A selective assay for prephenate aminotransferase activity in suspension-cultured cells of Nicotiana silvestris

Prephenate aminotransferase in Nicotiana silvestris Speg. et Comes is highly stable to thermal treatment. This property was exploited to obtain, by treatment at 70° C for 10 min, a residual level (1–4%) of aspartate aminotransferase activity that proved to be catalyzed exclusively by prephenate aminotransferase. The latter enzyme was the most mobile of all aspartate aminotransferase bands during polyacrylamide-gel electrophoresis conducted under non-denaturing conditions. This methodology for convenient assay of prephenate aminotransferase in crude extracts, as demonstrated for N. silvestris, may generally apply to higher plants since prephenate aminotransferase from a variety of plant sources has been found to exhibit high thermal stability.Abbreviations AGN L-arogenate - AT aminotransferase - ASP L-aspartate - GLU L-glutamate - HPP 4-hydroxyphenylpyruvate - 2-KG 2-ketoglutarate - OAA oxaloacetate - PPA prephenate - PPY phenylpyruvate Florida Agricultural Experiment Station, Journal Series No. 8286     

Purification and characterization of prephenate aminotransferase from Anchusa officinalis cell cultures

Prephenate aminotransferase (PAT) from rosmarinic acid-producing cell cultures of Anchusa officinalis has been purified to apparent electrophoretic homogeneity using a combination of high-performance anion-exchange, chromatofocusing, and gel filtration chromatography. The purified enzyme has a native molecular weight of 220,000 and subunit molecular weights of 44,000 and 57,000, indicating a possible alpha 2 beta 2 subunit structure. The purified PAT displays high affinity for prephenate (Km = 80 microM) but could also utilize other aromatic alpha-keto acids at less than 20% the rate with prephenate. L-Aspartate (Km = 80 microM) is about three times as effective as L-glutamate as amino-donor substrate. Anchusa PAT is not subject to feedback inhibition from L-phenylalanine or tyrosine, but its activity is affected by a rosmarinic acid metabolite, 3,4-dihydroxyphenyllactic acid.     

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M _r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M _r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K _m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K _m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]_0.5) value was 2.5 mM.Abbreviations DEAE diethylaminoethyl - M _r molecular weight     

Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis

Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. Here we present the X‐ray crystal structures of the wild‐type and various mutants of PAT from Arabidopsis thaliana (AtPAT). Steady‐state kinetic and ligand‐binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution of PAT activity among class Ib AAT enzymes. Structures of AtPAT mutants co‐crystallized with either α‐ketoglutarate or pyridoxamine 5′‐phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor of PAT from A. thaliana and Antirrhinum majus plants as well as the bacterium Chlorobium tepidum, uncovering a potential new effector of PAT.     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Floral determination in axillary buds of Nicotiana silvestris

At anthesis of the terminal flower the developmental fates of axillary buds of the long-day plant Nicotiana silvestris were assessed in situ and in isolation. The in situ developmental fate was assessed by decapitating the plant above the bud in question and letting the bud mature. The developmental fate of isolated buds was assessed by removing the bud from the main axis, rooting it, and letting it mature. The number of nodes below the terminal flower of the mature shoot was indicative of the developmental fate of the bud. Terminal meristems of rooted axillary buds exhibited two patterns of development: (1) Their developmental fate was the same as that of in situ buds at the same node or (2) their developmental fate was the same as that of seed-derived plants. For example, terminal meristems of rooted buds from the fourth node below the inflorescence produced either 15 to 19 nodes or 36 to 40 nodes. In situ fourth buds produced 12 to 14 nodes while seed-derived plants produced 33 to 39 nodes. Terminal meristems of rooted axillary buds that exhibited the same developmental fate as that of in situ buds were determined for floral development. Although determined buds produced a terminal flower, all but one had abnormal inflorescences. That is, in the place of floral branches determined buds produced vegetative branches. Four buds that were not determined for floral development had their shoot tips rooted each time the plant bolted. Only when the plants were allowed to grow without being rerooted did they flower. These results indicate that roots may prevent and/or destabilize floral determination in N. silvestris.     

Purification and characterization of tyrosine aminotransferase activities from Anchusa officinalis cell cultures

Three activities of tyrosine aminotransferase (TAT; EC 2.6.1.5), the enzyme which catalyzes the first step of the tyrosine pathway leading to the formation of rosmarinic acid (alpha-O-caffeoyl-3,4-dihydroxyphenyllactic acid), have been extensively purified from cell suspension cultures of Anchusa officinalis L. and subsequently characterized. TAT-1, TAT-2, and TAT-3 differ slightly in native molecular weights (180,000-220,000) and are composed of subunits (4 X 43,000 for TAT-1 and 4 X 56,000 for TAT-2). All three enzymes show a pronounced preference for L-tyrosine over other aromatic amino acids, but TAT-2 and TAT-3 can also effectively utilize L-aspartate or L-glutamate as a substrate. For amino acceptor cosubstrates, either oxaloacetate or alpha-ketoglutarate can be utilized equally well by TAT-1, while the former is the most effective alpha-keto acid for TAT-2 and the latter is the best for TAT-3. All the TAT activities display high pH optima (8.8-9.6), and are inhibited by the tyrosine metabolite 3,4-dihydroxyphenyllactate. TAT-2 and TAT-3 are also inhibited by rosmarinic acid.     

Purification and properties of a DNase inhibitor from Nicotiana tabacum cell cultures

Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE-cellulose and gel filtration resulted in a homogeneous protein (Mr = 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1:1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor X DNase-I complex have been calculated. This interaction is strongly temperature-dependent; at 20 degrees C and 26 degrees C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.     

Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK_a values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO⁻CH₂NH₂⁺CH₂PO₃²⁻, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K_i = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K_i′ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate].     

Chromosome stability of cell suspension cultures of Nicotiana spp.

Cell suspension cultures of Nicotiana were initiated using conditions designed to selectively favor stable chromosome number. These conditions included use of leaf explants to initiate cultures, growth of cells in culture medium containing 2,4-D, and transfer of cells with short subculture intervals. Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number. Each line could be regenerated to recover plants that retained the somatic chromosome number during culture. Establishment of haploid and diploid cell lines with stable chromosome number is important for mutant isolation and protoplast fusion.     

Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by l-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by l-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia

The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 M Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 M Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 M Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.     

Sesquiterpenoid phytoalexins from suspended callus cultures of Nicotiana tabacum

Treatment of suspended callus cultures of Nicotiana tabacum with commercial cellulase elicited four principal stress metabolites including the phytoalexin capsidiol and a second eremophilane-type diol, shown on the basis of chemical and spectroscopic evidence to be 4-epieremophil-9-ene-11 /gx, 12-diol (without assignment of absolute configuration). This diol appears to be structurally identical with debneyol isolated from N. debneyi (see accompanying paper). Among minor metabolites were an isomer and a dehydro-analogue of the diol. GC/MS of cyclic derivatives (boronates and di-t-butylsilylene derivatives) of vicinal diols was useful for their detection and characterisation. The remaining two major metabolites appeared to be phytuberol and phytuberin.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

A Novel abscisic acid metabolite from cell suspension cultures of Nigella damascena

The structure of a novel abscisic acid metabolite isolated from cell suspension cultures of Nigella damascena fed [2-¹⁴C]abscisic acid was iden     

Nicotine demethylation in Nicotiana cell suspension cultures: N'-formylnornicotine is not involved

Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N'-formylnornicotine and N'-carboethoxynornicotine. While cotinine was formed from [(13)C,(2)H(3)-methyl]nicotine without dilution of label, N'-formylnornicotine was labelled at only about 6% of the level of nicotine and N'-carboethoxynornicotine was unlabelled. Feeding with [1'-(15)N]nornicotine resulted in incorporation without dilution of label into both N'-formylnornicotine and N'-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N'-formylnornicotine and N'-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation.