDH in 'Chikusha' w a s 6.2, 6.4 and 8.0 when measured in glycine, Tris-HC1 and pottasium phosphate buffer, respectively, where- a s t h a t in 'N:CO' was 6.2, 7.0 and 8.0. The optimum temperature for MDH in two varieties was 45 "C in pottasium phosphate buffer. The optium pH for PEPC in 'Chikusha' was 7.0, 7.5 and 8.0 whenmeasured in glycine, Tris-HC1 and pottasium phosphate buffer, respectively, whereas that in 'N:CO' was 8.5, 7.5 and 6.5. The optimum temperature for PEPC in two varieties was 40 "C in pottasium phosphate buffer. The optimum pH for PEPCK in 'Chikusha' was 6.5, 7.5 and 8.0 when measured in glycine, Tris-HC1 and pottasium phosphate buffer, respectively, whereas that in 'N:CO' was 7.0, 7.5and 7.5. The optimum tem- perature for PEPCK in the two varieties was 40 "C. The Km value for MDH, PEPC and PEPCK in the two varieties was 5.0, 12.5 and 4.0 mM, respectively.
Seasonal changes in MDH, PEPCK and M E activities in leaves
Malate dehydrogenase (MDH). The activities of MDH in the leaves taken from three positions in the plant a r e shown in Fig. 1. In 'Chikusha', the activities of MDH increased in the threeleaves until August and then decreased. The MDH activity in the top leaf was about 2.5 times as high a s that in the basal in August and it was higher than that of the rniddIe leaves (significantly different a t 1% level when cal- culated similarly to the previous paper (lo)). The profile of the MDH activity in 'Chikusha, was almost the same a s that in 'N:CO' but there were no significant differences in the MDH activities in the three leaves of 'N:CO' harvested in August. The MDH activity in the leaves of 'N:CO' was about 1.7 times as hihg a s that of 'Chikusha' in August. No d a t a in the basal leaf for 'Chikusha' were obtained because leaves fall off in December.
178 Toshiyuki MATSUI : Photosynthetic Enzyme Activity m Sugarcane.
0 1 ' I I I I
June July Aug. Sep. Oct. Nov Dec. June July Aug. Sep. Oct. Nov. Dec.
Fig.1. Seasonal changes in malate dehydrogenase Fig.2. Seasonal changes in phosphoenolpyruvate activity in sugarcane leaves cv Chikusha and carboxykinase activity in the leaves of two N:CO.O:top part, x:middle part,@:basal part. sugar cane varieties.
Values a r e means with S.E. (n=3). Symbols a s shown in Fig.1.
Phosphoenolpyruvate carboxylase (PEPC) The top and middle leaves of 'Chikusha' showed highest PEPC activity in August and the basal in September, whereas all three leaves in 'N:CO' showed highest activity in August. The PEPC activity in the leaves of 'N:CO' was about 1.7 times that of 'Chikusha' in August (Fig. 2). The phenomenon may be explained a s follows: COa entering the leaf of 'N:CO' was used more effectively than in 'Chikusha'. 'N:CO' showed higher growing rate than 'Chikusha'.
Ph~sphoenolpyruvate carbosykinase (PEPCK) The highest PEPCK activity in 'Chikusha' was observ- ed in the top and middle leaves in August and in the basal in September, whereas that in 'N:CO' was observed in the three leaves in August. The PEPCK activity in the leaves for 'N:CO' was about 1.5 times a s high a s that of 'Chikusha' in August (Fig. 3). The PEPCK activity was apt t o be higher in 'N:CO' than in 'Chikusha' but there were no significant differences in the MDH, PEPC and ME activitiesbetween the two varieties.
Mahc enzyme (ME) The highest ME activity in 'Chikusha' was observed in the middle and basal leav- es in August and the top in September, whereas that in 'N:CO' was observed in the top and basal leaves in September and in the middle of August.
No MDH, PEPC, PEPCK and ME activities were found in the stalk and root of sugarcane.
Seasonal changes in weight and malic acid content of sugarcane leaves Fig.4 and 5 show the seasonal changes in leaf weight and malic acid content of sugarcane leaves, respectively. Leaf weight increased rapidly in July to October in both varieties. In 'Chikusha' it increased m the middle and basal leaves in September and then decreased. In 'N:CO' it reached the highest value in November. The top and middle leaves of 'N:CO' weighed about two times that of 'Chikusha' and the basal leaf about 1.5 times. The stalk weight of 'N:CO9 w a s about two times a s large a s that of 'Chikusha' a t the harvest period('-4! Fig.6 shows the seasonal changes in malic acid content of sugarcane leaf. The highest malic acid content of the top
Tech. Bull. Fac. Agr. Kagawa Univ., Vo1..42, No.2 (1990)
Fig.3.. Seasonal changes in phosphoenolpyruvate June July Aug. Sep. Oct. Nov. Dec..
carboxylase activity in the leaves of two Fig.4. Seasonal changes in malic enzyme activity for two varieties.. in the leaves of two sugarcane varieties. Symbols a s shown in Fig..l. Symbols a s shown in Fig.1.
50
-
'Chikusha' 40 20 2 0 I I I I I bn?!
-
E 30-
20 .2 4 200-
3
100-
n 0June July Aug.. Sep. Oct.. Nov.. Dec.. June July Aug. Sep. Oct. Nov.. Dec.
Fig.5. Seasonal changes in leaf weight of the Fig..G.. Seasonal changes in malic acid content of leaves of two sugar cane varieties. the leaves of two sugarcane varieties. Symbols a s shown in Fig.1.. Symbols a s shown in Fig.1.
leaf of 'Chikusha' was observed in August and in the basal and middle in July, while in 'N:CO' it was ob- served in August in the middle and basal leaves and in July in the top leaf. Although the malic acid con- tent in the two varieties was almost the same a t harvest period, the leaf weight of 'N:CO' was higher than that of 'Chikusha' Therefore, the difference in weight of the two varieties is due to the differences in MDH, PEPC, PEPCK and ME activities. MDH, PEPC and PEPCK activities in the top and middle leave- es of the two varieties peaked in August As temperature and daylength during the developing period incre-
180 Tosiyuki MATSUI : Photosynthetic Enzyme Activity in Sugarcane.
ased, photosynthesis and organic metabolism In the leaves appeared t o Increase. In general, the stalk welght of 'N:CO' 1s hlgher than that of 'Chlkusha' and the growth r a t e of the stalk was faster durlng August and October (Iw4) The PEPC and PEPCK activities in the mlddle and basal leaves of 'N:C09 were hlgher than those of 'Chlkusha' and the MDH and ME actlvltles in the same leaf were also hlgher than those of 'Chlkusha' (slgnlflcant difference a t 5% level) I t may be concluded t h a t thedlfference in weight of the two varletles depends on these enzyme actlvltles
Acknowledgement The author wlshes t o thank emer ltus Prof. M. KURETANI of Kagawa Unlverslty for helpful suggestions in the cultivation of sugarcane, a n d also thanks Mr. H.YONEDA of Glnso Co Ltd for technical assistance.
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