Protein Metabolism in Cultured Plant Tissues

Rates of protein synthesis in normal callus tissues (either tight or loose morphological form), in crown gall callus tissues and in cultured pith cells were measured for both the lower surface cells (those in contact with the original growth medium) and upper surface cells (those never in contact with the growth medium until labeling). Cells of both surfaces of loose and crown gall callus and the upper-surface cells of tight callus had similar rates of protein synthesis, 29–31 mg of protein synthesized × (g protein)⁻¹× h⁻¹. The lower surface cells of tight callus had a 35% lower rate of synthesis, 20 mg × g⁻¹× h⁻¹. Pulse-chase experiments suggested that rates of protein degradation for all tissues were the same, 21–23 mg protein × (g protein)⁻¹× h⁻¹. Thus, there probably was no accumulation of protein in the lower surface cells of tight callus tissue, but the other tissues had rates of accumulation equaling 10 mg × (g protein)⁻¹× h⁻¹. Autoradiography and electron-microscopic examination of cells in tight callus labeled with ³H-leucine show that: (a) the lower-surface cells were more degenerate than cells within the callus or on the upper surface; and (b) the first few cell layers nearest the medium were preferentially labeled. Pulse-chase experiments were also used to quantitate the nonprecursor pool (defined as that tritium in the soluble amino acid pool that does not equilibrate with protein during a pulse-chase experiment). The nonprecursor pool increased linearly with time at the same rate as incorporation of ³H-leucine into protein. Furthermore, the nonprecursor pool copurified with leucine and was probably either D- or L-leucine.     

The Metabolism of Acetaldehyde by Plant Tissues

Apples and oranges can absorb appreciable amounts of acetaldehydevapour, but acetaldehyde does not accumulate in the tissues,in the presence or absence of oxygen. When the fruit is givenacetaldehyde the loss of carbon as CO₂ + ethanol equals thatlost from carbohydrate + acid+acetaldehyde. Part of the addedacetaldehyde is reduced to ethanol by NADH₂ and the rate ofglycolysis is increased; the remainder is oxidized to CO₂. Respiration of apples is limited by oxygen; the output of CO₂is only slightly increased at the beginning of the treatmentwith acetaldehyde, but afterwards it declines; acetaldehydespares oxidation of carbohydrate. In oranges, availability of substrate limits respiration, andacetaldehyde stimulates CO₂-production and O₂-uptake. In theabsence of oxygen, acetaldehyde is reduced to ethanol, and thequotient CO₂/ethanol falls.     

The Metabolism of 3-Indolylacetic Acid in Plant Tissues3

The nature of metabolic products of 3–indolylacetic acid(IAA) extracted from potato tuber disks treated with aeratedIAA solution has been investigated. Two major products, knownat first as ‘V’ and ‘P’ in these studieshave been isolated and ‘V’ has been identified as3-indolylacetylaspartic acid (IacAsp). The rapid uptake of IAA is inhibitited by metabolic poisonssuch as 10^–3 M. cyanide. The maximum mean internal concentrationexceeds the external concentration well–aerated cultures.The mean internal concentration, however only remains for aperiod and then falls off rapidly as a result of extrusion ofabsorbed IAA into the external solution. This extrusion is notinhibited by 10^-3 cyanide; when the mean internal IAA concentrationis 150 µ mol/ml. and the localized IAA concentration musttherefore exceed this value. We conclude therefore that theIAA concentration in the sites where it has accumulated exceedsthe concentration of IAA outside. Uptake of IAA and also its further conversion are inhibitedby indolylacetonitrile and promoted by aspartate, but this promotionis not associated with any gain in amount of indolylacetylaspartate(IacAsp). The data suggest that IacAsp may be formed in tissue from ‘boundIAA’ rather then free IAA. The ‘accelerator ’ found in potato and beans whichhas similar R_F to IAcAsp has been shown definity to be someother substance or substances and not IAcAsp as was at firstthought possible.     

Studies in the Respiratory and Carbohydrate Metabolism of Plant Tissues1

Oxygen at pressures of 2 and 3 atmospheres caused an initialincrease in CO₂ output of strawberry leaves followed by a decrease.In oxygen at 2 atmospheres, but not in oxygen at 3 atmospheres,the increase in CO₂ output could be attributed to an increasein glucose-6-phosphate and in fructose diphosphate; in oxygenat 3 atmospheres the increase may be due to an increased accessibilityof substrates and enzymes or to other causes. The decline inCO₂ output in oxygen at both 2 and 3 atmospheres was associatedwith large decreases in glucose-6-phosphate and 3-phosphoglycerate,probably due to a large decrease in adenosine triphosphate relatedto a ‘block’ of the tricarboxylic acid cycle.     

Studies in the Respiratory and Carbohydrate Metabolism of Plant Tissues2

Feeding strawberry and poplar leaves with iodoacetate, arsenite,or fluoride decreased the contents of sucrose and ascorbate.The content of hexose increased in high concentrations of iodoacetatebut decreased in moderate strengths. Iodo-acetate increasedthe rate of CO₂ output and caused a large decrease in phospholipidwith increase of inorganic phosphate. When iodoacetate was fedin nitrogen, CO₂ output did not increase. An explanation of the results is that the sucrose and ascorbateof the cell are mainly confined to the vacuole and that theabove poisons accelerate the leakage of these compounds fromthe vacuole so that they are metabolized more rapidly in thecytoplasm. An additional possibility is that iodoacetate ‘uncouples’oxidative phosphorylation, thereby stimulating respiration andmore rapid metabolism of both sucrose and ascorbate.     

The Relationship between Metabolism and the 'Exchangeability' of Ions in Plant Tissues2

The uptake of the rubidium ion by well-washed disks of carrotparenchyma has been determined at 0·2° C and 25°C., in the presence and absence of 10^–4M. dinitrophenol,from solutions of rubidium chloride containing 0·5 and5·0 meq./l. Readily-exchangeable and non-exchangeablefractions were separately indentified. The lowering of temperature and application of DNP reduced themagnitude of both ion fractions at each of the two concentrationsof rubidium chloride. Despite the fact that the uptake of ionsinto an exchangeable form at 5·0 meq./l. was about 3times greater, the combined effect of low temperature and thepresence of DNP reduced this fraction by a relatively constantabsolute amount. Under the same conditions the uptake of ionsinto a non-exchangeable form from each concentration was reducedby approximately the same percentage. Over a 6-hour period the rate of uptake of rubidium into a non-exchangeableform at 25° C. was relatively constant, whereas at 0·2°C. there was an initial rapid uptake lasting for about 60 minutesfollowed by a slow steady uptake. The Q₁₀ of this latter processmeasured after 360 minutes was 2·3. It is concluded that an appreciable part of the capacity ofthe tissue to hold ions by exchange is dependent on concurrentmetabolism. The significance of measurements of exchangeability in the interpretationof mechanisms of ion uptake is discussed.     

Occurrence and Metabolism of Indoleacetaldehyde in Certain Higher Plant Tissues Under Aseptic Conditions

Qualitative and quantitative evidence based on thin-layer chromatography and the Avena curvature test respectively, are presented for the normal and natural occurrence of indoleacetic acid (IAA), indoleacetaldehyde (IAAld) and tryptophol in the etiolated shoots of Pisum sativum, grown, harvested and extracted under aseptic conditions. Non-aseptic pea shoots contain much more IAA and IAAld than aseptic ones. Extraneous contribution to the indole pool of pea plants grown under non-sterile atmosphere, other than by the inherent agency of the plant itself, is therefore not excluded. Indoleacetaldehyde metabolizing activity of the tissues and cell-free preparations of etiolated Avena seedlings, leading to the production of IAA and tryplophol, is unaffected by the antibiotics actinomycin and streptomycin over a wide range of concentrations. Formation of IAA from IAAld is suppressed by a small degree (10 to 15 %) by chloramphenicol. But this antibiotic does not influence the concurrent production of tryptophol. It is deduced that epiphytic bacteria play little role in the transformation of IAAld to IAA and tryptophol by Avena tissues.     

A Transient Study of Formation of Conjugates during TNT Metabolism by Plant Tissues

The formation of TNT-derived conjugates was investigated in hairy root tissue cultures of Catharanthus roseus and in aquatic plant systems of Myriophyllum aquaticum. The temporal profiles of four TNT-derived conjugates, TNT-1, 2A-1, TNT-2 and 4A-1, were determined over 3 to 16-day exposure durations. When axenic C. roseus roots were exposed separately to 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene, the array and levels of conjugates varied. Exposure of axenic roots to either 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene resulted in the formation of only 4A-1 and 2A-1, respectively, and not TNT-1 and TNT-2. However, amendment of previously unexposed roots with TNT produced all four conjugates. The conjugates were preferentially accumulated within the biomass phase of root cultures. Significantly, conjugates TNT-1 and TNT-2 were observed in the biomass phase of intact M. aquaticum plants exposed to TNT. The results clearly indicate the presence of common TNT transformation products in two diverse plants species and tissue type. The distribution of conjugates formed via monoamine derivatives of TNT, however, may be a function of several factors, including the starting xenobiotic type and/or level. Initial bulk rate constants for disappearance of 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were also determined. Their magnitude followed the order: TNT >> 4-A-2,6-DNT > 2-A-4,6-DNT.     

Mordanting Plant Tissues

Selected current ideas on the mordanting of plant tissues are presented in the hope that they will be of practical assistance, especially to the beginner. The nature of the mordanting process and the results which may be expected following mordanting are briefly described. Specific technics are presented for the mordanting of natural dyes, synthetic dyes generally, the basic synthetic dyes and the acid synthetic dyes.     

Electroosmotic Phenomena in Plant Tissues

The effect of a direct electric current on electrolyte transport through plant tissues was studied by applying it to 10-mm segments of the mesocotyls of etiolated maize seedlings, similar segments of one-year linden shoots with the normal conducting system and without vascular bundles, and isolated elements of the xylem and cell wall segments. At current densities of 9–38 A/mm² (10–20 V), electrolyte solutions in plant tissues always moved toward the cathode. The results suggest that electroosmosis is one of the factors responsible for changes in solution transport through the conductive plant tissues that occur under the effect of electric current.     

Freeze-Sectioning of Plant Tissues

Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or glycerol (named in order of rapidity of equilibration). Pretreatment in 10% DMSO is usually for 1-6 hr at 2 C for histochemical studies; or in 10% ethanediol or glycerol for 15-24 hr at either room temperature or 37 C for morphological studies. These methods permit serial cryostat sections free from freezing and thawing artifacts to be cut as thin as 2 μ.     

Softening Paraffin-Embedded Plant Tissues

Soaking paraffin-embedded plant specimens 2-3 days at 37°C. in a mixture of glycerol, 10 ml., Dreft, 1 g., and water 90 ml. is an effective means of softening them prior to sectioning. One side of the paraffin block must be pared away to expose the tissue before immersion in the softening solution.     

Regulators of Cell Division in Plant Tissues XIV. The Cytokinin Activities and Metabolism of 6-Acylaminopurines

Certain 6-acylaminopurines have been shown to exhibit activity in several cytokinin bioassays. The active compunds included 6-N,2′-O-dibutyryladenosine 3’:5′-cyclic monophosphate, but adenosine 3′:5′-cyclic monophosphate was inactive. The metabolites formed from [2,8-³H] 6-benzoylaminopurine by radish seedlings and excised radish cotyledons were investigated. When compared with zeatin, this amide showed considerable stability in vivo. Conversion to 6-benzylaminopurine and its riboside was not detected but slight degradation to adenine was indicated. The principal metabolite was an unidentified compund.     

Freezing-Sectioning of Plant Tissues1

An improved and simple method is described by which serial 10µ frozen sections of plant tissues may be obtained. Thevalue of this method for use in plant histochemistry is discussed.