Isoenzymes of Acid Phosphatase and Non-specific Esterases in Cultures of Neoplastic and Normal Tobacco Tissues

Axenic cultures of normal, habituated and crown gall teratoma tissues were grown under varying conditions to examine the effects of environment on the expression of neoplastic character. Acid phosphatase patterns on polyacrylamide gels did not vary greatly among tissues although there were differences in acid phosphatase activity between various strains of Agrobacterium tumefaciens , the bacteria which cause crown gall. Certain esterase isoenzymes were found only in tissues grown on specific media, while others were tissue-specific but independent of the nature of the medium. Comparisons of liquid and solid grown cultures revealed that culture conditions also influence esterase expression. Both sunflower and tobacco crown gall tissue contained an esterase not found in habituated or normal tissues, and similar in electrophoretic mobility to an esterase found in extracts of the bacteria that had induced the tumors. The basic difference between the three tissue types studied is the manner in which they respond to a given environment.     

Free amino Acid pools and enzymes in teratonia and habituated tobaceo tissue cultures

The free amino acid content of habituated (normal) and teratoma (abnormal) tobacco tissue cultured on white's medium were compared. Significant qualitative differences (twofold or more) were observed for serine, proline, alanine, leucine, phenylalanine, and lysine. There were qualitative differences in several unidentified ninhydrin sensitive compounds. One unknown which co-chromatographed with homo-serine was present in high concentrations (9.96 μmol/g dry weight) in habituated but was lacking in teratoma tissue, Isoenzymes of an esterase, acid phosphatase, and oxidase were demonstrated in both tissues. The distinct differences in the isoenzyme pattern of acid phosphatase suggests a differential regulatory capacity of certain phosphomonoester intermediates and P(1) . The invertase activity was higher in habituated tissue, implying a greater capacity to utilize the sole carbon source, socrose.     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

Chlorophyllous Nature and Growth Characteristics of Teratoma and Habituated Tissue Cultures of Tobacco

The growth characteristics of three types of tissue cultures of tobacco have been investigated. They are (i) pigmented and differentiated teratoma tissue; (ii) pigmented and non-differentiated habituated tissue and (iii) non-pigmented and non-differentiated habituated tissue. All the tissues were grown on a 10 × White's medium without any exogenous supply of auxin. The comparative growth rate of the tissues was related to their nature of differentiation, viz., either organelle differentiation with chloroplast or organ differentiation with buds, leafy appendages, etc. Teratoma and pigmented habituated tissues had normal chlorophyll and carotenoid contents and showed more favorable growth in light than in the dark. The pigmented tissues showed a higher protein content than the non-pigmented tissues irrespective of whether they were of tumorous or normal origin. The pigmented tissues showed less dependence on sucrose availability in the medium than the non-pigmented tissues. Recovery after sucrose starvation was higher in the pigmented tissues than in the non-pigmented tissues. The results indicate that the growth rate of highly differentiated tissues is much slower than non-differentiated tissues and that the pigmented tissues photosynthesize and survive on reserve food products manufactured as a result of photosynthetic activity.     

Evidence for Compartmentation of Tryptophan in Cultured Plant Tissues: Free Tryptophan Levels and Inhibition of Anthranilate Synthetase

1. Anthranilate synthetase activity in crude extracts from tissue cultures of Daucus carota L. (carrot), Nicotiana tabacum L. (tobacco; cv. Wisconsin 38 and xanthi), Glycine max Merr. (soybean) and Oryza sativa L. (rice) was completely inhibited by l -tryptophan (5 to 50 μM). Mutant carrot and tobacco lines, capable of growth in the presence of 5-methyltryptophan, required 500 to more than 1000 μM tryptophan for complete inhibition of enzyme activity, respectively. 2. Except for the mutant tobacco line, the concentrations of free tryptophan in all tissue cultures tested were greater than the levels necessary to completely inhibit the respective anthranilate synthetase activities in vitro. These findings would indicate that much of the free tryptophan is compartmentalized away from the regulatory enzyme, anthranilate synthetase. This could implicate compartmentalization of the inhibitor as a biosynthetic control mechanism. 3. During the growth of normal and mutant carrot tissues the anthranilate synthetase enzyme must be at least 7.8 and 10.8% active, respectively, in order to accumulate the amount of tryptophan found in the tissues. 4. Of the substrates and cofactors required for anthranilate synthetase activity in vitro, Mg²⁺ and glutamine were present at near optimal levels in the carrot and tobacco tissues, but chorismate was found to be significantly below the optimal concentrations.     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

Role of sucrose-phosphate synthase in partitioning of carbon in leaves

Variations in leaf starch accumulation were observed among four species (wheat [Triticum aestivum L.], soybean [Glycine max L. Merr.], tobacco [Nicotiana tabacum L.], and red beet [Beta vulgaris L.]), nine peanut (Arachis hypogea L.) cultivars, and two specific peanut genotypes grown under different nutritional regimes. Among the genotypes tested, the activity of sucrose phosphate synthase was correlated negatively with leaf sucrose content in seven of the nine peanut cultivars as well as the two peanut cultivars grown with different mineral nutrition. The peanut cultivars differed in the effect of 10 millimolar sucrose on sucrose phosphate synthase activity in leaf extracts. Enzyme activity in crude leaf extracts was inhibited by sucrose (10-42%) in four of the cultivars tested whereas five cultivars were not. Overall, the results suggest that a correlation exists between the activity of sucrose phosphate synthase and starch/sucrose levels in leaves.     

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.

In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [¹⁴C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.

It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants.

The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined.

     

Morphogenic responses of debudded tobacco plants to gibberellic Acid and indole-3-acetic Acid

Stem applications of indole-3-acetic acid (IAA) or gibberellic acid (GA) did not prevent or alter tumor or teratoma formation in debudded tobacco plants (Nicotiana tabacum L., var. One Sucker). The materials produced intense (in case of GA) and moderate (in case of IAA) stem proliferations when applied to debudded plants but were without effect on intact plants.

The results suggest that debudding-tumors are probably not related to or a result of an auxin or gibberellin deficit and that total debudding has a marked physiological effect on the plant. The altered physiological condition of the debudded plant, indicated by its responses to IAA and GA, may likely be related to tumor and teratoma formation.

     

Termination of nutrient import and development of vein loading capacity in albino tobacco leaves

The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with ¹⁴CO₂ and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.

To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [¹⁴C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves.

     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

(Na+ + K+)-activated adenosinetriphosphatase: fluorimetric determination of the associated K+ -dependent 3-O-methylfluorescein phosphatase and its use for the assay of enzyme samples with low activities.

Data are presented which prove that 3-O-methylfluorescein phosphate is a substrate for the K⁺-dependent phosphatase that is associated with Na⁺,K⁺-ATPase. Conditions for the continuous fluorimetric assay of 3-O-methylfluorescein phosphatase are described. Enzyme preparations from three different tissues with widely different specific activities exhibit similar K_m values for 3-O-methylfluorescein phosphate. Correlation between Na⁺,K⁺-ATPase activity and K⁺-dependent 3-O-methylfluorescein phosphatase activity is demonstrated in several partially purified enzyme preparations and crude tissue fractions. When the K⁺-dependent 3-O-methylfluorescein phosphatase of a crude rat-brain homogenate is assayed, the activity is a linear function of the amount of homogenate added to the assay mixture. The equivalent of 10 μg of brain tissue may be assayed under the conditions used. The potential value of this highly sensitive fluorimetric method for the assay of enzyme in small samples of various tissues is suggested.     

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.     

Transfer ribonucleic Acid modification and its relationship to tumorous and nontumorous plant growth

Detailed analyses of tRNA hydrolysates from four tissue types of Nicotiana tabacum, pith from intact plants, pith growing in culture, habituated tissue in culture, and crown gall tumor tissue in culture, revealed significant qualitative and quantitative differences in the pattern of methylation. Although pith from intact plants and pith growing in culture possessed seven different methylated nucleosides, only two were found in habituated and tumorous tissues in culture. Four of the five compounds accounting for the difference were tentatively identified as methylated guanosines. Evaluation of results in terms of several parameters, including growth rate, the tumorous state, habituation, tissue culture, and potential for differentiation, indicate that the extent of tRNA methylation may be correlated with the potential for differentiation of a particular tissue.     

Some properties and immunological relationship of acid phosphatases from gramineae

Two acid phosphatases have been found in crude extracts of seeds, coleoptiles and leaves of various grass species by means of crossed immunoelectrophoresis.The enzymes, cross-reacting with antibodies raised against proteins of Poa pratensis seeds differ in their binding to con A. The use of affinity chromatography on con A-Sepharose has separated the acid phosphatases into two fractions: the non-binding (acid phosphatase A) and the con A-binding (acid phosphatase B). The con A-binding acid phosphatase B from all tissues was further purified by gel filtration on Biogel P-100 and hydrophobic interaction chromatography on phenyl-Sepharose. Two isoenzymes: acid phosphatase B₁ and B₂ were obtained. The isoenzymes are glycoproteins containing D-mannose or D-glucose in their carbohydrate moiety. They retained the enzyme activity after binding in macromolecular complex with antibodies or con A. The purified acid phosphatases from all tissues cross-react with monospecific antibodies raised against P. pratensis seeds acid phosphatase B₁ indicating the antigenic relationship between the enzymes of various grass species.     

Possible correlation between increased vigour and chitinase activity expression in tobacco

Nicotiana tabacum (tobacco) plants with several—fold increasedchitinase enzyme activity levels were obtained through transformationwith a Zea mays (maize) chitinase cDNA clone, Ch2, under thecontrol of the CaMV 35S promoter. There was a linear correlation(r=0.74, P = 0.009) between the endochitinase activity and theweight of homozygous seedlings grown in the greenhouse in someexperiments. Progeny seedlings of self-pollinated regenerants(heterozygous for Ch2) grown for 18 d in Petri plates couldbe separated into vigorous and non-vigorous categories basedon size, which were predicted to be 2n or 1,n and null, respectively,for Ch2. Testing showed that the visually estimated vigour ofthese progeny co-segregating for Ch2 and npfll generally correlatedwith resistance to kanamycin and chitinase activity. There wasalso a correlation between growth and chitinase activity insuspension cultures derived from transgenic and wild-type whenmeasured after 20 d. Twelve-day-old wild-type and transformedseedlings grown in soil under culture room conditions in closedplastic containers were visibly taller and had higher seedlingweights than their counterparts grown under mist in the greenhouse.Likewise, the chitinase activity in the cell wall-bound proteinsof the faster growing seedlings was more than double that ofthe greenhouse-grown seedlings. While these studies do not always demonstrate the correlationbetween vigour and chitinase activity, overall, they do supportthe hypothesis that chitinases have a role in plant growth. Key words: Chitinase, maize endochitinase, growth, tobacco     

Serological Characterization of the Glycolate-oxidizing Enzymes from Tobacco, Euglena gracilis, and a Yellow Mutant of Chlorella vulgaris

An antiserum to tobacco glycolate oxidase has been prepared by injection of the purified enzyme into rabbits. Double gel diffusion tests between the antiserum and purified antigen and also with a crude tobacco preparation gave a single immunoprecipitation band. Crude extracts of Euglena gracilis Z Klebs, containing glycolate dehydrogenase, and of Chlorella vulgaris 211-11h/20, containing glycolate oxidase, also formed single bands with the tobacco antiserum. The algal bands were identical and showed partial identity with the tobacco band. The antiserum inhibited the glycolate oxidase activities of the tobacco and Chlorella extracts but did not affect Euglena glycolate dehydrogenase activity.     

Mannityl opine accumulation and exudation by transgenic tobacco

Three genes from the T_R region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.