oxygenase in leaves of C3, C4, and C3−C4 intermediate species of Flaveria (Asteraceae)

Biochem. Physiol. Pflanzen 179, 253-268 (1984)

Photosynthetic Enzyme Activities and Immunofluorescence Studies on the Localization of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase in Leaves of C3, C4, and C3-C4 Intermediate Species of Flaveria (Asteraceae) HERMANN BAUWE

Zentralinstitl1t fur Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, DDR - 4325 Gatersleben Key Term Index: C4 photosynthesis, C3 -C 4 intermediate species, photosynthetic enzymes, enzyme compartmentation, immunofluorescence; Flavcria spec.

Summary Activities of ribulose-1,5-bisphosphate carboxylase (rubisco), phosphoenolpyruvate carboxylase (PEPC), aspartate aminotransferase, alanine aminotransferase, NADH-malate dehydrogenase, NADPH-malate dehydrogenase, NAD-malic enzyme, NADP-malic enzyme, and pyruvate phosphate dikinase (PPDK) were measured in leaf extracts of Fla.veria cronquistii (C 3 ), F. pubescens, F. anomala (C 3 -C4 ), F. brownii, F. palmeri, F. trinervia, and F. australasica (C 4). The two C3-C4 intermediate species show activities of PEPC, both aminotransferases, NADPH·malate dehydrogenase, NADPmalic enzyme, and PPDK intermediate between those of C3 and C4 Flaveria species. The Km values of rubisco for CO 2 exhibit distinct differences with higher values for the C4 species. PPDK is light activated reaching its maximum activity at noon in F. brownii and, in contrast, very early after the beginning of the light phase in F. pubescens. PEPC from both F. palmeri, F. trinervia, and F. australasica is activated about twofold by NADH but there is no effect to the enzyme of all other Flaveria species. This activation might be a more general featnre of PEPC from NADP-malic enzyme type C4 plants. An immunofluorescence investigation of the intercellular compartmentation of rubisco revealed an intermediate distribution of this enzyme in leaves of F. pubescens and F. anomala. Typical immunofluorescence patterns with nearly exelusive localization of rubisco in the mesophyll or bundle sheath cell chloroplasts, respectively, were found in the C3 and C4 Flaveria species. The data are discussed with respect to the evolution of C4 photosynthesis from C3 photosynthesis via C3 -C 4 intermediate types of photosynthetic carbon assimilation.

Introduction

Since the discovery by KENNEDY and LAETSCH (1974) of photosynthesis characteristics intermediate between the Ca and C4 type of carbon assimilation of higher plants in Mollugo verticillata there is a growing number of reports about cases of Ca-C4 intermediacy also in species from other genera. Up to date, Ca-C4 intermediate species are Abbreviations: Ala-AT, alanine aminotransferase; Asp-AT, aspartate aminotransferase; FITC, fluorescein isothiocyanate; NAD(P)H-MDH, NAD(P)H-malate dehydrogenase; NAD(P)-ME, N AD(P)-malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate phosphate dikinase; rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; Tn' CO 2 compensation concentration at 21 % O2 17 Biochem. Physio!. Plianzen, Bd. 179

254

H.BAUWE

known in at least four genera, Mollngo, Panicnm, Moricandia, and Flaveria. Today, the term "Ca-C4 intermediacy" is well established in photosynthesis research to reflect the fact that the border lines between C3 and C4 photosynthesis are not so clear cut as it was assumed in the early seventies. Primarily, this term often is used to describe the existence of modifications of the photosynthetic gas exchange which are most easily recognized by measuring the CO 2 compensation concentration of actively photosynthesizing leaves. The Ca typical range for the CO 2 compensation concentration at 21 % 02 (r21 ) is above 40 ,ull-l whereas under normal conditions the corresponding value for C4 plants falls below 10,uII-l. Species with r 21 between these two ranges and which, in addition, show a biphasic oxygen dependence of the CO 2 compensation concentration with a C4 1ike oxygen response below 10 to 15 % O2 and C3 typical behaviour above 20 % 02 are called C3-C4 intermediate species. Although this definition is restricted to only one parameter of CO 2 exchange and, of course, there is a number of other features which can be expected to be expressed in some plants in a Ca-C4 intermediate manner, e.g., leaf anatomy, enzyme activities, kinetic properties and compartmentation of enzymes, etc., it is quite suitable to use r 21 as a primary criterion for the classification into Ca, C4 , and Ca-C4 intermediate c~rbon assimilation type. Currently, the metabolic base of Ca-C4 intermediate gas exchange is subject to intensive research work. Up to now, most studies have dealt with Panicum milioides which is still the best characterized intermediate species both biochemically and by gas exchange measurements (BROWN and BROWN 1975; KESTLER et al. 1975; KECK and OGREN 1976; Ku et al. 1976; Ku and EDWARDS 1978; EDWARDS et al. 1982). In contrast to earlier work RATHNAM and CHOLLET (1978, 1979a, 1979b) in a series of papers presented evidence for the existence of a limited C4 pathway of CO 2 assimilation in this species. These results, however, very recently have been questioned both by the same laboratory (HOLADAY et al. 1982) and by others (EDWARDS et al. 1982). Besides work with P. milioides now there is also an increasing amount of investigations concerning gas exchange and biochemistry of carbon metabolism as well as the evolution of Ca-C4 intermediate species in Moricandia (Brassicaceae, APEL and OHLE 1979; BAUWE 1979a and 1983; BAUWE and APEL 1979; HOLADAY et al. 1981 and 1982). This genus, however, is only partly suitable for a study of the evolution of the C4 pathway of photosynthesis because according to current knowledge it includes no C4 species. As to Mollugo, it was questioned recently that any Mollugo species is a C4 plant (ZIEGLER et al. 1981). Therefore, we have started to investigate photosynthetic carbon assimilation and related features in several species of Flaveria (Asteraceae). Flaveria, besides Panicum, belongs to those very few genera which are known today to include both Ca, C4 , and Ca-C4 intermediate species and, therefore, are especially well suited for studies concerning details of the evolution of C4 photosynthesis. The systematics of Flaveria is well known from recent work by POWELL (1978) and earlier studies by JOHNSTON (1903) and RYDBERG (1915). There are also some reports about leaf anatomy (SMITH and TURNER 1975), carbon isotope discrimination, and gas exchange (APEL and MAASS 1981) in Flaveria. Enzyme data up to now are completely lacking.

Enzyme Activities and Localization in Flaveria Species

255

In this paper I present a survey of activities of severa] enzymes of potentia] importance in C4 photosynthetic carbon assimilation under inclusion of one Ca species (F. cronquistii), two Ca-C4 intermediate species (F. pubescens and F. anomala), and four C4 species (F. brownii, F. palmeri, F. trinervia, and F. australasica). From these data which are supported by an estimation of kinetic parameters and intercellular distribution of rubisco it is concluded that Ca-C4 intermediate species of Flaveria, in contrast to those of both Panicum and Moricandia, possess a sufficiently and Ca-C4 intermediately active enzyme complement for C4 photosynthetic carbon assimilation which might allow CO 2 to be fixed partially by a limited NADP-malic enzyme type CIl pathway. Material and Methods Plants were grown in a glasshouse under natural illumination as previously described (BAUWE 1984). Enzyme extraction and activity measurements were performed as described earlier (BAUWE 1984) with the exception of NAD-ME which was extracted and assayed according to HATCH et al. (1982). The optical assay system for PEPC contained (in mM): 50 Tricine-KOH, pH 8.0; 5 sodium glutamate; 2.5 dithioerythritol; 6 Mg C1 2 ; 3 tricyclohexyl-ammonium phosphoenolpyruvate; 0.2 NADH, and 1 unit malate dehydrogenase. For the determination of K m values for CO 2 and O2 rubisco was purified (BAUWE 1979b) and kinetic measurements were performed following the recommendations given by LORIMER et a!. (1977). Rubisco content of leaves was determined electrophoretically accord-. ing to BAUWE (1979b). The amount of chlorophyll in the extracts was determined in 80% acetone (AnNON 1949). For immunofluorescence studies small leaf pieces (about 2 x 5 mm) were fixed for 24 h in a mixture of 50 % ethanol, 5 % acetic add and 3 % formaldehyde. Thereafter, the pieces were transferred into paraffine by subsequent immersion into 70 % ethanol, 96 % ethanol, propanol, xylene, and paraffine-saturated xylene for 24 h in each case followed by paraffine for 48 h at 58 cC. The pieces were embedded into paraffine blocks and 10 {tm thin leaf cross sections were cut with a microtome and mounted on standard microscope slides. The paraffine was removed completely by subsequent washes in xylene for 15 min and in propanol, 96% ethanol, 70% ethanol, and 40% ethanol for 5 min in each case followed by 0.2 M potassium phosphate in 0.1 M NaCl, pH 7.5, for 10 min. After immersion once more for 10 min into the phosphate buffered saline the mounted sections were incubated for 1 h at room temperature with 1: 10 diluted anti-barley-rubisco serum from rabbits. After washing with phosphate buffered saline for 15 min with three changes of buffer the sections were covered with fluorescein isothiocyanate (FITC) labelled anti-rabbit-immunoglobulin solution (Staatliches Institut fur Immunpraparate und Nalmnedien, Berlin, G.D.R.) and incubated for 1 h in the dark. After further 3 washes with phosphate buffered saline (each time for 5 min) the cross sections were covered with 50 % glycerol and immunofluorescence was observed within some hours with a "Fluoval" epifluorescence microscope set up with narrow band filters for FITC fluorescence. Permanent visual records were kept as colour transparencies from ORWO-CHROM reversal film DT 18. From these transparencies black and white copies were made on negative film ORWO NP 15 and enlargements were prepared under carefully controlled and standardized conditions.

Results and Discussion

During their survey of Asteraceae SMITH and TURNER (1975) found Kranz anatomy in 4 species of Flaveria. Subsequent studies by BROWN (cf. POWELL 1978) recognized the Kranz syndrome in 5 further species and furthermore led to the conclusion that certain Flaveria species (e.g., F.oppositifolia, F.linearis, F. floridana) are intermediate 17'

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Fig. 1. Immunofluorescent labeling of ribulose-] ,5-bisphosphate carboxylase/oxygenase in leaf crosssections of Flaveria cronquist-ii (C3 ).

Cross-sections of 10/-tm thickness were cut with a microtome from paraffine embedded small leaf pieces. Fluorescent labelling was performed by subsequent incubation with anti-barley ribulose-l,5bisphosphate carboxylase/oxygenase from rabbits and fluorescein isothiocyanate labeled anti-rabbit Immunoglobulin. Black and white enlargements were kept from colour transparencies. 200 x,

between Kranz and non-Kranz anatomy and, therefore, seem to be in the process of evolution from non-Kranz to Kranz physiology. Detailed experiments to locate individual enzymes of C4 photosynthesis at the cellular level nearly exclusively have been performed after cell separation mainly on plant leaves exhibiting typical Kranz anatomy. Applying fluorescent antibodies against rubisco to thin leaf cross-section HATTERSLEY et al. (1977) have provided additional evidence for the localization of rubisco in C4 plant leaves and extended these studies also to plants with non-classical Kranz-anatomy. In P. milioides they could show that rubisco occurs in chloroplasts of both mesophyll and bundle sheath cells. We have used a very similar immuno-labeling approach differing from the above mentioned technique only by the preparation of leaf slices by microtome cutting of paraffine embedded leaf sections. In F, cronquistii rubisco is exclusively located in the mesophyll cell chloroplasts (Fig. 1). Bundle sheath cells of this species contain some chloroplasts, too, which, however, do not exhibit any specific fluorescence. In contrast, F. pubescens (Fig. 2) and F. anomala (Fig. 3) leaf cross-sections show specific fluorescence in both mesophyll and bundle sheath cells indicating the localization of rubisco in both cell types. In F. palmeri (Fig. 4) and F. trinervia (Fig, 5) rubisco is nearly exclusively localized in the bundle sheath cell layer with only slight fluorescence in the mesophyll cells which was weak relative to

Enzyme Activities and Localization in Flaveria Species

257

Fig. 2. Immunofluorescent labeling of ribulose-l ,5-bisphosphate carboxylase/oxygenase in leaf crosssections of Flaveria pubescens (0 3 -04 ), 200 x.

Fig. 3. Immunofluorescent labeling of ribulose-l,5-bisphosphate carboxylase/oxygenase in leaf crosssections of Flaveria anomala (0 3 -04 ), 200 x.

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Fig. 4. Immunofluorescent labeling of ribulose-l,5-bisphosphatc carboxylase/oxygenase in leaf crosssections of Flaveria palmeri (04). 200 x.

Fig. 5. Immunofluorescent labeling of ribulose-l,5-bisphosphate carboxylase/oxygenase in leaf crosssections of Flaveria trinervia (04). 200 x.

Enzyme Activities and Localization in Flaveria Species

259

Fig. 6. Immunofluoreseent labeling of ribulose-l,5-bisphosphate carboxylase/oxygenase in leaf eross· sections of Flaveria browl/ii (0 4 ). 200 x.

Fig. 7. Autofluorescenee of leaf eross-sections of Flaveria brownii. lOllm thick leaf cross-sections were prepared as for the immunofluorescent labeling, but, after reo moving of the paraffine they were rinsed with phosphate buffered saline only and mounted in 50 % glycerol. 200 x .

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Fig. 8. Fluorescence of normal serum-treated leaf cross-sections of F. brownii. 10,urn thick leaf cross-sections were prepared as for immunofluorescent labeling, followed by incubation with normal serum (instead of anti·barley ribulose-1,5-bisphosphate carboxylase/oxygenase) and FITe-labeled anti-rabbit immunoglobulin. 200 x.

that in the bundle sheath and only somewhat more intensive relative to the normal serum control. The occurrence of more or less intensive non-specific fluorescence is a general problem in immunofluorescence work with plant material which often ncgatively influences the quantitative evaluation of immunofluoresccnce patterns. Therefore, we generally have compared the fluorescence of specifically labelled leaf cross-sections with the fluorescence of non-serum treated cross-sections and a normal serum control. In contrast to the technique used by HATTERS LEY et al. (1977) the technique used here leads to preparates completely devoid of chlorophyll, Therefore, the typical red chlorophyll fluorescence was lacking. Nevertheless, for all species there was a weak non-specific fluorescence in both the autofluorescence control and the normal serum control very similar in colour to the FITC fluorescence. Taking this into account, to prevent any additional artefacts it was necessary to control carefully all conditions during the reproduction of black and white negatives from our colour transparencies and during the subsequent black and white enlargement procedure. As examples, Figs. 6-8 demonstrate the differences in fluorescence intensity between the two controls and anti-rubisco-serum treated leaf cross-sections from F. brownii. There is a slight but distinct autofluorescence (Fig. 7). which is only somewhat increased by treatment with normal serum folIowed by FITC-Iabelled anti-rabbit-immunoglobulins (Fig. 8) but distinctly lower than the rubisco specific fluorescence (Fig. 6). Nevertheless, this nonspecific fluorescence limits thc quantitative evaluation of the specific fluorescence distribution and prevents a definite conclusion about the occurrence of rubisco in the

261

Enzyme Activities and Localization in Flaveria Species

mesophyll chloroplasts of C4 Flaveria species. F. brownii is the only C4 representant of the Plaveria species investigated so far with an additional cell layer surrounding the bundle sheath and containing significant amounts of rubisco. The main bulk of rubisco in this species is concentrated in the bundle sheath, too. Possibly, this special spatial arrangement can be taken as an indication of a still incomplete transfer of Calvin cycle enzymes to the bundle sheath in the sense that F. brownii is one of the more ancient C4 species of Flaveria. This interpretation corresponds with some data given by POWELL (1978) who classified F. brownii as perennial C4 species in contrast to the annual C4 species F. palmeri, F. trinervia, and F. australasica. Possibly, it corresponds also with the close relationships between F. brownii and F. floridana (F. floridana belongs to those Flaveria species which are intermediate between Kranz and non-Kranz anatomy) as assumed by POWELL (1978). In all C4 and C3-C4 intermediate Flaveria species the chloroplasts of the bundle sheath cells are located centripetally. According to GUTIERREZ et al. (1974) this arrangement of bundle sheath chloroplasts is typical for dicotyledonous C4 species of the NADPME type. Our data support the findings mentioned above regarding a state of leaf anatomy intermediate between Kranz and non-Kranz anatomy for some Flaveria species. Furthermore, they show that C3 -C4 intermediate leaf anatomy at least in F. pubescens and F. cmomala is connected with a C3-C4 intermediate distribution of rubisco. Table 1. Chlorophyll content and chlorophyll ajb ratios in leaves of C3 , C3 -C4 intermediate, and C4 species of Flaveria. Numbers in parentheses refer to the number of experiments. Data were calculated according to AR~o~ (1949)

Species (Classification)

g Chi m- 2

ChlajChlb

F. cronquistii F. pubescens F. anomala F. broU'nii F. palmeri F. trinervia F. australasica

0.62 (7) 0.46 (7) 0.43 (9) 0.44 (5) 0.46 (7) 0.46 (10) 0.54 (3)

2.51 2.63 2.80 2.98 3.02 3.07 3.44

(C 3 ) (C 3 -C4 ) (C 3 -C4 )

(C4 ) (C4 ) (C4 ) (C4 )

(7) (7) (9) (5) (7)

(10) (3)

Table 1 summarizes chlorophyll contents and chlorophyll alb ratios of Flaveria leaves. The leaf area related chlorophyll content is significantly higher in F. cronquistii as compared with all C3-C4 intermediate and C4 Flaveria species except F. australasica where the chlorophyll content is relatively high, too. Chlorophyll alb ratios in Flaveria reflect the well known relative reduction in chlorophyll b with decreasing 21 (CHANG and TROUGHTON 1972, e.g.). Ratios for the two C3-C4 intermediates are between F. cronquistii and the C4 Flaveria species. The relative reduction in chlorophyll bin NADPmalic enzyme species is thought to be associated with a corresponding reduction of total chlorophyll associated with the light-harvesting-chlorophyll-protein and, consequently, a higher portion of chlorophyll in the chlorophyll-protein-complex I (HILLER

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Table 2. Leaf content, K m values for CO z and 0z, and whole leaf extract act£vity of rubisco from Ca,

Ca-C4 l:ntermediate, and C4 species of Flaveria.

Numbers in parentheses refer to the number of experiments. Kinetic data and activities were determined at 25 DC. n.d. means not determined. Species (Classification)

ribulose-1,5-bisphosphate carboxylase-oxygenase Leaf content

F. cronquistii F. pubescens F. anomalc! F. brownii F. palmeri F. trinervia F. australasica

(C a) (C a-C4) (C a-C 4 ) (C 4 ) (C4 ) (C4 ) (C4 )

K m values

Whole leaf extract activity

g m- z

fiJI CO 2 fdI0 2

flmol mg Chl- 1 h- 1

1.9 (3) 0.9 (:2) 1.2 (2) 0.6 (1) O.G (2) 0.6 (3) n.d.

25 (7) 24 (11) 24 (4) 31 (8) 32 (5) 28 (7) 49 (3)

3GO (6) 3GO (5) 480 (7) 260 (4) 180 (6) 190 (8) 230 (3)

360 (G) 460 (8) n.d. 450 (5) 340 (1) 360 (5) 430 (3)

and GOODCHILD 1981). Furthermore, lower chlorophyll alb ratios indicate a favouring of noncyclic over cyclic photophosphorylation (ED\VARDS and HUBER 1981). If this should be true for Flaveria C4 species, too, one might expect also intermediate ATP/ NADPH requirements of F. anomala and F. pubescens carbon assimilation which possibly is balanced via a limited NADPH generation by action of NADP-malic enzyme. Rubisco data are shown in Table 2. The decrease in rubisco content with decreasing F21 connected with intermediate values for F. pubescens and F. anomala completely corresponds to tendencies which can be observed also with Ca and C4 species from other genera (Ku et al. 1979, e.g.). Rubisco activities are relatively high in Ca and Ca-C4 intermediate Flaveria species as compared with the C4 species and fall in a Ca typical range. K m values for oxygen of rubisco yielded no large differences, but, the K m values for CO 2 of rubisco (as measured in a 60 s fixcd-time-assay) show distinct differences. The Km-values for CO 2 of rubisco from F. cronquistii, F. pubescens, and F. anornala fall in a relatively narrow range around 25,uM CO 2 , The K m values for the enzyme from F. brownii, F. palmeri, and F. trinervia with about 30,uM are somewhat higher. Rubisco from F. austmlasica which according to POWELL (1978) belongs to the most derived specics in Flaveria, exhibits the lowest CO 2 affinity with a K m of 49,uM CO 2 , This value is similar to those recently mcasured with rubisco from C4 Gmmineae of the NAD-ME and NADP-ME types of C4 photosynthesis (YEOH et al.1980). These authors argue that following evolution of C4 plants from Ca ancestors with high CO 2 affinity of rubisco, with development of an over-riding CO 2 concentrating mcchanism, the cnzyme's CO 2 affinity decreased again. Interestingly, accepting this argumcntation, all C4 Flaveria species investigated here seem to have reduced the CO 2 affinity of their rubisco to a more or less extent. This could be due to a more efficient equipment of these species relative to Ca and Ca-C4 intermediate Flaveria species to concentrate CO 2 and/or to prevent CO 2 leakage from the bundle sheath as proposed in the paper cited above. Extending this line of argumentatiOll, F. australasica could be regarded as one of those C4

F. F. F. F. F. F. P.

cronquistii pubesccns anomala brownii palmeri trinervia auslralasica

Species PEPO optical assay 24 (5) 39 (5) 70 (7) 460 (4) GOO (6) 560 (8) 870 (3)

PEPO 140-assay

19 (G) 37 (5) 90 (7) 490 (4) 350 (6) 290 (8) 460 (3)

89 (G) 340 (5) 380 (7) 910 (4) 580 (G) 440 (8) 700 (3)

Asp-AT

Enzyme activities in f£mol h -1 mg Ohl- 1

170 (G) 340 (5) 490 (7) 1430 (4) 1200 (6) 710 (8) 1100 (3)

Ala-AT

4340 (6) 5920 (5) 4580 (6) 4270 (3) 2G30(5) 2190 (6) 3230 (3)

NADH· MDH 19 (6) 54 (5) 87 (6) 270 (3) 300 (5) 220 (6) 230 (a)

NADPHMDH

NADP-ME

15 (6) 25 (5) 69 (7) 230 (4) 190 (6) 230 (8) 240 (a)

NAD-ME

13 (3) 20 (4) 17 (4) 21 (5) 20 (3) 15 (5) 27 (a)

Table 3. Activities of enzymes of potential imporlance in 0 4 photosynthesis in Flaveria. Numbers in parentheses refer to the number of experiments. All activities were measured at 25 °0 with whole leaf extracts.

3 (3) 14 (7) 9 (4) 44 (9) 43 (5) 44 (5) 54 (4)

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Flaveria species which had completed the evolution of a fully effective mechanism of concentrating CO 2 at the ribulose-1,5-bisphosphate carboxylating site in the bundle sheath chloroplasts relatively early as compared with other C4 species in Flave1'ia. It will have to be examined whether the Km for CO 2 of rubisco can be used as an indication of the intrageneric evolutionary age of different C4 species. The lack of a corresponding change in the oxygen affinity of rubisco suggests a relative constancy of the active site of rubisco with regard to the oxygenase reaction as opposed to a greater evolutionary flexibility of the carboxylase reaction. Possible selective advantages of the decrease in CO 2 affinity of rubisco during evolution of C4 photosynthesis in certain genera have been discussed by YEOH et al. (1980). Flaveria seems to be a suitable object for an experimental check of the existence of such advantages. Table 2 also contains whole leaf extract activities of the carboxylase activitity of rubisco. The activities in Ca and Ca-C4 intermediate Flaveria species are distinctly higher than in the C4 species. Activities of further enzymes, especially of those of potential importance in C4 photosynthesis are shown in Table 3. Chlorophyll related PEPC activities of F. anomala and F. pubescens are intermediate between those of F. cronquistii and the C4 Flaveria species. Notably, PEPC in F. anomala is about twice as active as in F. pubescens and four times as active as in F. cronquistii. This corresponds to a similar trend with respect to both the NADP-MDH and NADP-ME. As to the PEPC, apparently there are differences in kinetic properties of this enzyme in Flaveria species of different photosynthesis type. According to the activity ratios measured in two different assay systems, a 14C02 fixation fixed-time assay and an optical assay system including NADH and additional NAD-MDH, respectively, one can subdivide into two groups of PEPC isoenzymes. F. cronquist'ii, F. anomala, F. pubescens, and F. brownii yielded activity ratios of about one whereas in all other C4 Flaveria species investigated during this study the activities are nearly two times higher in the optical assay. This seems to be a more general feature of PEPC from NADP-malic enzyme C4 species. In contrast to the NAD-ME species Eleusine coracana and Atriplex spongiosa where the activity ratios are about one in both cases (substrate saturated activities are 890 Jlmol mg Chl-1 h-1 and 630 Jlmol mg Chl- 1 h- 1 , respectively) PEPC from the NADP-ME species Digitaria sanguinalis and Zea mays yielded activity ratios of 12 and 3, respectively, (substrate saturated activities in the optical assay are 1,500 JlMol mg Chl- 1 h-1 and 700 Jlmol mg Chl-1 h-1, respectively). Obviously, this behaviour is due to an activating effect of NADH. Addition of 0.2 M NADH to the HC-assay system leads to a similar increase of PEPC activity from F. trinervia, F. palmeri, and F. austmlasica in contrast to F. brownii where NADH addition is without effect. With respect to this feature PEPC from F. brownii is more closely related to the Ca and Ca-C4 intermediate type of PEPC in Flaveria. This relationship corresponds to a phylogenetic scheme of evolution of the genus Flaveria proposed by POWELL (1978) on the base of both morphological and ecogeographical considerations and interspecific cross experiments. In this scheme, F. brownii beside F. pubescens and several further species - classified as Ca plants on the base of carbon isotope discrimination and the lack of Kranz anatomy - is the only C4 species with 5 to 6 phyllaries whereas all other C4 Flaveria species and F. anomala belong to a second line

Enzyme Activities and Localization in Flaveria Species

265

with only 3-4 phyllaries. As already mentioned F. bt'ownii is also the only perennial C4 species of the genus which perhaps can be taken as an indication of a relatively ancient origin. By the same author it was concluded that C4 metabolism seems to have evolved at least twice in Flaveria. Our data support this sub-classification of C4 Flaveria species and show that evolution probably also led to the appearance of isoenzymes of PEPC with different catalytic and regulatory properties adapted to the new function in C4 photosynthetic carbon assimilation. NADP-ME is the dominant decarboxylating enzyme in Flaveria C4 species as well as in the C3-C4 intermediate species. It is most active with Mn2+ in contrast, e.g., to maize where Mg2+ is more effective. Mn2+ dependent activities of this enzyme in F. pubescens and F. anomala are intermediate between the activities in F. cronquistii and in the C4 Flaveria species. NAD-malic enzyme activity is very low without any group-specific differences. This activity is not stimulated by 8 mM ammonium sulfate as NAD-ME from most NAD-ME species. Therefore, C4 species of Flaveria have to be classified most appropriately as NADP-ME type C4 plants. This classification contrasts to the high activity of both aspartate and alanine aminotransferase. Normally, these two aminotransferases are most active in NAD-ME type C4 plants where they are involved in the amination of oxaloacetic acid derived from the PEPC reaction and in balancing the fluxes of amino nitrogen between mesophyll and bundle sheath cells. Although both aminotransferases function also in NADP-ME type and in phosphoenolpyruvate carboxykinase type C4 plants as well as in C3 species their activities in non-NAD-ME species normally are very low. Insofar, the photosynthetic carbon metabolism in C4 Flaveria species possible resembles that of Gomphrena celosioides. This species has been classified as NADP-ME type C4 species, however, like Flaveria C4 species it contains high Asp-AT and Ala-AT activities. Despite the high activity of both aminotransferases malate is the major C4 acid labeled initially after a 14C0 2 pulse and label was lost rapidly from the C-4 of malate during a chase with 12C0 2 like in other NADP-malic enzyme type plants (HATCH et al. 1975; REPO and HATCH 1976). Pyruvate phosphate dikinase drives the synthesis of phosphoenolpyruvate and, therefore, is an essential part of the C4 pathway of carbon assimilation. Flaveria C3-C4 intermediate species contain low but distinctly higher activities of PPDK than F. cronquistii which are about 20% of those found in C4 Flaveria species. This is different to C3-C4 intermediate species of other genera. C3-C4 intermediate species of Moricandia have C3 typical very low activities of PPDK (BAUWE 1984) and in the case of P. milioides there are several contradictory reports. Whereas RATHNAM and CHOLLET (1979a) reported about a relatively high capacity of this pathway of phosphoenolpyruvate biosynthesis these measurements very recently in accordance with our own results (about 2,umol mg Chl-1 h-1) were questioned by several authors including CHOLLETS laboratory itself (EDWARDS et al. 1982; HOLADAY et al. 1982). At low rates, especially in coupled assay systems it is sometimes difficult to derive definite conclusions about the enzymic base of the observed reaction. Therefore, to get additional certainty, we have examined light activation of PPDK in F. pubescens and F. brownii (Fig. 9). In F. brownii the PPDK activity raises from zero at the transition from dark to

266

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.

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of F.

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Plants were kept overnight in a greenhouse under a black cloth which was removed at zero time (about 8 a.m.). Thereafter the plants were illuminated by sun-light.

light (the plants were kept overnight under a black cloth) up to a maximum of about 70,umol mg Chl-1 h-1 at noon. In F. pubescens the initial activity is zero, too, but maximum activitiy (about 9-10,umol mg Chl-1 h-1) is reached very fast already 1 h after the onset of illumination. Thereafter, the activity remains nearly unchanged. It needs to be elucidated, if this increase in activity is caused by de novo synthesis of PPDK itself, of an activating protein factor or, alternatively, whether PPDK is subject to energy charge regulation (SUGIYAMA and HATCH 1981). Our data show that the Ca-C4 intermediate F. pubescens exhibits light activation qualitatively similar to C4 plants. This property is supplemented by the intermediate level of maximum activity of PPDK in both Ca-C4 intermediate Flaveria species dealt with in this paper. Summarizing the data presented here it can be stated that Flaveria seems to be the only genus known up to date to include Ca-C4 intermediate species with an intermediate between Ca and C4 plants enzymatic potential of phosphoenolpyruvate regeneration via this C4 typical pathway. This feature is complemented by a Ca-C4 intermediate expression of the capacity also of other reactions of potential importance in C4 photosynthesis including PEPC, Asp-AT, Ala-AT, NADP-ME, and NADPH-MDH. One can conclude that F. anomala and F. pubescens are "ideal" Ca-C4 intermediate species in the sense defined by RATHNAM-CHAGUTURU (1981), i.e., they show a whole set of structural, biochemical, and physiological traits definitively half-way between those exhibited by Ca and C4 species.

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Acknowledgements I gratefully acknowledge the technical assistance by Mrs. UTE RIEDEL, Mrs. ELlS FRAUST, and Mrs. HEIDI HAUGK. I am also indebted to Dr. RENATE MANTEUFFEL for preparation of the antiserum.

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Received June 15, 1983; accepted September 21, 1983 Author's address: Dr. HERMANN BAUWE, Zentralinstitut fUr Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, DDR - 4325 Gatersleben. CorrensstraBe 3