Regulation of Carbon Partitioning to Respiration during Dark ...

Received for publication October 10, 1989 and in revised form January 11, 1990

Plant Physiol. (1990) 93, 166-175 0032-0889/90/93/0166/1 0/$01 .00/0

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum' David H. Turpin*, Frederik C. Botha, Ronald G. Smith, Regina Feil, Anne K. Horsey, and Greg C. Vanlerberghe Department of Biology, Queen's University, Kingston, Ontario K7L 3N6 Canada (D.H.T., R.G.S., R.F., A.K.H., G.C.V.); and Department of Botany, University of the Orange Free State, Bloemfontein, South Africa (F.C.B.) cytosolic G6P, which is thought to stimulate sucrose phosphate synthase activity, increasing sucrose synthesis (34). Although providing us with insights into the partitioning of carbon to sucrose, this work has not considered the regulatory mechanisms responsible for partitioning of mobilized starch down glycolysis both for use as a respiratory substrate and for provision of intermediates in biosynthetic reactions. It is well established that nitrogen exerts a major influence on respiratory carbon metabolism (1, 9, 15, 16, 31, 35-37, 41). The assimilation of NH4' enhances starch breakdown and the flow of carbon into TCA cycle intermediates which are then used in amino acid biosynthesis. At present there is no model which provides an integrative picture of the mechanism by which NH4' assimilation regulates this carbon partitioning. In the present study we report changes in cellular metabolites during the dark assimilation of NH4' by the Nlimited green alga Selenastrum minutum. We also report preliminary data on the localization and regulation of PFK, PFP, and FBPase in this organism. Together, these results allow us to propose an integrative model for the control of carbon partitioning from starch to respiration during NH4' assimilation by this organism.

ABSTRACT The assimilation of NH4 causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoonolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH4 assimilation causes a transient 60% increase in fructose2,6-bisphosphate. Given the sensitivity of the cytosolic fructose1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid

MATERIALS AND METHODS Materials The green alga Selenastrum minutum (Naeg.) Collins (UTEX 2459) was cultured in N03-limited chemostats as previously described (8). The medium was buffered to pH 8.0 with 25 mM Hepes-KOH. The steady-state growth rate of cells for metabolite experiments was 0.3 d-' and for enzyme extraction and purification was 1.2 d-'. Sodium pyrophosphate, ATP, and Fru-2,6-P2 were from the Sigma Chemical Company, while auxiliary enzymes and other cofactors were from Boehringer-Mannheim Biochemicals. DEAE-Sephacel, Qsepharose and Sephadex G-25 were obtained from Pharmacia Fine Chemicals. P- 11 cellulose phosphate was obtained from Whatman. The IgG fraction of antibodies against potato PFK (anti-PFK46) was purified (24) and supplied by N. J. Kruger (Rothamsted, UK). The IgG against cucumber plastid PFK was obtained and purified as described previously (6).

biosynthesis.

Over the last decade there has been a significant increase in our knowledge of the control of carbon partitioning from starch to sucrose in photosynthetic tissue (34). In the proposed models, most of the carbon exported from the chloroplast is TP (see Table I for complete list of abbreviations). In the cytosol, Fru-2,6-P2 plays an important role in regulating the conversion of TP to sucrose through its effects on cytosolic FBPase and PFP. Decreased Fru-2,6-P2 levels result in an increase in FBPase activity and a decrease in PFP activity. These changes favour gluconeogenesis, causing a build-up in

Extraction and Partial Purification of Enzymes All procedures were carried out at 4°C. Approximately 15 g fresh mass of S. minutum cells were suspended in extraction

Supported by the Natural Sciences and Engineering Research Council of Canada. 166

REGULATION OF CARBON PARTITIONING DURING N ASSIMILATION

Table I. Abbreviations Used in This Paper TP, Triosephosphate Fl P, Fructose-1 -phosphate F6P, Fructose-6-phosphate FBP, Fructose-1 ,6-bisphosphate Fructose-2,6-bisphosphate Fru-2,6-P2, FBPase, Fructose-1,6-bisphosphatase (D-fructose1,6-bisphosphate 1 -phosphohydrolase EC 3.1 .3.1 1) Gi P, Glucose-1 -phosphate G6P, Glucose-6-phosphate Glu, Glutamate Gln, Glutamine PDC, Pyruvate dehydrogenase complex PGA, 3-Phosphoglycerate PEP, Phosphoeno/pyruvate Pyr, Pyruvate PK, Pyruvate kinase PFK, 6-Phosphofructokinase (ATP: D-fructose-,6phosphate 1- phosphotransferase EC 2.7.1.11) PFP, Pyrophosphate dependent 6-phosphofructokinase (PPi,:D-fructose-6-phosphate 1phosphotransferase EC 2.7.1.90) 2-OG, 2-Oxoglutarate Sucrose phosphate synthase SPS,

buffer in a 1:1 ratio and lysed with two passes through a French pressure cell (18,000 psi). The extract was centrifuged at 25,000g for 20 min and the resulting supernatant used in all subsequent steps. PFK was extracted in a 100 mm sodium phosphate buffer (pH 7.2) containing 2 mM EDTA and 5 mM MgCl2. The protein fraction precipitating between 35 and 60% ammonium sulfate saturation was collected by centrifugation at 30,000g for 15 min. The pellet was dissolved in 10 mL extraction buffer and then dialyzed for 14 h against 20 mM sodium phosphate buffer (pH 7.5) containing 2 mm EDTA and 5 mM MgCl2 (buffer A). The extract was centrifuged at 30,000g for 30 min and the supernatant applied to a DEAESephacel column (1.5 x 20 cm) equilibrated with buffer A. The column was washed with three bed volumes of equilibration buffer and then developed with a 120 mL linear gradient of KCI (0-500 mM) in the same buffer. The flow rate was 60 mL- h-' and 1.5 mL fractions were collected. FBPase was extracted in a 100 mM Hepes-NaOH buffer (pH 7.5) containing 10 mM EDTA, 10 mM MgCl2, 10 mM DTT, and 2 mM PMSF. After centrifugation the supernatant was directly applied to a DEAE-Sephacel column (1.5 x 20 cm) equilibrated with 20 mm Hepes-NaOH buffer (pH 7.5) containing 2.5 mM EDTA, 2 mM MgCl2, and 2 mm DTT. The column was washed with one bed volume of equilibration buffer and then developed with a linear KCI gradient as described above. PFP was extracted as previously described (19, 23). In recovery experiments 1 IU of partially purified potato PFP (specific activity 6.8 ,umol.mg-' protein-min-') was added per g of cells prior to extraction. Enzyme Assays

All assays were carried out at room temperature in a total volume of 1 mL. Auxiliary enzymes were dialyzed against 5

167

mM Hepes-NaOH (pH 7.5) before use. NADP+ reduction and NADH oxidation were recorded at 340 nm. PFK was assayed in 100 mm imidazole (pH 7.2) containing 1 mI MgCl2, 0.1 mM NADH, 5 mm F6P, 0.5 mm ATP, 1 IU aldolase, 10 IU TP isomerase, and 1 IU glycerol-3-P dehydrogenase. The reaction was started with ATP. In experiments where the effect of Pi and/or PEP was determined, the PFK preparation was desalted on a Sephadex G-25 column equilibrated with 50 mM imidazole (pH 7.3) immediately prior to use. Immunoremoval of PFK from crude extracts as well as from the partially purified PFK preparations were done as previously described (6). FBPase was assayed in 100 mM HEPES-NaOH (pH 7.5) containing 16 mM MgSO4, 10 mM EDTA, 2 mM DTT, 0.5 mm NADP+, 0.4 mm FBP, 1 IU G6P dehydrogenase, and 2 IU hexose-P isomerase. The reaction was started with the FBP. PFP was measured as described by Kombrink et al. (23). Metabolite Extraction

All metabolite experiments were with cells in the dark. Cells were harvested from chemostats and concentrated by centrifugation (4400g, 7 min) and resuspended at approximately 60 ,ug Chl-mL-1 in the supernatant medium. They were then preincubated in the dark, while air bubbled and stirred for 40 min in a water-jacketed (20°C) cuvette. Following preincubation, 1 mL samples were taken at the times described in the results. NH4Cl (5 mM) was added at the time indicated and in most cases the next sample taken 5 s later. NH4+ remained saturating throughout the experiment. The samples were immediately frozen in liquid N2 and lyophilizedd The lyophilized samples were resuspended in a mixture containing 1.5 mL CHC13, 3.5 mL methanol, and 0.6 mL 20 mM Hepes (pH 8.5) with 5 mm EGTA, and 50mM NaF. They were then incubated at 4°C for 40 min. After adding 3 mL H20 the samples were centrifuged and the upper aqueous phase dried (10 min, 38°C) by rotary evaporation and resuspended in water. For most metabolites the reported values represent the means of duplicate experiments. ATP, ADP, and AMP were

analyzed from lyophilized samples of triplicate experiments which were extracted in 10% HC104 and neutralized with SN KOH/ 1 M triethanolamine. Amino acid analyses were carried out on samples from triplicate experiments which were immediately killed in 10% HC104 and neutralized as above. All metabolites were stored in liquid N2. Metabolite Analysis

Fru-2,6-P2 was measured enzymatically as outlined by Van Shaftingen et al. (40) using a Milton Roy Spectronic 3000 diode array spectrophotometer equipped with a multiple cuvette holder. The assay buffer contained 91 mM Tris Acetate (pH 8.1), 4.5 mm Mg-acetate, 2.1 mM F6P, 260 ,M NADH, and 520 ,M PPi. For each determination, two or three internal standards (0.0625-2.0 pmol) and a blank (derived from an acid-treated sample) were run simultaneously with the sample. (33; M Stitt, personal communication). The F6P was pretreated with HCI to eliminate any contaminating Fru-2,6-P2 (40). The PFP used in the assay of Fru-2,6-P2 was partially purified (235-fold) from 1 kg of potato (Solanum tuberosum)

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TURPIN ET AL.

tubers, using a P- l I Cellulose Phosphate column (Whatman) and a Q-Sepharose (1 x 6.5 cm) column (Pharmacia) (44). The active fractions of the latter column were collected and concentrated to 0.9 mL using an Amicon YM-30 Ultrafilter, and made to 25% (v/v) glycerol to be stored in liquid N2. A standard solution of Fru-2,6-P2 (Sigma) was added to some samples before lyophilization, at various times during the experiment. Recovery of this internal standard was 80.8 + 4.0% (SE, n = 6). Amino acids were analyzed by Dr. K. Joy (Carleton University, Ottawa) by HPLC of ortho-phthalaldehyde derivatives on a C- 18 reverse-phase column. The remaining metabolites were measured using standard coupled enzymatic assays and a dual-wavelength spectrophotometer (ZFP 22, Sigma Instruments, FRG). 2-OG was assayed as in Bergmeyer (2) using 100 mm phosphate buffer (pH 7.5), 17 mm NH4C1, 50 FM NADH, and starting the reaction by the addition of 1.2 U of Glu dehydrogenase. Malate, citrate, Glu and sucrose were measured as in Bergmeyer (2). ATP, ADP, AMP, F6P, FBP, GIP, G6P, PGA, PEP, Pyr, and TP were analyzed using previously reported techniques (28, 42).

Table II. Effect of Fru-2,6-P2, AMP, and Pi on the Activity of the Two lsoenzymes of FBPase in S. minutum FBPase activity was assayed at pH 7.5 in the presence of 40 Mm FBP, 16 mm MgSO4, and 2 mm DTT. Activity Treatment

Peak 1

Control 4 Mm Fru-2,6-P2 4 AM Fru-2,6-P2 + 0.5 mm AMP

10mMPi

Peak 2

nmol-mg-' protein-min-' 50 78 44 26 8 40 14 39

Table Ill. Effect of Pi and PEP on S. minutum PFK Activity at pH 7.2 PFK activity was measured in the presence of 0.5 mm ATP, 1 mM MgCI2, and 0.25 mm F6P. Treatment

Activity

Control 0.5 mM PEP 10 mM Pi 10 mM Pi + 0.5 mM PEP

AMnol-mg'1 protein -min- 1 11.5 2.8 29.0 22.0

Carbon Flow to Cell Walls Cells were incubated in the light for 3 h in the presence of 20 mM H'4C03- (1 ,uCi/,mol). H'4CO3- was then removed by bubbling with air in the presence of carbonic anhydrase. Cells were then incubated in the dark for 3 h in the presence or absence of NH4'. The cell wall fraction was then isolated according to Takeda and Hirokawa (38). Radiolabel incorporation into the cell wall fraction was determined using liquid scintillation counting. Other Methods

Chl concentrations were estimated from the lower CHC13 phase of the CHCl3/Methanol extractions and the phaeophytin content of the HC104 pellet in the adenylate experiments. Protein was measured as described by Bradford (4). When used, CCCP was added to a final concentration of 6 AM. RESULTS

FBPase, PFK, and PFP

Selenastrum minutum exhibited a total FBPase activity of 527 Amol mg-' Chl * h-'. The FBPase activity eluted as two separate peaks from a DEAE-Sephacel column at 0.1 M and 0.2 M KCI, respectively. Peak 2 of the FBPase activity was strongly inhibited by Fru-2,6-P2 (Table II). The inhibition was enhanced by AMP. In contrast peak 1 was only slightly inhibited by Fru-2,6-P2, was only marginally sensitive to AMP, but was strongly inhibited by Pi. This isozyme (peak 1) had a broad pH optimum; Vmax at pH 7.5 and 8.8 in the presence of high Mg2' and DTT concentration differed with less than 10% (FC Botha, unpublished data). This was in contrast to peak 2 where activity at pH 8.8 was 30% of that atpH 7.5. In contrast, only one symmetrical peak of PFK activity

(700 umol mg-' Chl h-') was found. PFK from crude extracts was analyzed on a DEAE-Sephacel column equilibrated in buffer A over a range of pH from 6.8 to 8.0 at 0.2 pH intervals. In all cases the PFK activity eluted as a single peak. In the absence of Pi the PFK activity was unstable and 80% loss in activity occurred in 24 h. In contrast, there was no loss of activity in the presence of 50 FM Pi. The PFK of S. minutum cross-reacted with both the anti-PFK46 and anti-PFKp from cucumber. The efficiency of the two sera in removing the PFK activity was similar in crude and partially purified PFK preparations (data not shown). The partially purified PFK has a pH optimum of 7.2 (not shown) and activity is stimulated by Pi (Table III). PEP strongly inhibits the enzyme in the absence of Pi. The inhibition by PEP is alleviated by increasing the Pi concentration (Table III). Using the extraction procedures of Kiss et al. (19) and Kombrink et al. (23), no PFP activity was detected. In addition no PFP activity was detected in any of the crude extracts or column fractions during isolation of PFK and FBPase. When purified potato PFP was added to the S. minutum cells prior to extraction, 98.6 + 2% recovery of the potato enzyme was found. Metabolite Analyses Amino Acids, 2-OG and Citrate Addition of NH4' resulted in a decline in Glu and 2-OG and an increase in citrate (Fig. 1). During ammonium assimilation there was a sustained increase in gln and alanine (Table IV) while other amino acids did not change appreciably during the course of the experiment.

Adenylates NH4' addition resulted in an immediate decline in ATP (Fig. 2). There was a slight recovery in ATP by 2 min but it

REGULATION OF CARBON PARTITIONING DURING N ASSIMILATION

169

(Fig. 5). During the first several minutes of NH4' assimilation there was a transient increase in Fru-2,6-P2. Within 5 min, however, the levels had declined to slightly lower than those before NH4' addition (Fig. 6). 0'

1.5

GlP and G6P

E

E

1.0

Both G1 P and G6P declined immediately following NH4'

NH+

addition (Fig. 7). By the end of the experiment G1P had returned to near control levels. G6P remained depressed throughout the experiment. GlP/G6P remained relatively constant over the first 15 min of NH4' assimilation but increased slightly by the end of the experiment.

0.5

300,

2--OG

2001

i

100-

Metabolite Concentrations Table V provides a crude estimate ofthe range ofmetabolite concentrations which might be expected in vivo. The assumptions required for these calculations are outlined in this table.

NH$

E 600

+

NH4~ E

|Citrate|

ai_

300*

0 -15

Cell Wall Synthesis Radiolabel associated with the cell wall component of control cells remained constant over the duration of the experiment (34.6 ± 1.0 x 106 dpm; + SE, n = 10). In contrast, NH4' assimilation caused a 10% increase (3.4 ± 0.1 x 106/dpm; ± SE, n = 10) in radiolabel associated with cell walls over that in control treatments.

1

-10

5

o

lo

5

15

20

25

TIME (min)

Figure 1. Changes in glutamate, 2-oxoglutarate and citrate during dark ammonium assimilation.

remained below control levels for the duration of the experiment. ADP and AMP increased upon NH4+ addition. ADP returned to control levels within 2 min. AMP declined rapidly initially but did not return to control levels for 25 min (Fig. 2).

CCCP Experiment CCCP (6 ,lM) caused a 40% increase in ADP within 5 s of addition. This coincided with both a drop in PEP, an increase in Pyr and a 2.6-fold increase in the Pyr/PEP ratio.

DISCUSSION The onset of N assimilation results in major increases in respiratory carbon flow (9, 14, 41). The increase in Pyr/PEP (Fig. 3) and FBP/F6P (Fig. 5) indicate that the activity of PK and the conversion of F6P to FBP increase during N assimi-

TP, PGA, PEP, and Pyruvate

NH4' assimilation resulted in

rapid drop in PEP to approximately 15% of its original value (Fig. 3). This, combined with a transient increase in Pyr within 5 s of NH4' addition, resulted in a 6- to 8-fold increase in the ratio of Pyr/ PEP (Fig. 3). Over the duration of the experiment Pyr slowly declined towards its control value. PGA decreased from about 500 nmol -mg-' Chl to less than 200 nmol. mg-' Chl over a time frame similar to that of PEP (Fig. 4). TP increased approximately 3.5-fold to levels as high as 140 nmol mg-' Chl (Fig. 4), but its response was somewhat slower than PGA, PEP, and Pyr. a

F6P, FBP, and Fru-2,6-P2

NH4' assimilation initiated a drop in F6P and an increase in FBP (Fig. 5). These changes took longer to occur than those of PEP and pyruvate but resulted in an eight-fold increase in the FBP/F6P ratio 5 min following NH4' addition

Table IV. Changes in Amino Acids during Dark NH4' Assimilation The initial concentration represents the average of four determinations over a 20 min period prior to NH4+ addition for each of three experiments (n = 12). Each other time represents the average of three experiments (n = 3). Time after NH4+ addition (min) Amino Acid"

0

2

5

10

15

20

Mmol-mg' Chl Gln