Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis.

Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.

Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit nepsilon-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes methylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body. Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity. However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37. Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity. Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco.

Related alphaN- and epsilonN-methyltransferases methylate the large and small subunits of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is methylated at the ax amino position of the N-terminal methionyl residue of the processed and assembled form of the small subunit (SS), and is also methylated in some species at the epsilon-amino group of lysine-14 in the large subunit (LS). The gene (rbcMT-S) and cDNAs for the SS alphaN-methyltransferase (SSMT) from spinach (Spinach oleracea) have been cloned, sequenced, and expressed. The gene is closely related to a previously characterized LS methyltransferase (Rubisco LSMT) cDNA from pea (Rubisco LSMT) and a Rubisco LSMT gene from tobacco. Sequence analysis of the cDNA and transcript mapping experiments demonstrate that the rbcMT-S pre-mRNAs experience alternative 3' splice site selection, such that mRNAs for a long form with a four amino acid insertion and a short form are expressed at approximately equal abundance. The coding sequence of spinach SSMT includes a putative targeting presequence with sequence identity at a plastid processing site. A N-terminal truncated form of spinach SSMT was expressed and purified from E. coli cells. Both long and short forms of the cDNAs were shown to catalyze methylation of the a amine of the N-terminal methionine of the SS of Rubisco.

Functional analysis of the Rubisco large subunit ÂN-methyltransferase promoter from tobacco and its regulation by light in soybean hairy roots.

The ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) large subunit (LS) ɛ N-methyltransferase (Rubisco LSMT) catalyzes post-translational methylation of the ɛ-amino group of lysine-14 in the LS of Rubisco. The entire nucleotide sequence for the tobacco (Nicotiana tabacum) Rubisco LSMT (rbcMT-T) gene including the putative promoter region was recently reported, and sequence analysis of the promoter region revealed seven GT-1 motifs. The ability of several truncated rbcMT-T promoter fragments to confer light responsiveness to reporter gene expression in transgenic soybean (Glycine max) hairy roots was examined. Chimeric constructs consisting of the rbcMT-T promoter fused to a bacterial ß-glucuronidase (GUS) reporter gene and transferred to soybean via Agrobacterium rhizogenes were evaluated. The rbcMT-T promoter fragments conferred expression of the reporter gene in transgenic hairy roots, callus, and cell suspension cultures based on histochemical and fluorometric GUS analyses. The results suggest a quantitative role for the number of GT-1 motifs in determining differential expression between light and dark conditions.

Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase gene in tobacco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) epsilon N-methyltransferase (Rubisco LSMT) catalyzes the posttranslational methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco in many higher plant species including tobacco. The tobacco Rubisco LSMT gene (rbcMT-T) and its cDNA were isolated, sequenced, and characterized. The gene contains 6 exons and spans about 6 kb. Primer extension analysis indicated one transcription start site located 93 nt upstream of the translation initiation site. Sequence analysis of the 5'-flanking region suggests several potential binding sites for transcription factors, including 7 GT-1 elements and an HSP-70.5 element. Gene dosage analysis by Southern hybridization demonstrated that the tobacco rbcMT-T gene is present as a single copy in the tobacco haploid genome. The full-length cDNA for tobacco rbcMT-T is 1974 nt in length excluding the 3' poly(A)15 tail, and encodes a 491 amino acid polypeptide with a molecular mass of ca. 56kDa. The deduced amino acid sequence of tobacco Rubisco LSMT has 64.5% identity and 75.3% similarity with the sequence of pea Rubisco LSMT, and both proteins contain several copies of a conserved imperfect leucine-rich repeat motif.

Affinity purification of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT is described and characterized. Spinach (Spinacia oleracea L. cv Melody) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyvinylidene fluoride (PVDF) transfer membranes (Immobilon-P) and used as a ligand for the affinity purification of Rubisco LSMT from pea leaf extracts and chloroplast lysates. Pea Rubisco LSMT specifically bound to PVDF-immobilized spinach Rubisco but not to control PVDF membranes which contained immobilized BSA or pea Rubisco. Rubisco LSMT was not eluted by 1 M KCl but was specifically released by S-adenosyl-L-methionine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rubisco, and was therefore more efficient than elution by the competitive ligand spinach Rubisco. An increase in the specific activity of Rubisco LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein with a molecular mass of approximately 60 kDa.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) N-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco. With limited internal amino acid sequence information obtained from HPLC-purified peptic polypeptides from Rubisco LSMT, a full-length cDNA clone was isolated utilizing polymerase chain reaction-based technology and conventional bacteriophage library screening. The 1802 bp cDNA of Rubisco LSMT encodes a 489 amino acid polypeptide with a predicted molecular mass of ca. 55 kDa. A derived N-terminal amino acid sequence with features common to chloroplast transit peptides was identified. The deduced sequence of Rubisco LSMT did not exhibit regions of significant homology with other protein methyltransferases. Southern blot analysis of pea genomic DNA indicated a low gene copy number of Rubisco LSMT in pea. Northern analysis revealed a single mRNA species of about 1.8 kb encoding for Rubisco LSMT which was predominately located in leaf tissue. Illumination of etiolated pea seedlings showed that the accumulation of Rubisco LSMT mRNA is light-dependent. Maximum accumulation of Rubisco LSMT transcripts occurred during the initial phase of light-induced leaf development which preceded the maximum accumulation of rbcS and rbcL mRNA. Transcript levels of Rubisco LSMT in mature light-grown tissue were similar to transcript levels in etiolated tissues indicating that the light-dependent accumulation of Rubisco LSMT mRNA is transient. This is the first reported DNA and amino acid sequence for a protein methylase III enzyme.

Posttranslational modifications in the amino- terminal region of the large subunit of ribulose- 1,5-bisphosphate carboxylase/oxygenase from several plant species.

A combination of limited tryptic proteolysis, reverse phasehigh performance liquid chromatography, Edman degradative sequencing, amino acid analysis, and fast-atom bombardment mass-spectrometry was used to remove and identify the first 14 to 18 N-terminal amino acid residues of the large subunit of higher plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas reinhardtii, Marchantia polymorpha, pea (Pisum sativum), tomato (Lycopersicon esculentum), potato (Solanum tuberosum), pepper (Capsicum annuum), soybean (Glycine max), petunia (Petunia x hybrida), cowpea (Vigna sinensis), and cucumber (Cucumis sativus) plants. The N-terminal tryptic peptide from acetylated Pro-3 to Lys-8 of the large subunit of Rubisco was identical in all species, but the amino acid sequence of the penultimate N-terminal tryptic peptide varied. Eight of the 10 species examined contained a trimethyllysyl residue at position 14 in the large subunit of Rubisco, whereas Chlamydomonas and Marchantia contained an unmodified lysyl residue at this position.

Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase.

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P(2) carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-(3)H]-l-methionine to spinach ribulose-P(2) carboxylase. (3)H-Methyl groups incorporated into spinach ribulose-P(2) carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P(2) carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P(2) carboxylase LS (lysine) ;N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P(2) carboxylase and exhibited apparent K(m) values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P(2) carboxylase, a V(max) of 700 picomoles CH(3) groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P(2) carboxylase LS (lysine)(epsilon)N-methyltransferase was capable of incorporating 24 (3)H-methyl groups per spinach ribulose-P(2) carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P(2) carboxylase LS, but was inactive on ribulose-P(2) carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg(2+) and CO(2)) and unactivated forms of ribulose-P(2) carboxylase as substrates. However, complexes of activated ribulose-P(2) carboxylase with the reaction-intermediate analogue 2'-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P(2) carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS (epsilon)N-methyltransferase.

Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis.

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P(2) carboxylase under catalytic conditions (in the presence of CO(2), Mg(2+), and ribulose-P(2)) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO(2) and Mg(2+) only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

Early Inhibition of Photosynthesis during Development of Mn Toxicity in Tobacco.

Early physiological effects of developing Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) were examined in glass-house/water cultured plants grown at high (summer) and low (winter) photon flux. Following transfer of plants to solutions containing 1 millimolar Mn(2+), sequential samplings were made at various times for the following 9 days, during which Mn accumulation by leaves increased rapidly from approximately 70 on day 0 to approximately 1700 and approximately 5000 microgram per gram dry matter after 1 and 9 days, respectively. In plants grown at high photon flux, net photosynthesis declined by approximately 20 and approximately 60% after 1 and 9 days, respectively, and the onset of this decline preceded appearance (after 3 to 4 days) of visible foliar symptoms of Mn toxicity. Intercellular CO(2) concentrations and rates of transpiration were not significantly affected; moreover, the activity of the Hill and photosystem I and II partial reactions of chloroplasts remained constant despite ultimate development of severe necrosis. Though the activity of latent or activated polyphenol oxidase increased in parallel with Mn accumulation, neither leaf respiration nor the activity of catalase [EC 1.11.1.6] and peroxidase [EC 1.10.1.7] were greatly affected. These effects from Mn toxicity could not be explained by any changes in protein or chlorophyll abundance. Additionally, they were not a consequence of Mn induced Fe deficiency. Therefore, inhibition of net photosynthesis and enhancement of polyphenol oxidase activity are early indicators of excess Mn accumulation in tobacco leaves. These changes, as well as leaf visual symptoms of Mn toxicity, were less severe in plants cultured and treated at low photon flux even though the rates of leaf Mn accumulation at high and low photon flux were essentially equivalent.

Evidence for Effects on the in Vivo Activity of Ribulose-Bisphosphate Carboxylase/Oxygenase during Development of Mn Toxicity in Tobacco.

The progressive decrease in net photosynthesis accompanying development of Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) is a result of effects on in vivo activity of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco, EC 4.1.1.39). This conclusion is supported by: (a) decrease in rates of CO(2) depletion during measurements of CO(2) compensation, (b) increase in leaf RuBP concentrations, (c) progressive decreases in rate-constants of RuBP loss (light to dark transition analyses) with progressive increases of leaf Mn concentrations, and (d) restoration of diminished rates of net photosynthesis to control rates by elevated CO(2) (5%). Moreover, elevated CO(2) (1100 microliters per liter) during culture of Mn-treated plants decreased elevated RuBP concentrations to control levels and alleviated foliar symptoms of Mn toxicity. These effects of Mn toxicity on in vivo activity of rubisco were not expressed by in vitro kinetic analyses of rubisco prepared under conditions to sequester Mn or to adsorb polyphenols or their oxidation products. Similarly, the in vitro activity of fructose bisphosphatase (EC 3.1.3.11) was unaffected by Mn toxicity.

Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.

Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], but the stoichiometry of binding of the reaction-intermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was resistant. Three tryptic peptides were isolated and characterized after digestion of the activated enzyme; the tryptic-sensitive sites were identified at Lys-8, Lys-14, and Lys-466 of the large subunit. Tryptic digestion of the enzyme complexed with the reaction-intermediate analogue released only two peptides by hydrolysis at Lys-8 and at Lys-14. The loss of susceptibility of Lys-466 to trypsin may be the result of a conformational change that limits the accessibility of the carboxyl-terminal region after binding of the reaction-intermediate analogue. Analysis of the amino-terminal tryptic peptide by composition and fast atom bombardment mass spectrometry demonstrated that the actual amino-terminal residue is proline at position 3 of the DNA-deduced sequence and that this proline is blocked with an N-acetyl moiety. Thus, posttranslational processing of the chloroplast-encoded large subunit of the enzyme must occur to remove Met-1 and Ser-2 and to acetylate the amino terminus.

Effect of Triacontanol on Chlamydomonas: I. Stimulation of Growth and Photosynthetic CO(2) Assimilation.

Treatment of Chlamydomonas reinhardtii cells, cultured at 5% CO(2), with 1 to 1000 micrograms triacontanol (TRIA) per liter resulted in 21 to 35% increases in cell density, 7 to 31% increases in total chlorophyll, and 20 to 100% increases in photosynthetic CO(2) assimilation. The increase in CO(2) fixation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least one order of magnitude above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca(2+) and K(+) present in the Chlamydomonas growth medium. These ions caused flocculation of the colloidally dispersed TRIA in apparent competition with binding of [(14)C]TRIA to Chlamydomonas cells. Octacosanol inhibited the effect of TRIA on photosynthetic CO(2) assimilation. TRIA treatment did not alter the distribution of (14)C-label among photosynthetic products. The effect of TRIA on photosynthetic CO(2) assimilation increased with time after treatment up to 3 days. Chlamydomonas cells that had been grown at low-CO(2) (air) did not respond to TRIA, and transfer of high-CO(2) (5%) grown cells that had responded to TRIA to a low-CO(2) atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic CO(2) assimilation indicated that CO(2) is probably the species of inorganic carbon utilized by control and TRIA-treated Chlamydomonas cells.

Effect of Triacontanol on Chlamydomonas: II. Specific Activity of Ribulose-Bisphosphate Carboxylase/Oxygenase, Ribulose-Bisphosphate Concentration, and Characteristics of Photorespiration.

Increased photosynthetic CO(2) assimilation by Chlamydomonas reinhardtii cells treated with triacontanol (TRIA) was not due to changes in glycolate excretion, CO(2) compensation point, or the sensitivity of photosynthetic CO(2) assimilation to O(2). Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic CO(2) assimilation was a result of an increase in the apparent V(max) for intact cells. The total activity of ribulose-P(2) carboxylase/oxygenase was higher in cell lysates from TRIA-treated cells. However quantification of this enzyme concentration by binding of [(14)C]carboxyarabinitol-P(2) did not show an increase in TRIA-treated cells. Thus, there was an increase in the specific activity of ribulose-P(2) carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect on the activity of the enzyme in cell lysates from Chlamydomonas or purified from spinach (Spinacia oleracea L.) leaves.The ribulose-P(2) pool was 50 to 60% higher in cells treated with TRIA that were assayed for photosynthetic CO(2) assimilation at high- and low-CO(2). TRIA also increased ribulose-P(2) levels in the absence of CO(2) in the light with atmospheres of N(2) or N(2) with 21% O(2).