Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Enzymatic Properties of Phosphoenolpyruvate Carboxylase Isoforms in the CAM Plant Kalanchoe daigremontiana

Three isoforms (Types 1, 2 and 3) of phosphoenolpyruvate (PEP)carboxylase in young leaves of the Crassulacean acid metabolism(CAM) plant Kalanchoe daigremontiana were separated by DEAE-cellulosecolumn chromatography and preparative polyacrylamide-agarosegel electrophoresis, and their enzymatic properties were characterized. All three isoforms had similar molecular weights of about 234,000.At pH 8.0 Type 1 showed a high affinity to PEP, (K_m=0.08 mM),whereas Type 3 showed a low affinity (K_m=1.0mM). K_m values forMgCl₂ were 0.26 HIM in Types 1 and 3 and 0.5 nut in Type 2.All three types exhibited the same pH optimum at 8.0, but Type1 showed relatively low activity below pH 6.0, whereas Type3 showed high activity. Type 3 was more acid stable than theother forms. In the presence of glucose-6-phosphate, the K_mvalues of Types 1, 2 and 3 for PEP lowered to 0.027, 0.037 and0.044 mu at pH 8.0, respectively. Inhibition of activity byorganic acids such as malate and pyruvate was pronounced inType 3. Type 2 exhibited properties intermediate to Types 1and 3 with regard to pH curve, affinity to PEP and its effectof various metabolites. The physiological significance of PEPcarboxylase isoforms in CAM plants is discussed on the basisof these findings. ¹Present address: Agricultural Chemicals Research Lab., SankyoCo., Ltd., Yasu-cho, Yasugun, Shiga 520-23, Japan. (Received November 30, 1983; Accepted March 24, 1984)     

Polypeptide Composition of Envelope Membranes Isolated from Chloroplasts of C3, C4, and CAM Plants

Chloroplast envelopes were isolated from chloroplasts purifiedfrom Spinacea oleracea L. (C₃), Panicum miliaceum L. (NAD-malicenzyme-type C₁), Digitaria sanguinalis (L.) Scop. (NADP-malicenzyme-type C₄), Kalanchoe daigremontiana Hamet et Perrier (constitutiveCAM), and from Mesembryanthemum crystallinum L. (inducible CAM)performing either C₃ photosynthesis or Crassulacean acid metabolism(CAM). For each species, methods were developed to isolate chloroplastenvelopes free of thylakoid contamination. The polypeptidesof ribulose bisphosphate (RuBP) carboxylase which has been consistentlyreported in envelope preparations of spinach were not foundin envelope preparations of C₄ mesophyll chloroplasts. Silverstaining of envelope polypeptides resolved electrophoreticallyon sodium dodecylsulfate polyacrylamide gradient slab gels produceda more complex profile than did Coomassie staining which haspreviously been used with C₃ envelope preparations, even thoughsilver reacted poorly with polypeptides corresponding to thesubunits of RuBP carboxylase. All of the plants examined possesseda major polypeptide of 27 to 29 kilodaltons (kD) which was previouslysuggested to be the phosphate translocator in spinach. WithC₃ M. crystallinum, the 29 kD polypeptide stained most intensely.After induction of CAM, a 32 kD polypeptide also stained intensely,giving a profile similar to that obtained with the constitutiveCAM species. A 32 kD polypeptide was also prominent in C₄ envelopepreparations, suggesting that a 32 kD polypeptide may be a translocatorprotein which is required in Crassulacean acid metabolism andC4 photosynthesis, but not in C₃ photosynthesis. (Received April 25, 1983; Accepted July 9, 1983)     

Novel Light-Dark Change of Proline Levels in Halophyte (Mesembryanthemum crystallinum L.) and Glycophytes (Hordeum vulgare L. and Triticum aestivum L.) Leaves and Roots under Salt Stress

Proline accumulation was determined in a facultative halophyte,Mesembryanthemum crystallinum and glycophytes, barley (Hordeumvulgare L.) and wheat (Triticum aestivum L.) Proline accumulationpreceded the shift of CAM in M. crystallinum and did not occurin the continuous darkness. The novel light-dark change of prolinelevel (high in the light and low in the dark) was observed inleaves of all three plants. Proline levels of shoots in barleyand wheat also showed the same light-dark change, suggestingthat proline accumulated in the leaves in the light was nottranslocated to other tissues in the dark period. These resultssuggest that proline has a bifunctional role in the acclimationto high salt stress; an osmoregulant role in the light, anda substrate for dark respiration to supply energy to compartmentationof ions into vacuole in the dark. ¹Present address: Kyoto Biological Res. Lab., Bio-Chiba Inc.Watsuka,Soraku, Kyoto, 619-12 Japan ²Present address: Kobayashi Pharmaceutical Co., Ltd. Doshomachi,Chuo-ku, Osaka, 541 Japan     

Cloning, Expression, and Characterization of a Root-Form Phosphoenolpyruvate Carboxylase from Zea mays: Comparison with the C4-Form Enzyme

A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase(PEPC) was isolated. In the coding region, the root-form PEPCshowed 76 and 77% identity with the C₄- and C₃-form PEPCs ofmaize, respectively, at the nucleotide level. At the amino acidlevel, the root-form was 81 and 85% identical to the C₄- andC₃-form PEPCs, respectively. The entire coding region was insertedinto a pET32a expression vector so that it was expressed underthe control of T7 promoter. The purified recombinant root-formPEPC had a V_max value of about 28 mol min^–1(mg protein)¹at pH 8.0. The K_m values of root-form PEPC for PEP and Mg²⁺were one-tenth or less of those of C₄-form PEPC when assayedat either pH 7.3 or 8.0, while the value for HCO^–₃ wasabout one-half of that of C₄-form PEPC at pH 8.0. Glucose 6-phosphateand glycine had little effect on the root-form PEPC at pH 7.3;they caused two-fold activation of the C₄-form PEPC. The K_i(L-malate) values at pH 7.3 were 0.12 and 0.43 raM for the root-and C₄-form PEPCs, respectively. Comparison of hydropathy profilesamong the maize PEPC isoforms suggested that several stretchesof amino acid sequences may contribute in some way to theircharacteristic kinetic properties. The root-form PEPC was phosphorylatedby both mammalian cAMP-dependent protein kinase and maize leafprotein kinase, and the phosphorylated enzyme was less sensitiveto L-malate. ¹These authors contributed equally to this work. ²Present address: Otsuka Chemical Co. Ltd., 463 Kagasuno, Kawauchi-cho,Tokushima, 771-0130 Japan. ³Present address: Sumitomo Pharmaceuticals Research Center,1-98, Kasugade, Naka 3-cho-me, Konohana-ku, Osaka, 554-0022Japan.     

A Mutant of Arabidopsis thaliana That Defines a New Locus for Glycine Decarboxylation

A novel photorespiratory mutant of Arabidopsis thaliana, designatedgld2, was isolated based on a growth requirement for abnormallyhigh levels of atmospheric CO₂. Photosynthetic CO₂ fixationwas inhibited in the mutant following illumination in air butnot in atmosphere containing 2% O₂. Photosynthetic assimilationof ¹⁴CO₂ in an atmosphere containing 50% O₂ resulted in accumulationof 48% of the soluble label in glycine in the mutant comparedto 9% in the wild type. The rate of glycine decarboxylationby isolated mitochondria from the mutant was reduced to 6% ofthe wild type rate. In genetic crosses, the mutant complementedtwo previously described photorespiratory mutants of A. thalianathat accumulate glycine during photosynthesis in air due todefects in glycine decarboxylase (glyD, now designated gld1)and serine transhydroxymethylase (stm). Because glycine decarboxylaseis a complex of four enzymes, these results are consistent witha mutation in a glycine decarboxylase subunit other than thataffected in the gld1 mutant. The two gld loci were mapped tochromosomes 2 and 5, respectively. ³Present address: Department of Crop and Soil Sciences, MichiganState University, East Lansing, MI 48824, U.S.A. ⁴Present address: Department of Applied Bioscience, Facultyof Agriculture, Hokkaido University, Kita-Ku, Sapporo, 060 Japan ⁵Present address: Department of Biology, Carnegie Institutionof Washington, 290 Panama Street, Standford, CA 94305, U.S.A.     

The participation of phytochrome in the signal transduction pathway of salt stress responses in Mesembryanthemum crystallinum L.

Continuous irradiation of Mesembryanthemum crystallinum plantswith light of equal amounts of photosynthetically active radiation,but widely different red:far red ratios was used to intervenein phytochrome-mediated signal transduction pathways in thepresence and absence of salt stress. Light with a low ratioof red:far red (in contrast to light with a high ratio of red:farred), caused induction of PEP carboxylase activity, accumulationof the CAM isoform of PEP carboxylase, and the accumulationof malate anion. Taking these as indicators of CAM inductionit is concluded that phytochrome can participate in the signaltransduction pathway leading to CAM in M. crystallinum. A lowratio of red: far red light acted synergystically with saltstress in the induction of these CAM indicators. The simplestinterpretation of this interaction is that the phytochrome-mediatedeffects and salt stress effects acted on the same signal transductionpathway. The accumulation of pinitol was also increased by light witha low ratio of red:far red, consistent with the existence ofa stress syndrome in M. crystallinum which utilizes a commontransduction pathway. A low ratio of red:far red light induced a strong shade avoidanceresponse and, compared to light with a high red:far red ratio,modified chlorophyll content and betacyanin pigment complement. Plants grown in light with a low ratio of red:far red floweredearlier than plants grown in light with a high red:far red ratio. It is concluded that phytochrome can participate in the signaltransduction pathway leading to the induction of both CAM andthe processes which result in pinitol accumulation and pigmentationin M. crystallinum, as well as in the mediation of shade avoidanceand flowering responses. Key words: Mesembryanthemum crystallinum, CAM, phytochrome, signal transduction, drought stress     

Distinct Cellular and Organismic Responses to Salt Stress

We have compared metabolic effects of high salinity betweenplants and cell suspension cultures from the facultative halophyteMesembryanthemum crystallinum (common ice plant). This plantshows developmentally-programmed inducibility for a switch fromC₃-photosynthesis to CAM (Crassulacean Acid Metabolism). Themetabolic switch is enhanced by environmental factors such asdrought, low temperature, and, most effectively, soil salinity.CAM induction is dependent on organized leaf tissue and cannotbe elicited by salt stress in suspension culture cells. In contrast,the accumulation of proline [Thomas et al. (1992) Plant Physiol.98: 626] is induced by NaCl in cultured cells as well as inplants and must be considered a cellular response to stress.We have extended our observations to include another trait ofsalt- and low-temperature-stress responses in the ice plant,the accumulation of putative osmoprotective sugars and sugaralcohols. In whole plants the cyclic sugar alcohol, pinitol,accumulates to amounts that approach 1 M during stress, whilein suspension cells no increase in sugar alcohols is observed.The distribution of carbon to different sugars is markedly differentbetween cells and plants under stress. Particularly obviousis the distinction between cell types in the different compositionof sugars and polyols, as exemplified by the epidermal bladdercells of ice plants. Ion contents and the content of sugarsand sugar alcohols of bladder cells indicate that Na⁺, Cl^–,pinitol and an unknown carbohydrate compound provide osmoticpressure in these cells, while organic anion concentrationsare low. With the ice plant, we conclude that cells in culturemimic only partly the stress response mechanisms of intact plantsand we hypothesize that communication between different tissuesis required to mount a complete environmental stress response. ⁴ Present address: Department of Botany Oklahoma State University,Stillwater, OK, 74078, U.S.A.     

Photosynthetic and Photorespiratory Enzymes in Widely Divergent Plant Species with Special Reference to the Moss Ceratodon purpureus: Properties of Ribulose Bisphosphate Carboxylase/ Oxygenase, Phosphoenolpyruvate Carboxylase and Glycolate Oxidase

Rintamäki, E. and Aro, E.-M. 1985. Photosynthetic and photorespiratoryenzymes in widely divergent plant species with special referenceto the moss Ceratodon purpureus: Properties of ribulose bisphosphatecarboxylase/oxygenase, phosphoenolpyruvate carboxylase and glycolateoxidase.—J. exp. Bot. 36: 1677–1684. Km(CO₂) values and maximal velocities of ribulose bisphosphatecarboxylase/oxygenase (E.C. 4.1.1.39 [EC] ) were determined for sixplant species growing in the wild, consisting of a moss, a fernand four angiosperms. The maximum velocities of the RuBP carboxylasesvaried from 0.13 to 0.;62 µmol CO₂ fixed min^–1 mg^–1soluble protein and the Km(CO₂) values from 15 to 22 mmol m^–3CO₂. The highest Km(CO₂) values found were for the moss, Ceratodonpurpureus, and the grass, Deschampsia flexuosa. These plantsalso had the highest ratios of the activities of RuBP carboxylaseto RuBP oxygenase. Glycolate oxidase (E.C. 1.1.3.1 [EC] ) activitieswere slightly lower in D.flexuosa, but not in C. purpureus,than for typical C₃ species. Phosphoenolpyruvate carboxylase(E.C. 4.1.1.31 [EC] ) was not involved in the photosynthetic carboxylationby these two plants. However, another grass, Phragmites australis,was intermediate in PEP carboxylase activity between C₃ andC₄ plants The properties of RuBP carboxylase/oxygenase are discussedin relation to the activities of PEP carboxylase and glycolateoxidase and to the internal CO₂ concentration. Key words: RuBP carboxylase, oxygenase, Km(CO₂), moss     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Preferential Loss of Chloroplast Proteins in Nitrogen Deficient Euglena

Two dimensional gel electrophoresis was used to follow changesin the relative amounts of over 500 cellular proteins duringnitrogen deficiency and in light limited nitrogen sufficientstationary phase Euglena cultures. Of 53 polypeptides whoserelative amount decreased in nitrogen deficient cells, 37 werechloroplast proteins and only 11 were mitochondrial proteins.This corresponds to a decrease in the relative amounts of 77%of the chloroplast proteins and 31% of the mitochondrial proteinsfound on the two dimensional gel map. Over a similar time period,the relative amounts of only 1 chloroplast and 1 mitochondrialprotein decreased in light limited nitrogen sufficient stationaryphase cultures. Many of the chloroplast proteins whose leveldeclined during nitrogen deficiency were proteins whose lightinduced accumulation is independent of chlorophyll synthesis,photosynthetic CO₂ fixation and the developmental status ofthe chloroplast. Taken together, these results indicate thatnitrogen deficiency triggers a preferential loss of chloroplastproteins which can not simply be explained through a dependenceof protein stability on chlorophyll levels or the developmentalstatus of the chloroplast. ¹Present address: The Mycology Center, Washington UniversitySchool of Medicine, Box 8050 St. Louis, MO 63178, U.S.A. ²Present address: Department of Biology, University of Tampa,Tampa, Florida 33060, U.S.A. (Received March 23, 1988; Accepted June 20, 1988)     

Action Spectra Confirm Two Separate Actions of Phytochrome in the Induction of Flowering in Lemna paucicostata 441

Action spectra studies have shown that in the short day plant(SDP) Lemna paucicostat441 there are at least two actions ofphytochrome in the induction of flowering. At the beginningof the dark period far-red light inhibited flowering, and theaction spectrum corresponded to the absorption spectrum of P_FR,while at the middle of the inductive dark period both red andfar-red light were inhibitory. The action spectrum for the redlight corresponded to that of PR absorption, but there was activityin the region beyond 720 nm which exactly coincided with theabsorption by P_FR observed at the beginning of the dark period,indicating that at the middle of the dark period there was absorptionby both P_R and P_FR. The difference in quantum efficiency betweenthe red and far-red light effects was about 60-fold. These resultsare consistent with there being a stable pool of P_FR necessaryfor the induction of flowering and another pool of phytochromein a different cellular environment which participates in thenight-break reaction as P_R. ¹ Present address: School of Applied Biology, Faculty of Science,Lancashire Polytechnic, Preston PR1 2TQ, U.K. ² 2 Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabemachi, Tsukuba, Ibaraki305, Japan. ³ Present address: Division of Plant Biological Regulation,The Riken Institute for Frontier Research Program, Hirosawa,Wako-shi, 351-01, Japan. (Received December 13, 1986; Accepted July 17, 1987)     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

NO2 Suppression of Light-Induced Nitrate Reductase in Squash Cotyledons

NADH-nitrate reductase (NR) (EC 1.6.6.1 [EC] ) activity in the cotyledonsof squash (Cucurbita maxima Duch.) seedlings showed daily variationwhen the seedlings were subjected to an alternating light-darkcycle. When the seedlings were transferred into continuous darkness,NR activity rose at first and then decreased continuously. Irradiationafter continuous darkness induced a rapid increase in NR activity;this light induction of NR activity was inhibited completelyby fumigation with 4 ppm nitrogen dioxide (NO₂). This inhibitoryeffect of NO₂ was prominent even at 1 ppm and became more pronouncedas the concentration of NO₂ increased. NO₂ fumigation did notremarkably affect the content of reductant (NADH) in the cotyledons.The results of immunoblotting using anti-NR serum indicatedthat irradiation induced the increase in the NR-polypeptidecontent and NO₂ fumigation inhibited the increase, suggestingthat NO₂ put an inhibitory effect on the synthesis of NR inducedby irradiation. ⁴ Present address: College of Environmental Health, Azabu University,Fuchinobe, Sagamihara, Kanagawa 229, Japan ⁵ Present address: Faculty of Home Economics, Otuma Women'sUniversity, Sanban-cho, Chiyoda, Tokyo 102, Japan (Received October 21, 1987; Accepted January 13, 1988)     

Photorespiratory Enzyme Activities in C3 and CAM Forms of the Facultative CAM Plant, Mesembryanthemum crystallimum L.

The extent to which photorespiration occurs in CrassulaceanAcid Metabolism (CAM) plants has received limited attention.No comparative studies of C₃ and CAM development have been made.To address this problem, activities of several photorespiratoryenzymes were measured in a facultative CAM plant—Mesembryanthemumcrystallinum L.—during induction of CAM by water stress(NaCl-treatment). Salt-treatment over a 22 d period produceda progressive change in metabolism from C₃ to CAM. This wasconfirmed by (I) changes in gas exchange from C₃ fixation tothe characteristic CAM pattern of nocturnal CO2 uptake; (2)increases in did acid fluctuation and (3) a 30-fold increasein phosphoenol pyruvate (PEP) carboxylase activity. In contrast,no significant changes were observed in the activities of glycollateoxidase (GO), NAD-dependent-hydroxypyruvate reductase (HPR),glutamine synthetase (GS) or glutamate dehydrogenase (GDH) whenplants were induced into CAM. Ion exchange chromatography onDEAE Sephacel detected only one GS isoenzyme (GS2, chloroplastic)at all stages of CAM induction, Western blot analysis, however,detected an additional, although minor, band of GS1 (cytosolic),in C₃ plants, which disappeared following CAM induction. Ourresults show that, after development of CAM, these plants stillretain the capacity to photorespire. This may be an essentialrequirement of CAM plants growing in habitats with variablerainfall. When water availability is high, stomata may openin the light allowing ribulose bisphosphate carboxylase oxygenase(Rubisco) activity and photorespiration to occur. The inherentcapacity to photorespire would allow plants to survive variableperiods of rainfall. Key words: Mesembryanthemum, C₃, CAM, photorespiration     

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of Panicum hians and Panicum milioides

Enzymes of the C₄, C₃ pathway and photorespiration have beenanalyzed for P. hians and P. milioides, which have chlorenchymatousbundle sheath cells in the leaves. On whole leaf extracts thelevels of PEP carboxylase are relatively low compared to C₄species, RuDP carboxylase is typical of C₃ species, and enzymesof photorespiratory metabolism appear somewhat intermediatebetween C₃ and C₄. Substantial levels of PEP carboxylase, RuDPcarboxylase, and photorespiratory enzymes were found in bothmesophyll and bundle sheath cells. Low levels of C₄-acid decarboxylatingenzymes may limit the capacity for C₄ photosynthesis in P. hiansand P. milioides. The results on enzyme activity and distributionbetween mesophyll and bundle sheath cells are consistent withCO₂ fixation via C₃ pathway in these two species. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and bythe University of Wisconsin Research Committee with funds fromthe Wisconsin Alumni Research Foundation; and by the NationalScience Foundation Grant BMS 74-09611. (Received September 16, 1975; )     

Pinitol, a Compatible Solute in Mesembryanthemum crystallinum L.?

The irrigation of Mesembryanthemum crystallinum L. plants with400 mol m^–3 NaCl to induce crassulacean acid metabolism(CAM) was accompanied by the accumulation of pinitol. Pinitolconstituted 71% of the soluble carbohydrate fraction and 9.7%dry weight in the CAM form. Pinitol in the C₃ form did not exceed5% of the soluble carbohydrate fraction. Pinitol appeared metabolicallyinert: it was not readily degraded during 96 h of darkness inthe CAM form or during CAM deinduction. Preparations of CAMM. crystallinum protoplasts, vacuoles and chloroplasts showedpinitol to be chloroplastic at a concentration of about 230mol m^–3 and cytosolic at about 100 mol m^–3. No pinitolwas detected in vacuoles. CAM leaf extracts possessed a highermyo-inositol phosphate synthesising capacity than C₃ extracts,revealing greater activity in the CAM form of glucose-6-phosphatecycloaldolase, an enzyme in the pathway of pinitol synthesis. Although pinitol accumulation and CAM induction could not beseparated and appeared to be specific responses to water stress,there may not be a causal link between them. Pinitol may functionas a compatible solute in the cytosol and especially the chloroplaststo counteract the presence of high concentrations of Na⁺ andCl^– ions in the vacuole. The accumulation of pinitol,though apparently not directly related to CAM may, like CAM,be viewed as an aspect of the adaptation of the plant to a reductionin water availability. Key words: pinitol, Mesembryanthemum crystallinum L, CAM, compatible solute     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )     

Metabolism of p-Hydroxybenzoate via Hydroxyquinol by Trichosporon cutaneum WY2-2: Characterization of the Pathway using Superoxide Dismutase as a Stabilizer of Hydroxyquinol

Trichosporon cutaneum WY2-2 was shown to metabolize p-hydroxybenzoatevia protocatechuate and hydroxyquinol. Using superoxide dismutaseas a stabilizer of hydroxyquinol, the conversion of protocatechuateto hydroxyquinol and the ring fission process of hydroxyquinolwere confirmed. Hydroxyquinol was chemically identified as theproduct of protocatechuate hydroxylase reaction. Partially purifiedprotocatechuate hydroxylase was highly specific for protocatechuate;its K_m values for protocatechuate and NADH were 17.6 and 12.4µM, respectively. It catalyzed equimolar CO₂ formation,NADH oxidation and O₂ consumption from protocatechuate. Hydroxyquinoldioxygenase was highly specific for hydroxyquinol, with a K_mof 2.9 µM. ¹A preliminary account of this work was presented at the 81stMeeting of the Chubu-branch of Agricultural Chemical Societyof Japan, Gifu, October, 1980. ²Present address: Biological Institute, Faculty of Science,Nagoya University, Nagoya 464, Japan. ³Present address: Shin Nihon Chemical Co. Ltd... 19-10, Showa-cho,Anjoh, Aichi 446, Japan. (Received November 15, 1985; Accepted August 27, 1986)