Photosynthesis Research 4, 151-1 70 (1983} © 1983, Martinus Ni]hoff/Dr W. Junk Publishers, The Hague. Printed in The Netherlands

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying COz compensation points

JOCELYNE ASCENCIO* and GEORGE BOWES

Department of Botany, University of Florida, GainesviUe, FA, USA

(Received 28 October 1982)

Key words: Aquatic plants, carboxylating enzymes, CO2 compensation ponts, enzyme kinetics, PEP carboxylase

Abstract. Incubation of the submersed aquatic macrophyte, Hydrilla verticillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions pro- duced high (130 to 150t~1 CO2/1) and low (6 to 8/A CO2/1) CO 2 compensation points iF), respectively. The activities of both ribulose bisphosphate (RuBP) and phos- phoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high I' plants to 0.2 in low F plants. These ratios resemble the values in terrestrial C3 and C4 species, respectively.

Kinetic measurements of the PEP carboxylase activity in high and low 17 plants indicated the Vmax was up to 3-fold greater in the low F plants. The Km (HCO~) values were 0.33 and 0.22mM for the high and low F plants, respectively. The Km (PEP) values for the high and low F plants were 0.23 and 0.40 mM, respectively; and PEP ex- hibited cooperative effects. Estimated Km (Mg 2+) values were 0.10 and 0.22 mM for the high and low F plants, respectively.

Malate inhibited both PEP carboxylase types similarly. The enzyme from low F plants was protected by malate from heat inactivation to a greater extent than the enzyme from high P plants. The results indicated that Ca acid inhibition and protection were not reliable methods to distinguish C3 and C, PEP carboxylases. The PEP carboxylase from low F plants was inhibited more by NaCI than that from high I' plants. These analyses indicated that Hydrilla PEP carboxyiases had intermediate characteristics between those of terrestrial C 3 and C, species with the low F enzyme being different from the high F enzyme, and closer to a C4 type.

Resumen. Ai incubar la macrofita acufitica sumergida (MAS) Hydrilla verticillata Royle, por un perfodo de hasta 4 semanas en cfimaras de crecimiento con condiciones seme- jantes a l a s de invierno (bajas temperaturas y dfas cortos) o de verano (altas tem- peraturas y dins largos), se originaron cambios en el punto de compensaci6n de CO2 (r) de las plantas a valores de 130 a 150~1CO~/1 y 6-8/~1 CO2/I, respectivamente. Las actividades tanto de la RiBP carboxilasa como de la PEP carbordlasa aumentaron durante la ineubaci6n, pero el aumento m~ts significativo fu6 de la actividad de la PEP carboxilasa en las plantas en la condici6n de verano. Este aumento reduj6 la relaci6n RiBP/PEP carboxilasa de 2.6 en las plantas con alto F a 0.2 en las plantas con bajo F. Estos valores se asemejan a los de especies C 3 y C4 terrestres, respectivamente.

*present address: Facultad de Agronomta, Departamento de Bothnica, Universidad Central de Venezuela, Maracay, Venezuela

151

152

Estudios de la cin6tica de la PEP carboxilasa en plantas de Hydrilla, indican una Vmax de la enzima en plantas con bajo F hasta tres veces mayor que la Vmax en plantas con alto F. Las Km (HCO~), Km (PEP) y Km (Mg 2÷) fueron diferentes en los dos tipos de plantas y la enzima mostr6 caractertsticas alost6ricas en presencia de uno de sus sub- stratos, PEP y del Mg : ÷.

La inhibici6n de la PEP carboxilasa de ambos tipos de plantas (alto y bajo F) fu6 similar en presencia de malato. E1 malato a su vez fu6 mils eficiente que el aspartato, en la protecci6n de la enzima de plantas con alto r, de su inactivaci6n pot altas temper- aturas. Los resultados indican sin embargo, que la inhibici6n de las PEP carboxilasas de especies C3 Y C4 en presencia de ~cidos de 4-C, no es un criterio aceptable para diferenciar entre las PEP carboxilasas de plantas C a y de plantas C 4 . La inhibici6n de la PEP carboxilasa en presencia de NaC1 fu6 un m6todo m~s preciso en la distincidn de ambos tipos de enzimas; la PEP carboxilasa de plantas de Hydrilla con bajo F mostr6 una mayor sensibilidad en presencia del NaC! que la enzima de plantas con alto F.

Estos estudios indican que la PEP carboxilasa de plantas de Hydrilla tienen carac- tertsticas que son intermedias entre la de las enzimas de especies terrestres C a y C 4 , siendo la enzima de las plantas con bajo I ~ mils parecida a la de las especies C4.

Introduction

It has been shown that environmentally related changes in the photosynthetic/

photorespiratory characteristics of submersed aquatic macrophytes (SAM species) occur in the lake and upon treatment of the plants in growth chambers [3]. These changes can be detected by changes in the F value of the

plants, which is higher in winter, or winter4ike conditions, than in summer or summer-like conditions [3, 13]. For Itydrilla, these changes have been cor- related with changes in the activity of PEP carboxylase, which appears to increase as the F value of the plant decreases [13].

The very low F values of terrestrial C4 species have been associated with

the activity of PEP carboxylase, which apparently acts as a CO2 pump to decrease the effect of CO: on the RuBP carboxylase/oxygenase in the bundle sheath cells. It has also been postulated (2) that in C4 species the PEP carboxylase refixes photorespired CO2, thereby keeping photorespiratory CO2 evolution to negligible proportions. Recently the importance of in vivo refixation in C4 species, as opposed to decreased photorespiratory CO2

formation has been questioned (29). The regulation and properties of PEP carboxylase from higher plants also

have been a source of increasing interest in recent years, including attempts to differentiate among alloenzymes of PEP carboxylase from plants belonging

to the various photosynthetic categories (Ca, C4 and CAM species). The importance of PEP carboxylase as a regulatory enzume has been

reported by several investigators. PEP carboxylase from higher plants show

cooperative properties in regard to one of the substrates, PEP [22, 37, 7, 33]. It also may exhibit allosteric properties with Mg 2÷, the major effector of this enzyme [33, 8]. Glucose-6-P is reported another allosteric effector of PEP

carboxylase. Oxaloacetate; malate, and aspartate, the end products of the PEP carboxylase and closely associated reactions, may function as important

153

controlling substances through feed-back inhibition of the PEP carboxylase reaction [9].

Further evidence for metabolic regulation had been demonstrated by examining the kinetic behavior of the enzyme, especially with regard to Michaelis-Menten hyperbolic plots and Hill coefficient values [18].

Different forms of the enzyme associated with different metabolic path- ways have also been reported. Ting and Osmond [34] have suggested that four different forms of the enzyme exist: a C4 photosynthetic PEP carboxy- lase; a C3 photosynthetic PEP carboxylase; a CAM type enzyme; and a non-green or nonautotrophic PEP carboxylase. These forms, which can be separated by DEAE column chromatography, show variety of kinetic pro- perties; for example, different 'Km' (HCO3), 'Km' (PEP), and 'Km' (Mg 2÷) values as well as different Vmax and Hill coefficients. More than one form of the enzyme has been reported to be present in some species. For example, Mukerjii and Ting [26] found three isoenzymes of PEP carboxylase in leaves of cotton. Hatch et al. [12] found two isoenzymes of PEP carboxylase in C4 Atriplex species.

In the CAM species Kalanchoe, Bryophyllum, Sedum and Opuntia, the kinetic properties appear to be remarkably constant, but different forms of PEP carboxylase have been differentiated by chromatography [34]. However, in the hybrids of Atriplex, produced by crossing C3 and C4 Atriplex species, the isoenzymes present in the hybrids exhibit the kinetic characteristics of the enzymes from both parents [34, 12]. In the CAM facultative species Mesembryanthemum crystallinum [11] treatment of the plant with NaC1 to induce the CAM mode caused a 15-fold decrease in the 'Km' (PEP). The authors suggested the possibility that these changes were associated with synthesis of a different isoenzyme of PEP carboxylase. In an analogous manner, synthesis of a new form of PEP carboxylase seems to occur during the development of the C4 photosynthetic pathway in greening maize tissue [34].

The inhibition of PEP carboxylase by a variety of metabolites has also been used to differentiate among the enzymes from different sources. Huber and Edwards [14] studied the competitive inhibition of PEP carboxylase by malate and aspartate; Rathman [28] examined the effects of several inhibitors in protecting the enzyme from heat inactivation and Osmond and Greenway [27] found that leaves of C4 species were extremely sensitive to high concen- trations of NaC1 whereas the enzyme extracted from leaves of C3 species was much less sensitive. The same response was later confirmed by Ting and Osmond [33] and later by Hatch et. al. [12].

This paper describes an investigation of the role that PEP carboxylase plays in controlling the variable CO2 compensation point (F) value of the submersed angiosperm Hydrilla verticiltata, and examines the various kinetic and regulatory properties of the enzyme from high and low F Hydrilla olants.

154

Material and methods

Plant material

The C3 species in the study were spinach (Spinacia oleraceae L.) and soybean (Glycine max L.); the C4 species were Sorghum bicolor (L.) Moench, slender- stem (Dig#aria pentzii Stent), and the SAM species Hydrilla verticillata Royle. The terrestrial C3 and C4 species were greenhouse grown. Hydrilla was col- lected throughout the year from Lakes Orange and Lochloosa, Florida, and used either immediately or after incubation for 10 days in growth chambers. Segments 10 to 15cm long were placed in a 5% v/v Hoagtand solution at pH 5.5 in gas washing bottles. The plants were incubated either in a growth chamber under a 14h 30°C day/20° C night or under a 9 h 12°C day/6° C night.

Enzyme assays

In all enzyme assays at least two different plant replicates were used and the assays were run in triplicate. For RuBP carboxylase 0.5 to 1.0 g fresh weight were ground with 50raM tris-HC1, 20mM MgC12, 0.1 mM EDTA and 5mM DTT at pH 8.5. Homogenates were centrifuged for 10min at 9,000g. The supernatants were used for the assays of enzyme activity in a solution con- taining at pH 8.5 50mM Tris-HC1, 20mM MgC12, 0.1 mM EDTA, 10mM DTT, 0.5mM RuBP, 20mM NaH14CO3 (0.2#Ci//aM) and 0.1 ml of leaf extract (previously activated) in a final volume of 1 ml. Reaction was halted after 2 rain with 0.1 N HC1 [23].

For the assays of PEP carboxylase activity, 0.5 to 1.0 g fresh weight were ground with 50mM Tris-HC1, 10mM MgC12, 0.1 mM EDTA, 10raM DTT, 5mM D4soascorbate and 1% w/v PVP-40 at pH 8.5. Homogenates were centrifuged at 9,000g and the supernatants used immediately for the assays of enzyme activity in a solution containing 50 mM Tris-HC1, 10 mM Nail 14 CO3 (0.2/2Ci//2M) and 0.1 ml of enzyme extract at pH 8.5. Reaction was halted after 4 min with 0.1 ml of 6N HCI saturated with 2,4-dinitrophenylhydrazine. Both RuBP carboxylase and PEP carboxylase were run at 30°C. Aliquots were placed in scintillation vials for radioactivity determinations by liquid scintillation spectroscopy.

In the determination of 'Kin' (PEP), 'Km' (HCO3) and 'Km' (Mg) different concentrations of PEP, Nail 14 CO3 and Mg were used in the assay solutions.

The degree of inhibition of PEP carboxylase activity by L-malic acid or L-aspartic acid [14] was determined using 0, 1.0, 5.0 and 10.0mM concen- trations of either malate or aspartate in the assay solution. In several experiments limiting concentrations of MgC12 and PEP, 2.0 and 0.3 raM, respectively, were used at pH 8.5 or 7.6.

The degree of inhibition of PEP carboxylase activity by NaC1 [ 12, 33] was determined using 0, 50, or 100 mM NaC1 in the assay solution. The grinding solution was as previously described except the pH was 7.6 and it contained

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Figure 1. The effect of HCO~ concentration on the activity of PEP carboxylase from Hydrilla plants with high and low F values.

2.0 mM MgC12. The assay solution was also at pH 7.6 with 2.0 mM MgC12 and 0.5 mM PEP.

The thermostability of PEP carboxylase in the presence and absence of C4 acids [28] was also tested. The supernatant from the leaf extract was in- cubated at either 40 or 55°C for 10 minutes with 10ram L-malate of L- aspartate, or water as a control. All treatments were then placed on ice and their PEP carboxylase activity determined immediately as previously described.

Infrared gas analyzer measurements

The CO2 compensation points (Y) of the plant were determined in a closed system as previously described (35).

Results

As was previously found by Bowes et al. [3] and Holaday [13], incubation of Hydrilla plants at 12 and 30°C with a short and long photoperiod, re- spectively, altered the P value of the plants (Table 1). Plants collected and measured directly from the lake had high Y values because collection was in the winte'r time. When incubated under the 12°C/9 h photoperiod the P of the plants did not significantly change. However, under the 30°C/14h photo- period there was a dramatic decrease in F even within 1 week. Table 1 also shows the activities of RuBP and PEP carboxylase as a function of incubation time under the two environmental treatments. Within the first week of

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Figure 2. Double reciprocal plot of the rate of ~4CO 2 incorporation versus HCO; con- centration for PEP carboxylase from Hydrilla plants with high and low I ~ values.

incubation there was some increase in RuBP carboxylase activity under both environmental treatments. In the treatment with a 12°C/9h photoperiod there was some indication of an increase in PEP carboxylase activity;however, in the treatment with 30°C/14h photoperiod PEP carboxylase showed a 9-fold increase in activity during the 3-week incubation period. Under the 12°C/9h photoperiod the RuBP carboxylase/PEP carboxylase ratio was around 1.5 and did not alter greatly during the incubation period. In contrast, the large increase in PEP carboxylase in the 30°C/14 h treatment resulted in a striking decrease in the ratio to 0.2 after 3 weeks.

Hydrilla plants with high (821xl CO2/1) and low 16/11 CO2/1) F values induced by incubation for 10 days were used to investigate in more detail the changes in PEP carboxylase that occurred during incubation.

The effect of HCO~ concentration on the PEP carboxylase from high and low F Hydrilla plants is shown in Figures 1 and 2. A hyperbolic curve, charac- teristic of enzymes that follow Michaelis-Mente.n kinetics, was observed for the enzyme from both plants. The Km (HCO~) value for the high F plants was somewhat higher than that for the low F plants (0.33 and 0.22mM HCO~, respectively). The Vmax values were 38.5 and 59.2/~mol CO2/mg Chl - h for the high and low P plants, respectively.

The effect of PEP concentration on PEP carboxylase is shown in Figure 3. As before, PEP carboxylase from the low F plants exhibited the highest reaction rate at all substrate concentrations except possibly the very lowest utilized, Saturation with PEP occurred at about 0.8 and 0.6 mM for the low

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and high I" plants, respectively. A Lineweaver-Burk plot (Figure 4) of the

values from Figure 3 showed significant deviations from a straignt line for both high and low P plants. This indicated a cooperative effect of the

159

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Figure 5. Hill plot of PEP carboxylase activity (expressed as log v/V-v) versus log PEP concentration for Hydrilla plants with high and low I" values V = the maximum velocity; and v = the rate at different PEP concentrations

substrate and prevented this type of graph from being used in calculations of the Km (PEP). Consequently the values were converted to a Hill plot (Figure 5). The Km (PEP) for the low P plants was almost twice that o f the high P plants (0.4 and 0.23 mM, respectively). The Vmax values were approximately 3-fold higher in the low [ ' plants as compared to the high P plants. Hill coefficients, (n), obtained directly from the slope of the line in the Hill plots can be used to determine the degree of substrate cooperativity. The n values were t .5 for low P plants and 1.6 for the high F plants. This indicated a similar positive cooperativity [30] between the substrate PEP and the enzyme from both types of plants.

Previous studies of the experimental parameters affecting the allosteric nature of PEP carboxylases from terrestrial plants have shown that a metal ion is required and that this requirement for a metal can be satisfied by either Mg 2+ or Mn 2+ [16]. Figure 6 shows the effect o f Mg 2+ on the PEP carboxylase from the low and high P Hydrilla plants at saturating substrate (PEP and HCO3) concentrations. The curves are somewhat sigmoidal although the effect of increasing Mg 2+ concentrations on the high F plants appears to be minimal; Ibr the tow F plants, Mg 2* concentrations up to 2.0 mM did not cause saturation. With both high and low 17 plants the enzyme rate was appreciably without any added Mg 2+.

It was thought that possibly the reason for a substantial reaction rate in the absence of added Mg 2+ could be the presence o f endogenous Mg z+ in the plant extract. Consequently, the crude extract was passed through a G-25 Sephadex column to remove any contaminating free Mg 2÷. Once again,

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Table 2. Kinetic parameters for PEP carboxylase from Hydrilla plants with high and low CO 2 compensation points (F)

Kinetic High r Low 17 Substrate parameters plants plants

HCO; Km 0.33 0.22 Vmax 38.50 59.20

PEP Km 0.23 0.40 Vmax 61.70 161.30 n 1.60 1.50

Mg 2. (crude extract) Km - 0.57 Vmax 42.40 96.80 n - 1 . 8 6

Mg 2÷ (sephadex treated) Km 0.05 0.14 0.22 Vmax 30.80 109.20 n 1.98 1.98

Note: Km values are expressed in mM and Vmax values in ~mol CO2/mg Chl. h. The high 17 value was 82 and the low F value 16#1 CO2/1.

despite the absence o f free endogenous Mg 2+, the enzyme rate was no t zero

in the absence o f added Mg 2+. Increasing Mg 2+ concent ra t ions had a greater

ef fec t on the react ion rate o f the low I ' plants and the rate was no t saturated

at 2 .0 raM Mg 2+. The react ion veloci ty o f the low F plants was higher at all Mg 2+ concen t ra t ions than that o f the enzyme from the high F plants.

Table 2 summarizes the kinet ic parameters for the PEP carboxylase from

high and low F plants. In all exper iments the low plants exhib i ted m u c h higher

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Vmax values. The Km values in all cases were different between high and low I" plants.

It has been reported in the literature that PEP carboxylase from terrestrial C3 species is not inhibited by malate or aspartate whereas the enzyme from terrestrial C4 species is substantially inhibited [14]. PEP carboxylase from high and low I" Hydrilla plants was inhibited by 5mM aspartate whereas the enzyme from the Ca and C4 terrestrial species showed no inhibition. The dif- ferent degrees of inhibition from these results are expressed as a histogram in Figure 7. For both malate and aspartate the enzymes from the two forms of Hydrilla tended to be more inhibited than the enzymes from terrestrial species.

It has been reported that PEP carboxylase from C4 species was effectively protected by malate or aspartate from heat inactivation and this has been suggested to be a method to differentiate between C3 and C4 PEP carboxy- lase enzymes [28]. The results of PEP carboxylase thermostability in high and low F Hydrilla as compared to terrestrial C3 and C4 species are shown in Table 3. Heat inactivation results are presented as percentages of the remaining activity. For all species aspartate provided negligible protection. Malate protected the enzyme from low F Hydrilla plants less than the enzyme from high F IIydrilla plants. The enzyme from the C4 Digitaria was com- pletely protected.

Another reported method to distinguish between the PEP carboxylase from terrestrial C3 and C4 species is the differential sensitivities of the

162

Table 3. Percentage of PEP carboxylase activity remaining after the enzyme was treated for 10 lnin with 10 mM malate or 10 mM aspartate at the temperature indicated

PEP Carboxylase activity as percentage of control (v/v)

Plant Material Temperature Malate Aspartate

C %

Hydrilla 55 60.1 0.11 (High F) Hydrilla 5 5 3 2.0 0.0 2 (Low r) Spinach 55 66.0 0.18 (C3) Soybean 55 34.3 0.92 (C3) Sorghum 55 2.7 0.02 (C4) Digitaria 55 104.7 0.00

Note: After treatment the enzyme was assayed as described in Material and methods.

enzymes to NaC1 [33, 12]. The PEP carboxylase from high and low P Hydrilla and also from spinach and Sorghum were assayed with limiting con- centrations of PEP and Mg z+ at pH 7.6 with 50 and 100mM NaCI (Table 4). At 50mM NaC1 the PEP carboxylase from both types of Hydrilla showed little or no inhibition. However, at a 100 mM NaC1 the PEP carboxylase from the low F Hydrilla was inhibited to a greater extent than the enzyme from the high P plant. The enzyme from the terrestrial Ca plants was more sensitive to NaC1 than that from the C3 plant when the activity was assayed either with 50 or 100mM NaC1.

Discussion

Previous work with submersed aquatic macrophytes has indicated that by changing the environment it is possible to change the photosynthet ic and photorespiratory characteristics o f these plants especially with regard to the relative rates at which the two processes operate. With the possible exception of facultative CAM species [32, 21, 20] this variability has not yet been re-

ported in terrestrial species. The present study has confirmed previous work by Bowes et al. [3] and

Holaday [13] on the relatively rapid nature of the change in F, and in addition, has shown that the change was maintained in the growth chambers for periods at least as long as four weeks. The F values of the high P plants were considerably higher than those typically found for terrestrial C3 species while F values, as low as 6/zl/CO2/1, for the low P plants approached the 0 to 5/.d/CO2 / 1 of terrestrial C4 species.

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A biochemical explanation for these large environmentally-induced alterations in P probably lies at least partially in the changes observed in the activity of the carboxylation enzymes. In both growth chamber treatments incubation of Hydrilla for one week produced a general increase in the activi- ties of both the carboxylation enzymes assayed but the increase in the PEP carboxylase activity under summer-type conditions was by far the most marked. The small general increase in carboxylation enzyme activity may be a consequence of the higher light and nutrient conditions in the growth chambers as compared to the lake especially with regards to RuBP carboxy- lase [36, 15]. The plants for these experiments were collected from the lake during the winter when Hydrilla typically grows at a depth where light penetration is low. Plants taken from the surface water of the lake during the summer do not exhibit much increase in RuBP carboxylase activity upon incubation (Ascencio, J. and G. Bowes, unpublished results). The changing light regime from lake to growth chamber cannot, however, explain the large differences in F and PEP carboxylase activity observed between the two growth chamber incubation treatments as the quantum flux density in both growth chambers was similar.

A major factor in the relationship between F and carboxylation activity appears to be the RuBP/PEP carboxylase ratio. Even though the activity of both RuBP and PEP carboxylases increases somewhat upon incubation, under the 12°C/9h photoperiod the RuBP/PEP carboxylase ratio was higher than 1.0 while under the 30°C/14 h photoperiod the ratio was as low as 0.2. A ratio of 4 to 21 is usually found in C3 species and a ratio of 0.05 to 0.56 occurs in C4 species (2). This indicates that the F values of the plants may be a conse- quence of the RuBP/PEP carboxylase ratio with lower ratios producing plants with a low P value. Whether this is the only factor involved cannot be ascertained from this study.

The changes in F were also associated with changes in the kinetic properties of PEP carboxylase. The most obvious difference between the PEP carboxy- lases from the high and low F plants was a large increase in the Vmax of the enzyme from the low F plants. The Vmax was 2- to 3-fold higher in the low F plants as compared to the high F plants.

It can be postulated then, that an increase in the Vmax of PEP carboxylase, relative to RuBP carboxylase, would potentially have several consequences. The proportion of CO~ being fixed into oxaloacetate hence into malate and/ or aspartate would increase at the expense of fixation into 3-P-glycerate via RuBP carboxylase. This would decrease the 02 inhibition of photosynthetic CO2 fixation as PEP carboxylase is not inhibited by 02 whereas RuBP carboxylase is inhibited [4]. A decreased 02 inhibition of photosynthesis has been found in the low P plants [ 1]. This rerouting of carbon would not in itself necessarily cause a reduction in P. A reduction in 12, solely as a result of an increased PEP carboxylase Vmax, would be likely due to more efficient refixation by PEP carboxylase of photorespired CO2 rather than decreased

165

photorespiratory CO2 formation. In terms of the two carboxylases, only a reduction in RuBP carboxylase/oxygenase activities could result in decreased carbon passing through the photorespiratory carbon oxidation cycle. From the data in Table 1 it is difficult to predict for each F value the relative con- tributions of refixation by PEP carboxylase as opposed to altered levels of RuBP oxygenase.

A further complicating factor is that with the high F plants (unlike terrestrial C3 species) the F value is not reduced to zero in low 02 [3]. This suggests that a 'dark' respiratory CO2 release process may also be a component of the P value.

The so-called C3-C4 intermediate species (such as Panicurn milioMes) also have P values which are lower than those of C3 species but not as low as C4 values being in the range of I3 [10] to 36//1 CO2/1 [17]. Hydrilla Pvalues can be in this range although as shown in Table 1 they may also be lower. The inhibition of photosynthesis by 02 in the C3-C4 intermediate is also less than that found with true C3 species [6]. The reduced P values and 02 inhibition in the C3-C4 intermediates have been attributed to an increased activity of PEP carboxylase [5]. The PEP carboxylase activity in these plants is higher than in C3 species but the RuBP carboxylase activity is still twice that of the PEP carboxylase [17]. The situation in Hydrilla with regard to PEP carboxylase appears to be far more extreme as the PEP carboxylase activity may be 3-fold greater than RuBP carboxylase. Furthermore, the C3-C4 inter- mediates so far described appear 'fixed' in that the F value of a particular plant is relatively constant. In Hydrilla, however, the F value of a particular plant can be easily and substantially altered.

Keck and Ogren [17] have questioned the importance of PEP carboxylase in lowering the 02 inhibition and P of the C3-C4 intermediates based on the fact that at F only 15% of the 14CO2 fixed is incorporated into C4 acids, such as malate and aspartate, and at 300 gl CO2 no C4 acids were synthesized [ 19]. They contend that changes in the activity and kinetics of RuBP carboxylase/ oxygenase may be as important as the increased PEP carboxylase activity. It appears unlikely that this criticism can be applied to Hydrilla, as firstly, the PEP carboxylase activity in the low F plants is substantially higher than the RuBP carboxylase, and secondly, over 60% of the 14CO2 initially fixed is incorporated into C4 acids [13].

The higher Vmax values of PEP carboxylase in the low F Hydrila plants may just be a consequence of an increased synthesis of the same enzyme found in the high F plants. However, no inferences can be drawn concerning the nature of enzyme by just examining Vmax values. At this stage two questions need to be posed: 1) Was there a different PEP carboxylase synthesized by the low F plants such as might occur by changing the com- position of isoenzymes of the C3 and C4 type? and 2) Do PEP carboxylases from the high and low P Hydrilla plants resemble the enzymes from C3 and C4 terrestrial species?

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167

An examination to the Km values with respect to HCO~, PEP, and Mg 2÷ for the PEP carboxylases from the high and low Y Hydrilla plants indicated that there were differences between the two plant types. This suggested that different enzymes were present. An analysis (Table 5) of the Km values for HCO3, PEP, and Mg 2+ (cited in the literature) for Ca and C4 terrestrial species [33, 7, 24] indicated lower values for the Km of the PEP carboxylases from Ca species as compared to C4 species. However, the range of variation among them is large and sometimes overlaps. The Km values for PEP carboxy- lase from both high and low YHydrilla plants were intermediate between the values for Ca and C4 terrestrial species. The high F plants showed lower values for PEP and Mg 2+ than the low F plants. This suggested that the PEP carboxy- lase from high Y Hydrilla was closer to the C 3 enzyme while the enzyme from the low Y Hydrilla closer to the C 4 type.

The Km (HCO]), however, was higher for the enzyme from the high 1 ~ plants than that from the low P plants. The significance of this fact may be interpreted in conjunction with the observed apparent Km (CO2) values for photosynthesis and the higher affinity for CO2 shown by the low P plants as compared to the high P plants.

Although it is known that the regulatory properties of PEP carboxylase change with assay conditions, only hyperbolic plots for HCO3 kinetics have so far been reported for terrestrial C3 and C4 species [37, 7, 33], and Hydrilla is no exception. However the kinetics with regard to PEP and Mg 2÷ appear to be more complex. Some workers [33, 16] have reported non- regulatory properties of the Michaelis-Menten type while others have found a regulatory or allosteric behavior of these substrates [8, 7, 33, 22]. These regulatory differences may be partly explicable by the different extraction and assay procedures utilized.

Although there is insufficient information concerning the regulatory function of PEP carboxylase in either C3 and C4 photosynthetic pathway, Ting and Osmond [33] showed that in Ca Atriplex the kinetics towards PEP and Mg 2+ were of the nonregulatory type while there were allosteric responses in the C4 Atriplex. No differences of this kind were detected between the enzymes from the high and low 1 ~ Hydrilla plants.

The possibility cannot be eliminated that a mixture of different isoenzymes of the C3 and C4 type are present in Hydrilla. This needs further investigation.

Although the protection by malate and aspartate of PEP carboxylase from heat inactivation has been reported to be a possible technique to distinguish between C3 and C4 PEP carboxylases [28], in this study it was not possible to differentiate between the terrestrial C3 and C4 species on this basis.

However, the fact that there was a significant difference in the response to this test of the enzymes from the high and low F Hydrilla plants is further evidence that the PEP carboxylases in the two Hydrilla types are different enzymes. This test, though, could not be used to assign them to a C3 and C4 enzyme category.

168

In regard to malate and aspartate inhibition, differences were again found among the terrestrial species tested as well as in high and low F Hydrilla plants. In the literature, however, it is suggested that only C4 species are inhibited by malate or aspartate [14]. The results of the present study con- tradict this. The PEP carboxylases from spinach (C3) and Sorghum (C4) were both inhibited by malate whereas the enzyme from Digitaria (C4) was un- affected by either malate and aspartate. Apparently this test also cannot be used to distinguish the two enzyme types. However, a significant difference was found in the degree of inhibition by aspartate of the PEP carboxylase from high and low I' Hydrilla plants suggesting that the enzymes are dif- ferent. The inhibition of PEP carboxylase by NaC1 was the most reliable of the three methods used to differentiate between the PEP carboxylases from terrestrial C3 and C4 species. The results in this study were consistent with the data reported in the literature which indicates that the enzyme from C4 species has a greater sensitivity to NaC1 [12, 33]. Also, the PEP carboxy- lase from the low F Hydrilla plants exhibited more inhibition than the enzyme from the high F plants. This may indicate that the enzyme from the low F plants more closely resembles the C4 enzyme while that from the high I" plants is closer to the C3 enzyme. The difference is response to NaC1 is further evidence that the enzyme f romthe two Hydrilla types are different.

From the results of this study it can be concluded that a dramatic change in photosynthesis/photorespiratory metabolism of Hydrilla is inducible by changes in the environment and that during the development of these metabolic changes the properties of PEP carboxylase in the plant apparently change. This alteration in the properties of PEP carboxylase enzyme can at least partially explain the different gas exchange characteristics of high and low I" Hydrilla plants. There are potentially two interacting effects in the PEP carboxylase changes: 1) An increased synthesis of PEP carboxylase under a higher temperature and a long photoperiod which would partly explain the increased Vmax in the low F plants, and 2) A change in the kinetic properties of the enzyme itself that reflects the different Km (HCOa), Km (PEP), and Km (Mg 2+) values in the high and low r' plants as well as the differential response of the enzymes in the presence of malate, aspartate, and NaC1. Both of these effects could be accounted for by the differential synthesis (and possibly degradation) of two isoenzymes of PEP carboxylase one exhibiting more C3 type properties and the other more akin to Ca. If the possession of isoenzymes by high and low F plants was not an 'either/or' response but rather one in which the plants contained variable proportions of the iso- enzymes then a continuous range of kinetic properties and P values could result. Whether PEP carboxylase (even from terrestrial species) can be definitely categorized into Ca and C4 types is open to question based on the results of this study.

169

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