Planta 142,269
Planta
274(1978)
9 by Springer-Verlag 1978
Photosynthetic Carbon Metabolism during Leaf Ontogeny in Zea mays L. : Enzyme Studies Larry E. Williams* and Robert A. Kennedy Department of Botany, University of Iowa, Iowa City, IA 52242, USA
Abstract. The activities of several enzymes, including ribulose- 1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z . mays. Mature leaf tissue had a R u D P carboxylase activity of 296.7 ~tmol CO2 g-1 fresh weight h 1 and a PEP-carboxylase activity of 660.6 gmol CO2 g ~ fresh weight h - t. In young corn leaves the activity of the two enzymes was I1 and 29%, respectively, of the mature leaves. In senescent leaf tissue, R u D P carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of N A D P malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and N A D malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and R u D P carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C4 acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total zgC remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in a2CO2. The activity of R u D P carboxylase during leaf ontogeny in Z. m a y s parallels the development of the activity of this enzyme in C3 plants. Key words: Carboxylases Photosynthesis (C4) - Zea.
Leaf development -
Introduction C4 plants are generally considered to be more efficient photosynthetically than C3 plants (Zelitch, 1973) primarily because of their lack of detectable photorespiration, low CO2 compensation point (Downton, 1975) * Present address: Dept. of Agronomy&Range Science Univ. of California, Davis, CA 95616, USA Abbreviations: RuDP = ribulose-l,5-diphosphate; PEP = phosphoenol pyruvate; PGA = 3-phosphoglycerate
and lack of an enhancement of photosynthesis under low oxygen tensions. During leaf ontogeny the efficiency of C4 photosynthesis appears to decline (Ludlow and Wilson, 1971; Nimbalkar and Joshi, 1974; Kennedy, 1976; Williams and Kennedy, 1977). In the C4 dicotyledon, Portulaca oleracea, PEP carboxylase activity decreased more in senescent leaves than did R u D P carboxylase (Kennedy, 1976), while in senescent corn leaves a CO2 compensation point of 24 ~tl/1 was found, in contrast to values of 0 10 tal/1 for most C4 plants (Williams and Kennedy, 1977). Both Z e a m a y s and P. oleracea were found to have an increased evolution of t4CO2 in the light using the 14C assay of photorespiration (Kennedy, 1976; Williams and Kennedy, 1977). In this paper, we report the results of studies on changes during leaf development in the activities of several enzymes involved in the C4 pathway of photosynthesis in Z. mays, as well as the relative turnover rate of photosynthetic products in mature and senescent corn leaves.
Material and Methods Plant Material Zea mays L. was grown, and the same ontogenetic series employed
as previously described (Williams and Kennedy, I977). Mature leaf tissue was taken from the 4th and 5th leaves from the base of the plant, midwayalong the leaf. Young leaf tissue was collected at the base of leaf blades 8 and 9, whichwere not yet fully expanded. Senescent leaf tissue was collected when it contained 10 20% of the chlorophyllcontent of mature leaf material. Additionally, tissue was used from whole leaf blades of corn seedlings 7 and 14 d after emergence. Soybean (Glycine max L.) was grown in soil in a naturally illuminated greenhouse under the same conditions as described before (Williams and Kennedy, 1977) and the youngest mature trifoliate leaf was used for experimentation. Enzyme Preparation and Assays Preparation of Crude Enzyme Extracts. Leaf tissue was cut into small pieces (1 mm2) and ground with sand in a chilled mortar.
0032-0935/78/0142/0269/$01.20
270
L.E. Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Zea
The grinding medium for PEP and RuDP carboxylase contained 195 mM phosphate buffer, pH 7.0; 10 mM MgClz; 20 mM 2-mercaptoethanol; 0.2 mM ethylenediamine tetraacetic acid (EDTA); and 16 g/1 polyvinylpyrrolidone (PVP) 10. The grinding medium for aminotransferase contained 150 mM phosphate buffer, pFI 7.3; 1.0 mM MgC12; 1.0 mM EDTA; 5.0 mM dithiothreitol and 20 g/ 1 PVP 10. Tissue for NADP-malic enzyme, NAD-malic enzyme and NAD-malate dehydrogenase determinations was ground in 100 mM tris(hydroxymethyl)aminomethane hydrochloride (TrisHCL), pH 8.3; 2.0 mM MgC1; 10 mM mercaptoethanoi; and 1.0 mM EDTA. One gram of leaf tissue was ground in 1.5 ml grinding medium for preparing PEP and RuDP carboxylase extracts, while 1 g leaf tissue per 3 ml of grinding medium was used in preparing the other extracts. The homogenate was filtered through 4 layers of cheesecloth, centrifuged for 20 min at 3,000 • g and 5~ C, and the supernatant used for enzyme assays. Chlorophyll and protein determinations were conducted according to the methods of Arnon (1949) and Lowry et al. (1951), respectively.
PEP Carboxylase (EC 4.1.1.31). The reaction mixture contained (in a final volume of 175 ~tl): 25 mM N-tris(hydroxymethyl) methyl glycine (Tricine), pH 8.0; 10 mM MgC12; 5 mM 2-mercaptoethanol; 15 mM PEP; 15 mM sodium glutamate; and 50 mM NaH14CO3 (1.0 gCi). The assays were initiated by the addition of 10-25 gI of leaf extract and run for 2-3 min. Assays were terminated by the addition of 100 gl 25% (v/v) acetic acid. Aliquots were sampled, dried and counted in an LS-100 C iiquid scintillation counter (Beckman Instruments, Fullerton, Cal., USA)
NAD Malate Dehydrogenase (EC 1.1.1.37). The assay medium contained 100 mM Tricine, pH 7.5; 0.2 mM EDTA; 1.0 mM oxaloacetate; 0.2 mM NADH and 5 15 gl enzyme extract in a total volume of 3 ml. Reactions were initated by addition of the enzyme extract and activities determined spectrophotometrically by the oxidation o f N A D H (A A at 340 nm) for 2 3 rain. Controls were run without NADH. Assays for PEP carboxylase and RuDP carboxylase were carried out at 29_+2 ~ C, with the other enzyme assays conducted at 27_+ 1~ C. All enzyme assays were linear with time and enzyme concentration. Reagents listed in the Methods section were obtrined from Sigma Chemical Co., St. Louis, Mo., USA. Pulse-chase Experiments Pulse-chase radiotracer studies were conducted using a 10-s pulse of 14CO2 as described in Williams and Kennedy, (1977), followed by a chase of up to 2 min in air. Separation and identification of labelled compounds was conducted using two-dimensional thinlayer electrophoresis and chromatography (Kennedy and Laetsch, 1973). Quantification of 14C was by liquid scintillation spectroscopy.
Results E n z y m e Activities
RuDP Carboxylase (EC 4.1.1.39). The reaction mixture contained (in a volume of 100 gl): 25 mM Tricine, pH 8.0; 10 mM MgClz; 5 mM 2-mercaptoethanol; 0.05 mM NADPH; 1.0 mM fructose1,6-diphosphate; 50 mM NaHI*CO3 (0.5 p,Ci); and 5.5 mM RuDP. The enzyme extract (10-25 tsl) was preincubated for 5 rain with all ingredients except RuDP The assay was initiated by the addition of RuDP and run 3 4 min. Termination and quantification of the assay were as for PEP carboxylase (above).
Aspartate Aminotransferase (EC 2.6.1.1). 25 mM Tricine, pH 8.0; 2.5 mM dithiothreitol; 1.25 mM e-ketoglutarate; 4.2 mM aspartate; 0.03 mM pyridoxal phosphate; 0.1 mM NADH; 2.0 mM EDTA; 1.5 units of malate dehydrogenase and 10-25 ~tl enzyme extract were contained in a total volume of 2.4 ml. The reaction was started after a 10-min preincubation by the addition of c~-ketoglutarate. Activities were determined spectrophotometrically by the oxidation of NADH at 340 nm for 3 min.
Alanine Aminotransferase (EC 2.6.1.2). The reaction mixture contained 25 mM Tricine, pH 7.25; 2.5 mM dithiothreitol; 2.5 mM c~ketoglutarate; 4.0mM alanine; 0.03 mM pyridoxal phosphate; 0.1 mM NADH; 2.0 mM EDTA; 1.5 units of lactate dehydrogenase and 10 25 gl enzyme extract in a total volume of 2.4 ml. Reactions were started and run as for aspartate aminotransferase. NADP-Malic Enzyme (EC 1.1.1.40). The assay mixture included 25 mM Tris-HC1, pH 8.3; 0.5 mM EDTA; 5.0mM MgC12; 2.5 mM malate, pH 8.3; 1.5 mM NADP and 10-20 gl enzyme extract in a total volume of 3 mi. The reaction was started by the addition of MgC12 or MnC12, and monitored spectrophotometrically at 340 nm for 3 min.
Carboxylases. T h e a c t i v i t i e s o f P E P c a r b o x y l a s e a n d RuDP carboxylase for the leaf tissue of various ages o f Z . mays, a l o n g w i t h t h e i r a c t i v i t i e s f o r s o y b e a n , a C3 p l a n t , a r e g i v e n in T a b l e 1. T h e r a t i o o f R u D P / P E P c a r b o x y l a s e in m a t u r e l e a v e s is 0.45, i.e., t h e a c t i v i t y o f P E P c a r b o x y l a s e is t w i c e as g r e a t as t h a t of RuDP carboxylase. In contrast, the RuDP/PEP r a t i o i n s o y b e a n l e a v e s is 10.82. I n Z . m a y s l e a v e s o f all o t h e r a g e s s t u d i e d t h e a b s o l u t e a m o u n t s o f both carboxylases were lower than those found in m a t u r e l e a f tissue. C o m p a r e d t o t h e m a t u r e l e a f tissue, y o u n g l e a f t i s s u e o f c o r n h a d 2 4 % o f t h e P E P carboxylase activity and 11% of the RuDP-carboxylase a c t i v i t y . T h e P E P - c a r b o x y l a s e a c t i v i t y i n s e n e s c e n t c o r n l e a f t i s s u e is 9 . 2 % o f t h e m a t u r e t i s s u e while the RuDP-carboxylase a c t i v i t y is o n l y 4 . 8 % . T h e R u D P / P E P c a r b o x y l a s e r a t i o (0.23) i n s e n e s c e n t Z. m a y s l e a v e s is a p p r o x i m a t e l y t h e s a m e as i n y o u n g leaves, but only about one-half that in mature ones. The activities of the two carboxylases of leaf b l a d e s o n 1- a n d 2 - w e e k - o l d c o r n p l a n t s w e r e a l s o d e t e r m i n e d ( T a b l e 1). A t e i t h e r age, t h e a c t i v i t i e s o f b o t h P E P c a r b o x y l a s e a n d R u D P c a r b o x y l a s e a r e less t h a n t h o s e f o u n d in m a t u r e l e a f tissue, b u t c o n s i d e r a b l y h i g h e r t h a n t h o s e f o u n d in y o u n g ( b a s a l ) t i s s u e o f t h e b l a d e o f a n o l d e r , m o r e e x p a n d e d leaf.
NAD-Malic Enzyme (EC 1.1.1.38). The reaction medium contained in a total volume of 3 ml: 25 mM Tris-HC1, pH 8.3; 0.5 mM EDTA; 5.0 mM MnC12; 2.5 mM malate pH 8.3; 2.0 mM NAD; 75.0 gM coenzyme A and 10-20 gl enzyme extract. The assay was carried out as described above.
Other Enzymes. T a b l e 2 s h o w s t h e a c t i v i t i e s o f t h e d e c a r b o x y l a t i n g e n z y m e in Z. m a y s d u r i n g l e a f d e v e l opment. Young and senescent tissue contained higher
L E . Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Z e a
271
14C02 I~C02 SO
Table 1, PEP carboxylase and RuDP carboxylase activities in several
stages of leaf development in Z e a m a y s
MATURE LEAF Age of leaf tissue
Young Mature ~ Senescent ! week 2 week Soybean
PEP carboxylase ~,b
% of mature
155.8_+11.2 660.6_+32.6 60.6_+ 6.2 288.0-+22,1 571.2_+48.0 36.7
RuDP carboxylase ~'b
23.6 100.0 9.2 32.6 86.5 -
33,2 -+ 1.5 296.7_+15.2 14.2-+ 1.4 125.4-+17.2 154.8 -+ 14,3 397.2
% of mature
RuDP/ PEP
TISSUE
70 "10
6o
11.2 0.21 100.0 0.45 4,8 0.23 42.4 0.z14 52,3 0.38 10.82
L,. O
513
o
%
N 4o
O
~o
3c
~ goleS
Data expressed as gmol g- ~ fresh weight h - t. Values represent the means of at least 4 trials run in duplicate -+ standard error u The specific activity of PEP carboxylase for young, mature and senescent leaf tissue was 4.21 _+0.80, 13.11_+1.68 and 2.17 _+0.63 gmol mg -~ protein h -~, respectively; specific activity of RuDP carboxylase for young, mature and senescent leaf tissue was 0.89 -+ 0.11, 5.89 _+0.80 and 0.51 _+0.02 Ixmol m g - 1 protein h - ~, respectively Enzyme activities of PEP and RuDP carboxylase expressed as lamol mg -~ chlorophyll h -1 were 1078.1 +51,9 and 484.2-+24.8, respectively
- - - e=-O.--r----- ~' -I0
I0
20
30
~
K 40
,
~
,
50
60
70
I10
120
Time (Seconds) Fig. 1, Pulse-chase labeling data of photosynthetic products for segments from mature Z . m a y s leaves. A pulse of i4COa for a 10-s period resulted in an average incorporation of 1 3 , 5 x ] 0 O c p m g ~ fresh weight, a ~4CO2 fixation rate of 78.57 gmol COz g - * fresh weight h L Each point represents the average of at Least two incorporations
Table 2. Decarboxylase activities during leaf development in Z e a mays
Age of leaf tissue
Young Mature ~ Senescent 1 week 2 week
NADP-malic enzyme "'b Mg 2+
Mn z+
377.9+28.0 1183.6+71.1 232.2_+ 5.7 1062.3_+55.3 1682.7_+66.2
418.5-+14.1 1016.3_+42.2 237.9+ 7.4 -
% of mature Mg2 +
NAD-malic enzyme a Mn 2+
31.9 100.0 19,6 89.8
43.4_+6.0 37.9_+4.8 -
142.2
-
Data expressed as gmol g- ~ flesh weight h - 1. Values represent the means of at least 4 trials run in duplicate -+ standard error b The specific activity of NADP-malic enzyme (Mg 2+) for young, mature and senescent leaf tissue was 5.11 +0.76, 11.77+ 1.61 and 2.55-+ 1.17 pmol mg ~ protein h - ~, respectively ~ Enzyme activity of NADP-maiic enzyme (Mg z+) expressed as btmol mg 1 chlorophyll h ~ was 1052.7 Table 3. Aspartate aminotransferase, alanine aminotransferase and
malate dehydrogenase enzyme activities in Z e a m a y s leaf tissues Age of 7eaf tissue
Aspartate aminotransferase"
% of mature
Alanine aminotransferase"
% of matare
Malate dehydrogenase ~
% of mature
Young
147.2 +8.9
26.2
39.4 -+3.6
53.5
3.98 -+0.38
73.8
Mature
562.9 +37.5
I00.0
73.7 +4.8
100.0
5.39 +0.56
I00.0
Senescent
84.1 + 3.4
14.9
14.2 _+3.2
19.3
1.67 _+0.20
31.0
activities of N A D P - m a l i c enzyme than either carboxylase, both on an absolute and comparative basis. N A D P - m a l i c enzyme activity in young leaf tissue was ca. 378 gmol g - 1 fresh weight h 1 (20% of mature). The activity of this enzyme in leaves from 1- and 2-week-old plants was similar to or greater than that of mature tissue, and higher than that of young leaf tissue taken from the base of leaves 8 and 9 in an older plant. The changes with leaf development in the activities of aminotransferase enzymes in Z. mays are shown in Table 3. Mature corn leaves have an aspartateaminotransferase activity of ca. 563 gmol g - 1 fresh weight h-1. The enzyme activities of both aminotransferases and N A D malate dehydrogenase seen in Table 3 for young and senescent leaf tissues are lower than those of mature leaf tissue, but these two stages of development still contained greater relative activities of these three enzymes than of either PEP carboxylase or R~zDP carboxyIase.
Pulse-chase Experiments
Data expressed as Itmol g 1 fresh weight h - 1 Values represent the means of at least 4 trials run in duplicate _+standard error b Data expressed as mmol g- 1 fresh weight h
Pulse-chase data for mature leaf tissue are shown in Figure 1. At the end of a 10-s pulse, radioactivity in the two C4 acids accounted for ca_ 73% of the total 14CO2 incorporated. During the subsequent chase, label in these acids decreased while that in PGA, sugar phosphates and sucrose increased. Ala-
272
L.E. Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Zea
nine, another product that becomes heavily labeled during the chase, accounts for almost 20% of the total 14CO2 incorporated from 60 to 120 s into the chase period. The percent label in sugar-phosphates is higher in senescent leaf tissue than that in mature leaves (Fig. 2), but radioactivity in PGA is lower and alanine never contains more than 4% of the total label in senescent leaf tissue at any one time. A comparison of the patterns of the C4-acid decarboxylation between mature and senescent leaf tissue is shown in Figure 3. In either tissue, label in C4 acids is lost during the 12CO2 chase, with the initial kinetics
14C02
12C02
8(
SENESCENT LEAF TISSUE 7(
~.%.
6c
c N o 40
p~_~h~ s
4-' 2
s..,~176
,o
Alanine .... -I0
0
i I0
20
l 30
I 40
I SO
I 60
I ,sf I 70 I10
I 120
Time (Seconds) Fig. 2. Pulse-chase labeling data of photosynthetic products for senescent Z. mays leaf tissue. A pulse of 14CO2 for a 10-s period resulted in an average incorporation of 2.6 x 106 cpm g 1 fresh weight, a ~4CO2 fixation rate of 15.11 lamol CO2 g- 1 fresh weight h - 1. Each point represents the average of at least three incorporations
of this loss between mature and senescent leaf material very similar. At the end of the 2-rain chase period, however, senescent leaves have twice as much radioactivity in C4 acids as found in mature corn leaves. Another difference between the two ages which becomes evident in the pulse-chase experiments is the predominant C4 acid. After 10-s, aspartate is the predominant C4 acid in the mature leaves, while malate is the major C4 acid in the senescent ones.
Discussion
Our study of enzymes of the photosynthetic carbonmetabolism pathway in Z. mays leaves of different developmental stages has shown that the activities of the non-chloroplastic enzymes increase, preceding those of the chloroplastic enzyme, RuDP carboxylase. A similar trend was observed in experiments on lightinduced enzyme development in C3 and C4 etioplasts (Bradbeer, 1969; Hatch et al., 1969; Graham et al., 1970). The apparent lag of RuDP-carboxylase activity in young leaf tissue and in leaf blades of 1- and 2week-old plants (Table 1) is, as reported in the literature, perhaps explained by the fact that the synthesis (Kleinkopf et al., 1970b) and activity of RuDP carboxylase is dependent on light (Huffaker et al., 1966) and leaf age (Obendorf and Huffaker, 1970; Dickman, 1971). We also found differences in enzyme activities between juvenile tissue taken from the base of leaf blades of older plants which were not yet fully expanded and whole leaf blades of 1- and 2-week-old corn seedlings. Although the former tissue comes from an older plant, it is perhaps physiologically less mature than that of the 1- and 2-week-old seedlings. The position of the young leaf tissue within
14C02
12C02
80
t2COz
14C02
SENESCENT LEAF TISSUE
70
"O ~ 0 o C
7C
"0
so
60
O
5C
c 40 Od ,r 3~
4(]
o
I.~
MATURE LEAF TISSUE
Total C4 Acids
Aspartate
zc
late
IC
-I0
J 0
, I0
, 20
, 50
, 40
50
Time (Seconds)
60
70
IlO
-
120
-I0
I0
20
30
40
50
60
70
IlO
120
Time (Seconds)
Fig. 3. C4 acid kinetics in segments from mature and senescent corn leaves during a 10-s pulse of in air
14CO2 followed
by 120 s chase
L.E. Williamsand R.A. Kennedy:PhotosyntheticCarbon Metabolismin Zea the shoot of the older plants, where it is still surrounded by the sheathing blades of lower leaves, may result in less light reaching this region and consequently lower amounts of chlorophyll being formed in this tissue (Williams and Kennedy, 1977). NADP-malic enzyme has been shown to be a bundle-sheath, chloroplastic enzyme (Slack et al., 1969; Rathnam and Edwards, 1975). Chollet and Ogren (1972) found that the activity of this enzyme progressively increased during development in both a virescent and a normal strain of maize. The relatively high activity of this enzyme in young, 1- and 2-weekold leaf seedlings found in our study, along with the results of Chollet and Ogren, indicate a relationship between NADP-malic enzyme synthesis or activation, light, or chloroplast development in Z. mays. It has been suggested that RuDP carboxylase is the major protein hydrolyzed during leaf senescence (Peterson and Huffaker, 1975). We also found that RuDP carboxylase lost activity in senescent leaf tissue to a greater extent than any of the other enzymes assayed. Similarly, investigations on leaves of tobacco (Kawashima et al., 1967) and Perilla (Kannangara and Woolhouse, 1968) have shown that RuDP carboxylase ("fraction I protein") was lost more rapidly during senescence than were lower-molecular-weight proteins. This pattern is different from that found in the succulent Ca dicotyledon, Portulaca oleracea; senescent leaves of this plant were shown to have relatively more RuDP-carboxylase activity than either PEP-carboxylase or malate-dehydrogenase activity when compared to mature leaf tissue (Kennedy, 1976). The activities of aminotransferase enzymes have been used as criteria for the preferential decarboxylation of one of the Ca acids in different Ca plants. Enzyme studies (Rathnam and Edwards, 1975), labelling patterns (Williams and Kennedy, 1977) and the data presented here show that aspartate is an important intermediate in the Ca pathway of photosynthesis at all stages of leaf development in Z. mays. The low levels of alanine aminotransferase and NAD-malic enzyme, and the rapid initial loss of label from malate druing the pulse-chase experiment with mature corn leaves (Fig. 3) indicate that, as proposed by Creach et al. (1974), aspartate may serve as an internal reservoir of CO2 in corn leaves. The transfer of CO2 to the Calvin cycle from aspartate in Z. mays could be accomplished via the scheme proposed for Gomphrena celosioides by Repo and Hatch (1976). The source of alanine found to be labelled during the chase period in mature corn leaves could be 3PGA as proposed by Smith et al. (1961) or again as in the scheme suggested for G. celosioides. We have reported earlier that senescent Z. mays leaf tissue has an elevated CO 2 compensation point
273
and releases recently assimilated 14CO2 in the light (Williams and Kennedy, 1977). The pulse-chase experiments described in this paper were conducted to determine if CO2 evolution in the light might be based upon decarboxylation of the C4 acids in senescent tissue, without subsequent refixation in the Calvin cycle, i.e., a lack of proper metabolic coordination such as that found by Bj6rkman et al. (1971) in Atriplex hybrids. The initial loss of label from C4 acids in senescent corn leaves (Fig. 3) was comparable to that of mature leaf tissue, but from 60 to 120 s into the chase, senescent corn leaves had twice as much label in C4 acids as did mature tissue. The higher percentage of label in Ca acids (particularly malate) in senescent leaf tissue during the chase may indicate a refixation of recently evolved CO2 or a reduced transfer of label between malate and PGA. The results reported in this paper show that the activities of several enzymes involved in the photosynthetic carbon metabolism in Z. mays vary during leaf ontogeny, as has been previously reported for C3 plants. These results are important since the photosynthetic carbon pathway of C4 plants is compartmentalized in two different cell layers, the bundle sheath and the mesophyll. These two cell layers have been shown to be differentially affected by water stress (Giles et al., 1974; Alberte et aI., 1977), with the mesophyll cells being more susceptible to damage than the bundle-sheath cells. Kennedy (1976) reported that the activity of PEP carboxylase, found in the mesophyll cytoplasm (Coombs et al., 1973 ; Gutierrez et al., 1974), declined more rapidly in senescent P. oleracea leaves than RuDP carboxylase, located in bundle-sheath chloroplasts (Gutierrez etal., 1974; Hattersley et al., 1977). The above results are consistent with the observations of Giles et al. (1974) and Alberte et al. (1977) and further demonstrate that the effects of water stress on many metabolic processes may be similar to those associated with senescence (Brady, 1973 ; Hsiao, 1973). RuDP carboxylase can account for 30-50% of the total soluble protein in leaves of C3 plants (Kawashima and Wildman, 1970; Kleinkopf etal., 1970a). Peoples and Dalling (1978) have shown that loss of RuDP carboxylase may be largely responsible for the high rate of protein loss in the primary leaf of wheat seedlings. In Z. mays the greater loss of activity by RuDP carboxylase when compared to the other enzymes studied indicates that RuDP carboxylase may also be the major enzyme hydrolyzed during leaf senescence in Ca plants, and a source of nitrogen for redistribution throughout the plant.
This worksupported by NationalScienceFomldationgrants BMS75-09931 and PCM-77-25100 to RAK.
274
L.E. Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Zea
References Alberte, R.S., Thornber, J.P., Fiscus, E.L.: Water stress effects on the content and organization of chlorophyll in mesophyll and bundle sheath chloroplasts of maize. Plant Physiol. 59, 351-353 (1977) Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949) Bj6rkman, O., Pearcy, R.W., Boynton, J.E., Nobs, M.A. : Hybrids between Atriplex species with an without fl-carboxylation photosynthesis. In: Carnegie Inst. Yr. Bk. for 1969, pp. 624-648 (1971) Bradbeer, J.W.: The activities of the photosythetic carbon cycle enzymes of greening bean leaves. New Phytol. 68, 233 245 (1969) Brady, C.J.: Changes accompanying growth and senescence and effect of physiological stress. In: Chemistry and biochemistry of herbage, vol. 2, pp. 317450, Butler, G.W., Bailey, R.W., eds. London: Academic Press 1973 Chollet, R., Ogren, W.L. : Greening in a virescent mutant of maize. II. Enzyme studies. Z. Pflanzenphysiol. 68, 45-54 (1972) Coombs, J., 13aldry, C.W., Bucke, C. : The C4 pathway in Penniseturn purpureum. I. The allosteric nature of PEP carboxylase. Planta 110, 95-107 (1973) Creach, E., Michel, J.P., Thibault, P. : Aspartic acid as an internal CO2 reservoir in Zea mays: Effect of oxygen concentration and of far-red illumination. Planta 118, 91 100 (1974) Dickman, D.I.: Chlorophyll, Ribulose-l,5-diphosphate carboxyIase and Hill reaction activity in developing leaves of Populus deltoides. Plant Physiol. 48, 143 145 (1971) Downton, W.J.S.: The occurrence of C4 photosynthesis among plants. Photosynthetica 9, 96-105 (1975) Giles, K.L., Beadsell, M.F., Cohen, D. : Cellular and ultrastructural changes in mesophyll and bundle sheath cells of maize in response to water stress. Plant Physiol. 54, 208-212 (1974) Graham, D., Hatch, M.D., Slack, C.R., Smillie, R.M.: Lightinduced formation of enzymes of the C4-dicarboxylic acid pathway of photosynthesis in detached leaves. Phytochemistry 9, 521-532 (1970) Gutierrez, M., Kanai, R., Huber, S.C., Ku, S.B., Edwards, G.E. : Photosynthesis in mesophyll protoplasts and bundle sheath cells of various types of C4 plants. I. Carboxylases and CO2 fixation studies. Z. Pflanzenphysiot. 72, 305-319 (1974) Hatch, M.D., Slack, C.R., Bull, T.A.: Light-induced changes in the content of some enzymes of the C4-dicarboxylic acid pathway of photosynthesis. Phytochemistry 8, 697-706 (1969) Hattersley, P.W., Watson, L., Osmond, C.B. : In situ immunofluorescent labelling of ribulose-l,5-bisphosphate carboxylase in leaves of C3 and C4 plants. Aust. J. Plant Physiol. 4, 523 540 (1977) Hsiao, T.C.: Plant responses to water stress. Ann. Rev. Plant Physiol. 24, 519-570 (1973) Huffaker, R.C., Obendorf, R.L., Keller, C.J., Kleinkopf, G.E.: Effects of light intensity of photosynthetic carboxylase phase enzymes and chlorophyll synthesis in greening leaves of Hordeum vulgare L. Plant Physiol. 41,913-918 (1966) Kannangara, C.G., Woolhouse, H.W.: Changes in the enzyme activity of soluble protein fractions in the course of foliar senescence in Perillafructescens (L.) Britt. New Phytol. 67, 533 542 (1968)
Kawashima, N., Wildman, S.G.: Fraction I protein. Ann. Rev. Plant Physiol. 21, 325-358 (1970) Kawashima, N., Imai, A., Tamaki, E. : Studies on protein metabolism in higher plant leaves. III. Changes in the soluble protein components with leaf growth. Plant & Cell Physiol. 8, 447458 (1967) Kennedy, R.A. : Relationship between leaf development, carboxylase enzyme activities and photorespiration in the C4-plant Portulaca oleracea L. Planta 128, 149 154 (1976) Kennedy, R.A., Laetsch, W.M.: Relationship between leaf development and primary photosynthetic products in the C~ plant Portulaca oleracea L. Planta 115, 113-124 (1973) Kleinkopf, G.E., Huffaker, R.C., Matheson, A. : A simplified purification and some properties of ribulose-l,5-diphosphate carboxylase from barley. Plant Physiol. 46, 204-207 (1970a) Kleinkopf, G.E., Huffaker, R.C., Matheson, A.: Light-induced de-novo synthesis of RuDP carboxylase in greening leaves of barley. Plant Physiol. 46, 416418 (1970b) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin reagent. J. Biol. Chem. 193, 262~75 (1951) Ludlow, M.M., Wilson, G.L.: Photosynthesis of tropical pasture plants. III. Leaf age. Aust. J. Biol. Sci. 24, 1077-1087 (1971) Nimbalkar, J.D., Joshi, G.: Photosynthesis and mineral metabolism in senescent leaves of sugarcane Var. Co. 740. Indian Agric. 18, 257~64 (1974) Obendorf, R.L., Huffaker, R.C. : Influence of age and illumination on distribution of several Calvin cycle enzymes in greening barley leaves. Plant Physiol. 45, 579 582 (1070) Peoples, M.B., Dalling, M.J. : Degradation of ribulose-l,5-biphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves. Planta 138, 153-160 (1978) Peterson, L.W., Huffaker, R.C. : Loss of ribulose-l,5-disphosphate carboxylase and increase in proteolytic activity during senescence of detached primary bean leaves. Plant Physiol. 55, 1009-1015 (1975) Rathnam, C.K.M., Edwards, G.E.: Intracellular localization of certain photosynthetic enzymes in bundle sheath cells of plants possessing the C, pathway of photosynthesis. Arch. Biochem. Biophys. 171, 214-225 (1975) Repo, E., Hatch, M.D.: Photosynthesis in Gomphrena celosioides and its classification amongst C4-pathway plants. Aust. J. Plant Physiol. 3, 863 876 (1976) Slack, C.R., Hatch, M.D., Goodchild, D.J.: Distribution of enzymes in mesophyll and parenchyma-sheath chloroplasts of maize leaves in relation to the C4-Dicarboxylic acid pathway of photosynthesis. Biochem. J. 114, 489498 (1969) Smith, D.C., Bassham, J.A., Kirk, M. : Dynamics of the photosynthesis of carbon compounds. II. Amino acid synthesis. Biochem. Biophys. Acta 48, 299-313 (1961) Williams, L.E., Kennedy, R.A. : Relationship between early photosynthetic products, photorespiration and stage of leaf development in Zea mays. Z. Pflanzenphysiol. 81, 314422 (1977) Zelitch, I. : Plant productivity and the control of photorespiration. Proc. Natl. Acad. Sci. USA 70, 579-584 (1973)
Received 31 March; accepted 9 June 1978
Planta
274(1978)
9 by Springer-Verlag 1978
Photosynthetic Carbon Metabolism during Leaf Ontogeny in Zea mays L. : Enzyme Studies Larry E. Williams* and Robert A. Kennedy Department of Botany, University of Iowa, Iowa City, IA 52242, USA
Abstract. The activities of several enzymes, including ribulose- 1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z . mays. Mature leaf tissue had a R u D P carboxylase activity of 296.7 ~tmol CO2 g-1 fresh weight h 1 and a PEP-carboxylase activity of 660.6 gmol CO2 g ~ fresh weight h - t. In young corn leaves the activity of the two enzymes was I1 and 29%, respectively, of the mature leaves. In senescent leaf tissue, R u D P carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of N A D P malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and N A D malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and R u D P carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C4 acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total zgC remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in a2CO2. The activity of R u D P carboxylase during leaf ontogeny in Z. m a y s parallels the development of the activity of this enzyme in C3 plants. Key words: Carboxylases Photosynthesis (C4) - Zea.
Leaf development -
Introduction C4 plants are generally considered to be more efficient photosynthetically than C3 plants (Zelitch, 1973) primarily because of their lack of detectable photorespiration, low CO2 compensation point (Downton, 1975) * Present address: Dept. of Agronomy&Range Science Univ. of California, Davis, CA 95616, USA Abbreviations: RuDP = ribulose-l,5-diphosphate; PEP = phosphoenol pyruvate; PGA = 3-phosphoglycerate
and lack of an enhancement of photosynthesis under low oxygen tensions. During leaf ontogeny the efficiency of C4 photosynthesis appears to decline (Ludlow and Wilson, 1971; Nimbalkar and Joshi, 1974; Kennedy, 1976; Williams and Kennedy, 1977). In the C4 dicotyledon, Portulaca oleracea, PEP carboxylase activity decreased more in senescent leaves than did R u D P carboxylase (Kennedy, 1976), while in senescent corn leaves a CO2 compensation point of 24 ~tl/1 was found, in contrast to values of 0 10 tal/1 for most C4 plants (Williams and Kennedy, 1977). Both Z e a m a y s and P. oleracea were found to have an increased evolution of t4CO2 in the light using the 14C assay of photorespiration (Kennedy, 1976; Williams and Kennedy, 1977). In this paper, we report the results of studies on changes during leaf development in the activities of several enzymes involved in the C4 pathway of photosynthesis in Z. mays, as well as the relative turnover rate of photosynthetic products in mature and senescent corn leaves.
Material and Methods Plant Material Zea mays L. was grown, and the same ontogenetic series employed
as previously described (Williams and Kennedy, I977). Mature leaf tissue was taken from the 4th and 5th leaves from the base of the plant, midwayalong the leaf. Young leaf tissue was collected at the base of leaf blades 8 and 9, whichwere not yet fully expanded. Senescent leaf tissue was collected when it contained 10 20% of the chlorophyllcontent of mature leaf material. Additionally, tissue was used from whole leaf blades of corn seedlings 7 and 14 d after emergence. Soybean (Glycine max L.) was grown in soil in a naturally illuminated greenhouse under the same conditions as described before (Williams and Kennedy, 1977) and the youngest mature trifoliate leaf was used for experimentation. Enzyme Preparation and Assays Preparation of Crude Enzyme Extracts. Leaf tissue was cut into small pieces (1 mm2) and ground with sand in a chilled mortar.
0032-0935/78/0142/0269/$01.20
270
L.E. Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Zea
The grinding medium for PEP and RuDP carboxylase contained 195 mM phosphate buffer, pH 7.0; 10 mM MgClz; 20 mM 2-mercaptoethanol; 0.2 mM ethylenediamine tetraacetic acid (EDTA); and 16 g/1 polyvinylpyrrolidone (PVP) 10. The grinding medium for aminotransferase contained 150 mM phosphate buffer, pFI 7.3; 1.0 mM MgC12; 1.0 mM EDTA; 5.0 mM dithiothreitol and 20 g/ 1 PVP 10. Tissue for NADP-malic enzyme, NAD-malic enzyme and NAD-malate dehydrogenase determinations was ground in 100 mM tris(hydroxymethyl)aminomethane hydrochloride (TrisHCL), pH 8.3; 2.0 mM MgC1; 10 mM mercaptoethanoi; and 1.0 mM EDTA. One gram of leaf tissue was ground in 1.5 ml grinding medium for preparing PEP and RuDP carboxylase extracts, while 1 g leaf tissue per 3 ml of grinding medium was used in preparing the other extracts. The homogenate was filtered through 4 layers of cheesecloth, centrifuged for 20 min at 3,000 • g and 5~ C, and the supernatant used for enzyme assays. Chlorophyll and protein determinations were conducted according to the methods of Arnon (1949) and Lowry et al. (1951), respectively.
PEP Carboxylase (EC 4.1.1.31). The reaction mixture contained (in a final volume of 175 ~tl): 25 mM N-tris(hydroxymethyl) methyl glycine (Tricine), pH 8.0; 10 mM MgC12; 5 mM 2-mercaptoethanol; 15 mM PEP; 15 mM sodium glutamate; and 50 mM NaH14CO3 (1.0 gCi). The assays were initiated by the addition of 10-25 gI of leaf extract and run for 2-3 min. Assays were terminated by the addition of 100 gl 25% (v/v) acetic acid. Aliquots were sampled, dried and counted in an LS-100 C iiquid scintillation counter (Beckman Instruments, Fullerton, Cal., USA)
NAD Malate Dehydrogenase (EC 1.1.1.37). The assay medium contained 100 mM Tricine, pH 7.5; 0.2 mM EDTA; 1.0 mM oxaloacetate; 0.2 mM NADH and 5 15 gl enzyme extract in a total volume of 3 ml. Reactions were initated by addition of the enzyme extract and activities determined spectrophotometrically by the oxidation o f N A D H (A A at 340 nm) for 2 3 rain. Controls were run without NADH. Assays for PEP carboxylase and RuDP carboxylase were carried out at 29_+2 ~ C, with the other enzyme assays conducted at 27_+ 1~ C. All enzyme assays were linear with time and enzyme concentration. Reagents listed in the Methods section were obtrined from Sigma Chemical Co., St. Louis, Mo., USA. Pulse-chase Experiments Pulse-chase radiotracer studies were conducted using a 10-s pulse of 14CO2 as described in Williams and Kennedy, (1977), followed by a chase of up to 2 min in air. Separation and identification of labelled compounds was conducted using two-dimensional thinlayer electrophoresis and chromatography (Kennedy and Laetsch, 1973). Quantification of 14C was by liquid scintillation spectroscopy.
Results E n z y m e Activities
RuDP Carboxylase (EC 4.1.1.39). The reaction mixture contained (in a volume of 100 gl): 25 mM Tricine, pH 8.0; 10 mM MgClz; 5 mM 2-mercaptoethanol; 0.05 mM NADPH; 1.0 mM fructose1,6-diphosphate; 50 mM NaHI*CO3 (0.5 p,Ci); and 5.5 mM RuDP. The enzyme extract (10-25 tsl) was preincubated for 5 rain with all ingredients except RuDP The assay was initiated by the addition of RuDP and run 3 4 min. Termination and quantification of the assay were as for PEP carboxylase (above).
Aspartate Aminotransferase (EC 2.6.1.1). 25 mM Tricine, pH 8.0; 2.5 mM dithiothreitol; 1.25 mM e-ketoglutarate; 4.2 mM aspartate; 0.03 mM pyridoxal phosphate; 0.1 mM NADH; 2.0 mM EDTA; 1.5 units of malate dehydrogenase and 10-25 ~tl enzyme extract were contained in a total volume of 2.4 ml. The reaction was started after a 10-min preincubation by the addition of c~-ketoglutarate. Activities were determined spectrophotometrically by the oxidation of NADH at 340 nm for 3 min.
Alanine Aminotransferase (EC 2.6.1.2). The reaction mixture contained 25 mM Tricine, pH 7.25; 2.5 mM dithiothreitol; 2.5 mM c~ketoglutarate; 4.0mM alanine; 0.03 mM pyridoxal phosphate; 0.1 mM NADH; 2.0 mM EDTA; 1.5 units of lactate dehydrogenase and 10 25 gl enzyme extract in a total volume of 2.4 ml. Reactions were started and run as for aspartate aminotransferase. NADP-Malic Enzyme (EC 1.1.1.40). The assay mixture included 25 mM Tris-HC1, pH 8.3; 0.5 mM EDTA; 5.0mM MgC12; 2.5 mM malate, pH 8.3; 1.5 mM NADP and 10-20 gl enzyme extract in a total volume of 3 mi. The reaction was started by the addition of MgC12 or MnC12, and monitored spectrophotometrically at 340 nm for 3 min.
Carboxylases. T h e a c t i v i t i e s o f P E P c a r b o x y l a s e a n d RuDP carboxylase for the leaf tissue of various ages o f Z . mays, a l o n g w i t h t h e i r a c t i v i t i e s f o r s o y b e a n , a C3 p l a n t , a r e g i v e n in T a b l e 1. T h e r a t i o o f R u D P / P E P c a r b o x y l a s e in m a t u r e l e a v e s is 0.45, i.e., t h e a c t i v i t y o f P E P c a r b o x y l a s e is t w i c e as g r e a t as t h a t of RuDP carboxylase. In contrast, the RuDP/PEP r a t i o i n s o y b e a n l e a v e s is 10.82. I n Z . m a y s l e a v e s o f all o t h e r a g e s s t u d i e d t h e a b s o l u t e a m o u n t s o f both carboxylases were lower than those found in m a t u r e l e a f tissue. C o m p a r e d t o t h e m a t u r e l e a f tissue, y o u n g l e a f t i s s u e o f c o r n h a d 2 4 % o f t h e P E P carboxylase activity and 11% of the RuDP-carboxylase a c t i v i t y . T h e P E P - c a r b o x y l a s e a c t i v i t y i n s e n e s c e n t c o r n l e a f t i s s u e is 9 . 2 % o f t h e m a t u r e t i s s u e while the RuDP-carboxylase a c t i v i t y is o n l y 4 . 8 % . T h e R u D P / P E P c a r b o x y l a s e r a t i o (0.23) i n s e n e s c e n t Z. m a y s l e a v e s is a p p r o x i m a t e l y t h e s a m e as i n y o u n g leaves, but only about one-half that in mature ones. The activities of the two carboxylases of leaf b l a d e s o n 1- a n d 2 - w e e k - o l d c o r n p l a n t s w e r e a l s o d e t e r m i n e d ( T a b l e 1). A t e i t h e r age, t h e a c t i v i t i e s o f b o t h P E P c a r b o x y l a s e a n d R u D P c a r b o x y l a s e a r e less t h a n t h o s e f o u n d in m a t u r e l e a f tissue, b u t c o n s i d e r a b l y h i g h e r t h a n t h o s e f o u n d in y o u n g ( b a s a l ) t i s s u e o f t h e b l a d e o f a n o l d e r , m o r e e x p a n d e d leaf.
NAD-Malic Enzyme (EC 1.1.1.38). The reaction medium contained in a total volume of 3 ml: 25 mM Tris-HC1, pH 8.3; 0.5 mM EDTA; 5.0 mM MnC12; 2.5 mM malate pH 8.3; 2.0 mM NAD; 75.0 gM coenzyme A and 10-20 gl enzyme extract. The assay was carried out as described above.
Other Enzymes. T a b l e 2 s h o w s t h e a c t i v i t i e s o f t h e d e c a r b o x y l a t i n g e n z y m e in Z. m a y s d u r i n g l e a f d e v e l opment. Young and senescent tissue contained higher
L E . Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Z e a
271
14C02 I~C02 SO
Table 1, PEP carboxylase and RuDP carboxylase activities in several
stages of leaf development in Z e a m a y s
MATURE LEAF Age of leaf tissue
Young Mature ~ Senescent ! week 2 week Soybean
PEP carboxylase ~,b
% of mature
155.8_+11.2 660.6_+32.6 60.6_+ 6.2 288.0-+22,1 571.2_+48.0 36.7
RuDP carboxylase ~'b
23.6 100.0 9.2 32.6 86.5 -
33,2 -+ 1.5 296.7_+15.2 14.2-+ 1.4 125.4-+17.2 154.8 -+ 14,3 397.2
% of mature
RuDP/ PEP
TISSUE
70 "10
6o
11.2 0.21 100.0 0.45 4,8 0.23 42.4 0.z14 52,3 0.38 10.82
L,. O
513
o
%
N 4o
O
~o
3c
~ goleS
Data expressed as gmol g- ~ fresh weight h - t. Values represent the means of at least 4 trials run in duplicate -+ standard error u The specific activity of PEP carboxylase for young, mature and senescent leaf tissue was 4.21 _+0.80, 13.11_+1.68 and 2.17 _+0.63 gmol mg -~ protein h -~, respectively; specific activity of RuDP carboxylase for young, mature and senescent leaf tissue was 0.89 -+ 0.11, 5.89 _+0.80 and 0.51 _+0.02 Ixmol m g - 1 protein h - ~, respectively Enzyme activities of PEP and RuDP carboxylase expressed as lamol mg -~ chlorophyll h -1 were 1078.1 +51,9 and 484.2-+24.8, respectively
- - - e=-O.--r----- ~' -I0
I0
20
30
~
K 40
,
~
,
50
60
70
I10
120
Time (Seconds) Fig. 1, Pulse-chase labeling data of photosynthetic products for segments from mature Z . m a y s leaves. A pulse of i4COa for a 10-s period resulted in an average incorporation of 1 3 , 5 x ] 0 O c p m g ~ fresh weight, a ~4CO2 fixation rate of 78.57 gmol COz g - * fresh weight h L Each point represents the average of at Least two incorporations
Table 2. Decarboxylase activities during leaf development in Z e a mays
Age of leaf tissue
Young Mature ~ Senescent 1 week 2 week
NADP-malic enzyme "'b Mg 2+
Mn z+
377.9+28.0 1183.6+71.1 232.2_+ 5.7 1062.3_+55.3 1682.7_+66.2
418.5-+14.1 1016.3_+42.2 237.9+ 7.4 -
% of mature Mg2 +
NAD-malic enzyme a Mn 2+
31.9 100.0 19,6 89.8
43.4_+6.0 37.9_+4.8 -
142.2
-
Data expressed as gmol g- ~ flesh weight h - 1. Values represent the means of at least 4 trials run in duplicate -+ standard error b The specific activity of NADP-malic enzyme (Mg 2+) for young, mature and senescent leaf tissue was 5.11 +0.76, 11.77+ 1.61 and 2.55-+ 1.17 pmol mg ~ protein h - ~, respectively ~ Enzyme activity of NADP-maiic enzyme (Mg z+) expressed as btmol mg 1 chlorophyll h ~ was 1052.7 Table 3. Aspartate aminotransferase, alanine aminotransferase and
malate dehydrogenase enzyme activities in Z e a m a y s leaf tissues Age of 7eaf tissue
Aspartate aminotransferase"
% of mature
Alanine aminotransferase"
% of matare
Malate dehydrogenase ~
% of mature
Young
147.2 +8.9
26.2
39.4 -+3.6
53.5
3.98 -+0.38
73.8
Mature
562.9 +37.5
I00.0
73.7 +4.8
100.0
5.39 +0.56
I00.0
Senescent
84.1 + 3.4
14.9
14.2 _+3.2
19.3
1.67 _+0.20
31.0
activities of N A D P - m a l i c enzyme than either carboxylase, both on an absolute and comparative basis. N A D P - m a l i c enzyme activity in young leaf tissue was ca. 378 gmol g - 1 fresh weight h 1 (20% of mature). The activity of this enzyme in leaves from 1- and 2-week-old plants was similar to or greater than that of mature tissue, and higher than that of young leaf tissue taken from the base of leaves 8 and 9 in an older plant. The changes with leaf development in the activities of aminotransferase enzymes in Z. mays are shown in Table 3. Mature corn leaves have an aspartateaminotransferase activity of ca. 563 gmol g - 1 fresh weight h-1. The enzyme activities of both aminotransferases and N A D malate dehydrogenase seen in Table 3 for young and senescent leaf tissues are lower than those of mature leaf tissue, but these two stages of development still contained greater relative activities of these three enzymes than of either PEP carboxylase or R~zDP carboxyIase.
Pulse-chase Experiments
Data expressed as Itmol g 1 fresh weight h - 1 Values represent the means of at least 4 trials run in duplicate _+standard error b Data expressed as mmol g- 1 fresh weight h
Pulse-chase data for mature leaf tissue are shown in Figure 1. At the end of a 10-s pulse, radioactivity in the two C4 acids accounted for ca_ 73% of the total 14CO2 incorporated. During the subsequent chase, label in these acids decreased while that in PGA, sugar phosphates and sucrose increased. Ala-
272
L.E. Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Zea
nine, another product that becomes heavily labeled during the chase, accounts for almost 20% of the total 14CO2 incorporated from 60 to 120 s into the chase period. The percent label in sugar-phosphates is higher in senescent leaf tissue than that in mature leaves (Fig. 2), but radioactivity in PGA is lower and alanine never contains more than 4% of the total label in senescent leaf tissue at any one time. A comparison of the patterns of the C4-acid decarboxylation between mature and senescent leaf tissue is shown in Figure 3. In either tissue, label in C4 acids is lost during the 12CO2 chase, with the initial kinetics
14C02
12C02
8(
SENESCENT LEAF TISSUE 7(
~.%.
6c
c N o 40
p~_~h~ s
4-' 2
s..,~176
,o
Alanine .... -I0
0
i I0
20
l 30
I 40
I SO
I 60
I ,sf I 70 I10
I 120
Time (Seconds) Fig. 2. Pulse-chase labeling data of photosynthetic products for senescent Z. mays leaf tissue. A pulse of 14CO2 for a 10-s period resulted in an average incorporation of 2.6 x 106 cpm g 1 fresh weight, a ~4CO2 fixation rate of 15.11 lamol CO2 g- 1 fresh weight h - 1. Each point represents the average of at least three incorporations
of this loss between mature and senescent leaf material very similar. At the end of the 2-rain chase period, however, senescent leaves have twice as much radioactivity in C4 acids as found in mature corn leaves. Another difference between the two ages which becomes evident in the pulse-chase experiments is the predominant C4 acid. After 10-s, aspartate is the predominant C4 acid in the mature leaves, while malate is the major C4 acid in the senescent ones.
Discussion
Our study of enzymes of the photosynthetic carbonmetabolism pathway in Z. mays leaves of different developmental stages has shown that the activities of the non-chloroplastic enzymes increase, preceding those of the chloroplastic enzyme, RuDP carboxylase. A similar trend was observed in experiments on lightinduced enzyme development in C3 and C4 etioplasts (Bradbeer, 1969; Hatch et al., 1969; Graham et al., 1970). The apparent lag of RuDP-carboxylase activity in young leaf tissue and in leaf blades of 1- and 2week-old plants (Table 1) is, as reported in the literature, perhaps explained by the fact that the synthesis (Kleinkopf et al., 1970b) and activity of RuDP carboxylase is dependent on light (Huffaker et al., 1966) and leaf age (Obendorf and Huffaker, 1970; Dickman, 1971). We also found differences in enzyme activities between juvenile tissue taken from the base of leaf blades of older plants which were not yet fully expanded and whole leaf blades of 1- and 2-week-old corn seedlings. Although the former tissue comes from an older plant, it is perhaps physiologically less mature than that of the 1- and 2-week-old seedlings. The position of the young leaf tissue within
14C02
12C02
80
t2COz
14C02
SENESCENT LEAF TISSUE
70
"O ~ 0 o C
7C
"0
so
60
O
5C
c 40 Od ,r 3~
4(]
o
I.~
MATURE LEAF TISSUE
Total C4 Acids
Aspartate
zc
late
IC
-I0
J 0
, I0
, 20
, 50
, 40
50
Time (Seconds)
60
70
IlO
-
120
-I0
I0
20
30
40
50
60
70
IlO
120
Time (Seconds)
Fig. 3. C4 acid kinetics in segments from mature and senescent corn leaves during a 10-s pulse of in air
14CO2 followed
by 120 s chase
L.E. Williamsand R.A. Kennedy:PhotosyntheticCarbon Metabolismin Zea the shoot of the older plants, where it is still surrounded by the sheathing blades of lower leaves, may result in less light reaching this region and consequently lower amounts of chlorophyll being formed in this tissue (Williams and Kennedy, 1977). NADP-malic enzyme has been shown to be a bundle-sheath, chloroplastic enzyme (Slack et al., 1969; Rathnam and Edwards, 1975). Chollet and Ogren (1972) found that the activity of this enzyme progressively increased during development in both a virescent and a normal strain of maize. The relatively high activity of this enzyme in young, 1- and 2-weekold leaf seedlings found in our study, along with the results of Chollet and Ogren, indicate a relationship between NADP-malic enzyme synthesis or activation, light, or chloroplast development in Z. mays. It has been suggested that RuDP carboxylase is the major protein hydrolyzed during leaf senescence (Peterson and Huffaker, 1975). We also found that RuDP carboxylase lost activity in senescent leaf tissue to a greater extent than any of the other enzymes assayed. Similarly, investigations on leaves of tobacco (Kawashima et al., 1967) and Perilla (Kannangara and Woolhouse, 1968) have shown that RuDP carboxylase ("fraction I protein") was lost more rapidly during senescence than were lower-molecular-weight proteins. This pattern is different from that found in the succulent Ca dicotyledon, Portulaca oleracea; senescent leaves of this plant were shown to have relatively more RuDP-carboxylase activity than either PEP-carboxylase or malate-dehydrogenase activity when compared to mature leaf tissue (Kennedy, 1976). The activities of aminotransferase enzymes have been used as criteria for the preferential decarboxylation of one of the Ca acids in different Ca plants. Enzyme studies (Rathnam and Edwards, 1975), labelling patterns (Williams and Kennedy, 1977) and the data presented here show that aspartate is an important intermediate in the Ca pathway of photosynthesis at all stages of leaf development in Z. mays. The low levels of alanine aminotransferase and NAD-malic enzyme, and the rapid initial loss of label from malate druing the pulse-chase experiment with mature corn leaves (Fig. 3) indicate that, as proposed by Creach et al. (1974), aspartate may serve as an internal reservoir of CO2 in corn leaves. The transfer of CO2 to the Calvin cycle from aspartate in Z. mays could be accomplished via the scheme proposed for Gomphrena celosioides by Repo and Hatch (1976). The source of alanine found to be labelled during the chase period in mature corn leaves could be 3PGA as proposed by Smith et al. (1961) or again as in the scheme suggested for G. celosioides. We have reported earlier that senescent Z. mays leaf tissue has an elevated CO 2 compensation point
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and releases recently assimilated 14CO2 in the light (Williams and Kennedy, 1977). The pulse-chase experiments described in this paper were conducted to determine if CO2 evolution in the light might be based upon decarboxylation of the C4 acids in senescent tissue, without subsequent refixation in the Calvin cycle, i.e., a lack of proper metabolic coordination such as that found by Bj6rkman et al. (1971) in Atriplex hybrids. The initial loss of label from C4 acids in senescent corn leaves (Fig. 3) was comparable to that of mature leaf tissue, but from 60 to 120 s into the chase, senescent corn leaves had twice as much label in C4 acids as did mature tissue. The higher percentage of label in Ca acids (particularly malate) in senescent leaf tissue during the chase may indicate a refixation of recently evolved CO2 or a reduced transfer of label between malate and PGA. The results reported in this paper show that the activities of several enzymes involved in the photosynthetic carbon metabolism in Z. mays vary during leaf ontogeny, as has been previously reported for C3 plants. These results are important since the photosynthetic carbon pathway of C4 plants is compartmentalized in two different cell layers, the bundle sheath and the mesophyll. These two cell layers have been shown to be differentially affected by water stress (Giles et al., 1974; Alberte et aI., 1977), with the mesophyll cells being more susceptible to damage than the bundle-sheath cells. Kennedy (1976) reported that the activity of PEP carboxylase, found in the mesophyll cytoplasm (Coombs et al., 1973 ; Gutierrez et al., 1974), declined more rapidly in senescent P. oleracea leaves than RuDP carboxylase, located in bundle-sheath chloroplasts (Gutierrez etal., 1974; Hattersley et al., 1977). The above results are consistent with the observations of Giles et al. (1974) and Alberte et al. (1977) and further demonstrate that the effects of water stress on many metabolic processes may be similar to those associated with senescence (Brady, 1973 ; Hsiao, 1973). RuDP carboxylase can account for 30-50% of the total soluble protein in leaves of C3 plants (Kawashima and Wildman, 1970; Kleinkopf etal., 1970a). Peoples and Dalling (1978) have shown that loss of RuDP carboxylase may be largely responsible for the high rate of protein loss in the primary leaf of wheat seedlings. In Z. mays the greater loss of activity by RuDP carboxylase when compared to the other enzymes studied indicates that RuDP carboxylase may also be the major enzyme hydrolyzed during leaf senescence in Ca plants, and a source of nitrogen for redistribution throughout the plant.
This worksupported by NationalScienceFomldationgrants BMS75-09931 and PCM-77-25100 to RAK.
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L.E. Williams and R.A. Kennedy: Photosynthetic Carbon Metabolism in Zea
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Received 31 March; accepted 9 June 1978
