Adenosine 3',5'-cyclic monophosphate in Chlamydomonas reinhardtii. Influence on flagellar function and regeneration.

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.

The action of antimitotic herbicides on flagellar regeneration in Chlamydomonas reinhardtii: a comparison with the action of colchicine.

The effect of the antimitotic herbicides amiprophosmethyl, trifluralin, and oryzalin upon flagellar regeneration in Chlamydomonas reinhardtii has been studied. After flagellar amputation colchicine completely blocks its regeneration at 2.5 mM whereas the herbicides do so at 5 microM. When applied to regenerating flagella, one hour after amputation, the action of the herbicides is different of that of colchicine. Colchicine stops further regeneration, whereas the herbicides induce a gradual shortening of the partly regenerated flagella. Fully developed flagella also shorten after herbicide application. The induction of shortening is concentration and temperature dependent. A 30 to 50% inhibition of photosynthesis and respiration as well as calcium uptake was caused by the herbicides, whereas colchicine did not affect these metabolic processes.

Developmental changes in the potential for h(2)s emission in cucurbit plants.

Based on results obtained with leaf discs exposed to sulfate, leaves on cucurbit plants (Cucurbita pepo L. cv Small Sugar Pumpkin and Cucumis sativus cv Chipper) 1 to 2.5 weeks old have a low potential for H(2)S emission (less than 10 picomoles per min per cm(2) leaf area) in response to sulfate, whereas discs from most of the leaves on plants 3 to 4 weeks old emit H(2)S at a higher rate (50 to 150 picomoles per min per cm(2) leaf area). This difference is determined by the age of the plant, and is independent of the leaves' age or developmental stage. In response to l-cysteine, however, discs from leaves on cucurbit plants 1 to 2.5 weeks old emit H(2)S at higher rates (15 to 50 picomoles per min per cm(2) leaf area) than in response to sulfate. Furthermore, the potential for H(2)S emission in response to l-cysteine decreases with increasing age of the individual leaf. Thus, most of the potential for H(2)S emission in response to l-cysteine is developed during germination and the early growth of cucurbit plants, but most of the potential for H(2)S emission in response to sulfate arises later in the development of the plants.The developmental changes in the potential for H(2)S emission in response to l-cysteine in vivo are paralleled by changes in the cysteine desulfhydrase activity extractable from the leaves. This extractable activity, which is thought to be responsible for the generation of H(2)S by leaf tissue in response to l-cysteine, can be increased by preincubation of leaf discs in l-cysteine. Overt cysteine desulfhydrase activity is up to 2-fold higher, and latent cysteine desulfhydrase activity is up to 4-fold higher, in leaves on cucurbit plants 1 to 2.5 weeks old than in leaves on plants 3 to 4 weeks old. Thus, most of the cysteine desulfhydrase activity develops during the early period of growth of a cucurbit plant. Overt cysteine desulfhydrase activity passes through a maximum value during the development of each leaf; total as well as latent cysteine desulfhydrase activities, however, decline with increasing age of the individual leaf in much the same way as H(2)S emission in response to l-cysteine declines.

Adenosine 3':5'-Cyclic Monophosphate in Chlamydomonas reinhardtii: Isolation and Characterization.

Chlamydomonas reinhardtii contains a factor that can replace adenosine 3':5'-cyclic monophosphate (cAMP) in the stimulation of rabbit-muscle protein kinase. The factor cochromatographs and coelectrophoreses with authentic cAMP, and is inactivated by beef heart cyclic nucleotide phosphodiesterase. When C. reinhardtii is exposed to aminophylline (theophylline(2) ethylenediamine), the concentration of the factor in the cells increases within 1 hr, from about 25 pmol of cAMP equivalents per g dry weight to more than 250 pmol. Cyclic nucleotide phosphodiesterase activity is present in crude extract of C. reinhardtii and is inhibited by theophylline. We conclude that cAMP occurs in C. reinhardtii and that the endogenous concentration is governed at least in part by a theophylline-sensitive cyclic nucleotide phosphodiesterase. These findings provide a sound basis for attributing the effects of methylxanthines on flagellar function and regeneration in C. reinhardtii to the resultant elevation of endogenous cAMP.

Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. Effects of sulfur and nitrogen sources on the formation and decay of the enzyme.

The ATP sulfurylase of cultured tobacco cells is repressed during growth on readily assimiliated sulfur sources, such as sulfate, L-cysteine, or L-methionine, but it is derepressed during growth on slowly assimiliated sulfur sources, such as L-djenkolate or glutathione, or during sulfur starvation. The enzyme is not induced by sulfate. The enzyme level in the cells begins to rise 12 to 24 h after the derepression conditions are initiated and continues to rise for 3 to 4 days, up to as much as 25 times above the initial specific activity. Addition of a repressing sulfur source to derepressed cells causes the enzyme to decay. Derepression by sulfur limitation does not occur in cells starved for nitrogen, a circumstance in which turnover synthesis of protein is known to continue. Upon addition of a nitrogen source to such cells, the development of the enzyme begins within 12 h, along with the resumption of growth and net protein synthesis. Derepression occurs in cells growing on the slowly assimilated nitrogen in urea, reaching specific activities very similar to those which develop in cells grown on nitrate, in spite of the lower protein accumulation rate on urea. Thus the ATP sulfurylase of tobacco cells appears to be regulated by both a negative feedback mechanism in which an end product of the sulfate assimilation pathway is the effector, and by a positive mechanism which serves to couple the regulation of the sulfate assimilation pathway to the cells' potential for nitrogen assimilation, i.e. net protein synthesis. The sulfur compounds which affect the development of ATP sulfurylase in vivo have no effect on the enzyme activity in vitro. Furthermore, extracts with high activity contain no activator and extracts with low activity contain no inhibitor of ATP sulfurylase. Cycloheximide, at a concentration which strongly inhibits amino acid incorporation into protein, inhibits derepression. ATP sulfurylase does not decay in cells inhibited by cycloheximide. Therefore, the changes in ATP sulfurylase of tobacco cells appear to involve changes in the rate of formation or degradation of the enzyme.

Resistance to acetohydroxamate acquired by slow adaptive increases in urease in cultured tobacco cells.

Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner 1980 Plant Phvsiol 65: 995-1003). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to about 4 days for 1U cells, while 1U and 12U cells double in 2 days on nitrate. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty per cent inhibition of growth by AHA occurred at 20 micromolar in 1U cells growing on urea and at 165 micromolar in 12U cells growing on urea, but at 600 micromolar for either 1U or 12U cells growing on nitrate. When 12U cells were grown on urea with 100 micromolar AHA, extractable urease activity decreased 80% within 2.5 hours and remained at this level for 2 weeks; the doubling time increased to 3.7 days, and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre-AHA level by incubation at 30 C after extraction and separation from free AHA. AHA inhibited incorporation of (15)N from [(15)N]urea into Kjeldahl nitrogen in the cells, in spite of the increased intracellular urea. These results indicate that AHA acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease.

Stimulation of h(2)s emission from pumpkin leaves by inhibition of glutathione synthesis.

The effect of inhibitors of glutathione (GSH) synthesis, namely gamma-methyl glutamic acid, d-glutamic acid, cystamine, methionine-S-sulfoximine (MSX), buthionine-S-sulfoximine, and GSH itself, on the emission of H(2)S was investigated. All these compounds stimulated H(2)S emission from pumpkin (Cucurbita pepo L. cv Small Sugar Pumpkin) leaf discs in response to sulfate. MSX and GSH were the most effective compounds, stimulating H(2)S emission from leaf discs of mature pumpkin leaves by about 80% in response to sulfate. Both inhibitors did not appreciably enhance H(2)S emission in response to l-cysteine and inhibited H(2)S emission in response to sulfite.Treatment with MSX or GSH enhanced the uptake of sulfate by pumpkin leaf discs, but did not affect the incorporation of sulfate into reduced sulfur compounds. Inhibition of GSH synthesis by MSX or GSH caused an increase in the pool size of cysteine, and, simultaneously, reduced the incorporation of labeled sulfate into cysteine. The incorporation of labeled sulfate into the sulfite and sulfide pools of the cells are stimulated under these conditions.These observations are consistent with the idea that inhibition of GSH synthesis leads to an elevated cysteine pool that inhibits further cysteine synthesis. The H(2)S emitted under these conditions appears to arise from diversion of a precursor of the sulfur moiety of l-cysteine. Therefore, stimulation of H(2)S emission in response to sulfate upon inhibition of GSH synthesis may reflect a role of H(2)S emission in keeping the cysteine concentration below a critical level.

Tubulin from cultured tobacco cells: isolation and identification based on similarities to brain tubulin.

The microtubule protein, tubulin, was isolated from most other proteins of cell suspension cultures of Nicotiana tabacum L. by its copolymerization with cow-brain tubulin. Cow-brain tubulin was added to the soluble protein fraction of extract from (35)S-labeled tobacco cells and subjected to two cycles of temperature-dependent assembly-disassembly (copolymerization). When analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) about 70% of the radioactivity in the twice copolymerized protein was found in a prominent doublet migrating close to the doublet of brain tubulin. When analyzed by two-dimensional isoelectric-focusing-SDS-PAGE the radioactive doublet behaved like the doublet of brain tubulin. Limited proteolysis of the individual polypeptides of the coublets showed that, while the peptide maps of the leading radioactive band and of the ß-subunit of brain tubulin were virtually indistinguishable, the maps of the trailing radioactive band and of the α-subunit of brain tubulin, though similar, were not identical. Most of the copolymerized (35)S-labeled protein also behaved like brain tubulin during gel filtration and ion-exchange chromatography. It is concluded that the doublet of radioactive polypeptides isolated by copolymerization with brain tubulin are tobacco tubulin polypeptides that have, in their native as well as denatured forms, properties very similar to, but not identical with, cow brain tubulin. Apparently, tubulin has been highly conserved during evolution.

The Kinetics of Chlorate Uptake by XD Tobacco Cells.

The uptake of [(36)Cl]chlorate by the 14U variant of the XD cell line of Nicotiana tobaccum L. cv Xanthi was investigated to examine the use of chlorate as a nitrate analog in transport studies. The kinetics of chlorate uptake against concentration was complex. Evidence was obtained, e.g., by means of nitrate competition, that these kinetics could be resolved into two components indicating the existence of two influx mechanisms: a saturable high affinity transport system (HATS) and a low affinity transport system (LATS) that showed first order kinetics. HATS has an apparent K(m) for chlorate of 0.3 millimolar, and a marked pH dependence. The V(max) dropped about fivefold as the pH was changed from the optimum pH (5.5-6.5), while the K(m) remained virtually unchanged. The activity of HATS was completely inhibited by 15 millimolar nitrate and was less sensitive to chloride. LATS was inhibited by chloride and showed some inhibition by nitrate. It was concluded that [(36)Cl]chlorate can be used as an analog for nitrate uptake studies only in a limited low concentration range where HATS is the main route for chlorate influx.

Participation of calcium in flagellar shortening and regeneration in Chlamydomonas reinhardii.

Cation chelators cause flagellar shortening in Chlamydomonas reinhardii. Most effective are EDTA and EGTA (1 mM) but pyrophosphate (10 mM) also is effective. Addition of 5 mM Ca2+ after shortening caused by 4 mM EGTA results in flagellar regeneration. Other divalent cations can replace Ca2+ with the following relative activities: Ca2+ greater than Sr2+ = Mn2+ much greater than Ba2+ = Mg2+. Although the specific ion requirement to reverse shortening is not clear, it is possible that all of the ions act by displacing one bound cation, presumably Ca2+. A specific requirement for Ca2+ in flagellar regeneration could be demonstrated, however, because as little as 50 microM EGTA in the presence of 500 microM Mg2+ delayed regeneration and prevented full regeneration. Ca2+ at 100 microM overcame this inhibition.

Structural and functional relationships of enzyme activities induced by nitrate in barley.

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH(2)-nitrate reductase and NADH-cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH-nitrate reductase (8S), one band of FMNH(2)-nitrate reductase activity (8S) and three bands of NADH-cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH-cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH-cytochrome c reductase band co-sediments with both NADH-nitrate reductase activity and FMNH(2)-nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH-cytochrome c reductase. 4. FMNH(2)-nitrate reductase activity is more stable (t((1/2)) 12.5min) than either NADH-nitrate reductase activity (t((1/2)) 0.5min) or total NADH-cytochrome c reductase activity (t((1/2)) 1.5min) at 45 degrees C. 5. NADH-cytochrome c reductase and NADH-nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH(2)-nitrate reductase activity. 6. Tungstate prevents the formation of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities, but it causes superinduction of NADH-cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH-cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH-nitrate reductase, FMNH(2)-nitrate reductase and NADH-cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH-cytochrome c reductase activity.

Regulation of sulfate uptake by amino acids in cultured tobacco cells.

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.

Slow adaptive changes in urease levels of tobacco cells cultured on urea and other nitrogen sources.

Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4-fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 x 10(-5), then multiplied 19% more rapidly than low urease cells. This frequency is about 10(3) greater than that of other biochemical variants previously isolated from XD cells. The high urease activity gradually declined in cells transferred from urea to other nitrogen sources, but rose rapidly when such cells were returned to urea, indicating the existence within the cells of some form of record of their ancestors' growth on urea. The data indicate the existence of a mechanism for generation, at unusually high frequency, of metastable variants with high urease activity. This mechanism, coupled with enrichment for the variants' progeny by virtue of their higher multiplication rate on urea, can account for the observed slow increase in urease activity of the population. It is suggested that the molecular basis of the urease increase may be gene amplification, based on animal cell models. An alternative hypothesis, namely a specific response induced in all cells by urea and manifested as a very slow adaptive increase in urease, has not been ruled out.

N-caffeoyl-4-amino-n-butyric acid, a new flower-specific metabolite in cultured tobacco cells and tobacco plants.

PUT cells were selected from the XD line of cultured tobacco cells (Nicotiana tabacum L. cv. Xanthi-nc) for the ability to utilize putrescine as sole nitrogen source. Previous work had indicated that hydroxycinnamoylputrescines (principally caffeoylputrescine) and 4-amino-n-butyric acid (GABA) are obligatory intermediates in the assimilation of putrescine by PUT cells. The apparent absence in these cells of diamine or polyamine oxidase and pyrroline dehydrogenase, enzymes which catalyze putrescine oxidation in some plant species, led us to propose the following pathway for putrescine oxidation in PUT cells: putrescine----hydroxycinnamoylputrescine----hydroxycinnamoyl - 4-aminobutyraldehyde----hydroxycinnamoyl-GABA----GABA. We tested the hypothesis by looking for the predicted compound, caffeoyl-GABA. A chemical synthesis was developed, and chromatographic and mass spectroscopic procedures were devised for identifying the compound in extracts of cells and plant tissues. Caffeoyl-GABA was found in extracts of PUT cells in micromolar concentrations but was not present in XD cells. Thus, its occurrence in PUT cells appears to be a direct result of selection for the ability to catabolize putrescine. Caffeoyl-GABA has the same distribution in tobacco plants as caffeoylputrescine, i.e. flower buds greater than open flowers greater than floral leaves, green fruit; absent in vegetative tissues.