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.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Effects of heat shock on amino Acid metabolism of cowpea cells

When cowpea (Vigna unguiculata) cells maintained at 26°C are transferred to 42°C, rapid accumulation of γ-aminobutyrate (>10-fold) is induced. Several other amino acids (including β-alanine, alanine, and proline) are also accumulated, but less extensively than γ-aminobutyrate. Total free amino acid levels are increased approximately 1.5-fold after 24 hours at 42°C. Heat shock also leads to release of amino acids into the medium, indicating heat shock damage to the integrity of the plasmalemma. Some of the changes in metabolic rates associated with heat shock were estimated by monitoring the ¹⁵N labeling kinetics of free intracellular, extracellular and protein-bound amino acids of cultures supplied with ¹⁵NH₄⁺, and analyzing the labeling data by computer simulation. Preliminary computer simulation models of nitrogen flux suggest that heat shock induces an increase in the γ-aminobutyrate synthesis rate from 12.5 nanomoles per hour per gram fresh weight in control cells maintained at 26°C, to as high as 800 nanomoles per hour per gram fresh weight within the first 2 hours of heat shock. This 64-fold increase in the γ-aminobutyrate synthesis rate greatly exceeds the expected (Q₁₀) change of metabolic rate of 2.5- to 3-fold due to a 16°C increase in temperature. We suggest that this metabolic response may in part involve an activation of glutamate decarboxylase in vivo, perhaps mediated by a transient cytoplasmic acidification. Proline appears to be synthesized from glutamate and not from ornithine in cowpea cells. Proline became severalfold more heavily labeled than ornithine, citrulline and arginine in both control and heat-shocked cultures. Proline synthesis rate was increased 2.7-fold by heat shock. Alanine, β-alanine, valine, leucine, and isoleucine synthesis rates were increased 1.6-, 3.5-, 2.0-, 5.0-, and 6.0-fold, respectively, by heat shock. In contrast, the phenylalanine synthesis rate was decreased by 50% in response to heat shock. The differential effects of heat stress on metabolic rates lead to flux and pool size redistributions throughout the entire network of amino acid metabolism.     

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost

Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, γ-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of γ-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development.     

Chilling-Induced Lipid Degradation in Cucumber (Cucumis sativa L. cv Hybrid C) Fruit

Chilling at 4°C in the dark induced lipid degradation in cucumber (Cucumis sativa L.) fruit upon rewarming at 14°C. Rates of ethane evolution by fruits rewarmed after 3 days of chilling were up to four-fold higher than those evolved by unchilled (14°C) fruits (0.02-0.05 picomoles gram fresh weight⁻¹ hour⁻¹). This potentiation of lipid peroxidation occurred prior to irreversible injury (requiring 3 to 7 days of chilling) as indicated by increases in ethylene evolution and visual observations. Decreases in unsaturation of peel tissue glycolipids were observed in fruits rewarmed after 3 days of chilling, indicating the plastids to be the site of the early phases of chilling-induced peroxidation. Losses in unsaturation of tissue phospholipids were first observed only after chilling for 7 days. Phospholipase D activity appeared to be potentiated in fruits rewarmed after 7 days of chilling as indicated by a decrease in phosphatidylcholine (and secondarily phosphatidylethanolamine) with a corresponding increase in phosphatidic acid. These results indicate that lipid peroxidation may have a role in conferring chilling injury.     

Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants

The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [³⁵S]Cys injected into endosperms of seedlings was partly metabolized to [³⁵S]sulfate. The scutella absorbed both [³⁵S]sulfate and [³⁵S]Cys and transformed 68 to 87% of the radioactivity into [³⁵S]GSH. [³⁵S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.     

An Extracellular Protein from Phytophthora parasitica var nicotianae Is Associated with Stress Metabolite Accumulation in Tobacco Callus

The most abundant extracellular protein produced by Phytophthora parasitica var nicotianae at early stages of rapid growth in culture has a molecular weight of 46 kilodaltons and has been designated Ppn 46e. Culture conditions for the production of this protein have been optimized and the protein has been purified by gel filtration and ion-exchange chromatography. Ppn 46e is a soluble, acidic protein (pI 4.67). The amino acids Asx (aspartic acid or asparagine), alanine, glycine, Glx (glutamic acid or glutamine), and serine are the most abundant at 13.4%, 12.3%, 12.1%, 9.3%, and 9.3% of the residues, respectively. The purified protein is, by weight, 1.8% glucose, 1.6% mannose, and 0.5% galactose. A bioassay for Ppn 46e based on tobacco callus has been developed. In this assay as little as 20 nanograms (4.3 × 10⁻¹³ mole) Ppn 46e causes the accumulation of the sesquiterpenoid phytoalexin, capsidiol, as estimated by gas chromatography. Levels of capsidiol of 25 micrograms per gram fresh weight were elicited by 80 nanograms Ppn 46e per callus piece. Pretreatment of the protein with either pronase or by boiling resulted in a loss of elicitor activity. Periodate treatment, which inactivates glucan elicitors, did not affect the ability of Ppn 46e to cause capsidiol accumulation. Monospecific antibodies to Ppn 46e were raised in mice. Western blotting experiments employing these antibodies showed that Ppn 46e was present in infected tobacco plants. Dot blotting experiments revealed the presence of the Ppn 46e epitope(s) in Phytophthora megasperma, P. cactorum, P. cinnamomi, and P. infestans but not in Fusarium.     

Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.     

Phosphorus stress effects on assimilation of nitrate

An experiment was conducted to investigate alterations in uptake and assimilation of NO₃⁻ by phosphorus-stressed plants. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were deprived of an external phosphorus (P) supply for 12 days. On selected days, plants were exposed to ¹⁵NO₃⁻ during the 12 hour light period to determine changes in NO₃⁻ assimilation as the P deficiency progressed. Decreased whole-plant growth was evident after 3 days of P deprivation and became more pronounced with time, but root growth was unaffected until after day 6. Uptake of ¹⁵NO₃⁻ per gram root dry weight and translocation of absorbed ¹⁵NO₃⁻ out of the root were noticeably restricted in −P plants by day 3, and effects on both increased in severity with time. Whole-plant reduction of ¹⁵NO₃⁻ and ¹⁵N incorporation into insoluble reduced-N in the shoot decreased after day 3. Although the P limitation was associated with a substantial accumulation of amino acids in the shoot, there was no indication of excessive accumulation of soluble reduced-¹⁵N in the shoot during the 12 hour ¹⁵NO₃⁻ exposure periods. The results indicate that alterations in NO₃⁻ transport processes in the root system are the primary initial responses limiting synthesis of shoot protein in P-stressed plants. Elevated amino acid levels evidently are associated with enhanced degradation of protein rather than inhibition of concurrent protein synthesis.     

Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to alpha-Methyltryptophan

Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by α-methyltryptophan (aMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 ± 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 ± 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 nanomoles per milligram protein per hour and 7.74 ± 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I₅₀) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I₅₀ for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I₅₀ greater than the average for the entire extract.     

Free space iron pools in roots: generation and mobilization

A rapid and simple method for the determination of a ferric iron pool in the free space of roots is described. Formation of this pool depended on the source of iron in the nutrient solution. During growth in water culture at pH 5 to 6 with Fe-ethylenediaminetetraacetate, a free space pool of 500 to 1000 nanomoles Fe per gram fresh weight was formed in the roots of bean (Phaseolus vulgaris L. var. Prélude), maize (Zea mays L. var. Capella), and chlorophytum (Chlorophytum comosum [Thunb.] Jacques). No significant pool (less than 100 nanomoles per gram fresh weight) was formed with ferrioxamine. Upon impending Fe deficiency, bean and chlorophytum were able to mobilize this pool. Fe-deficient bean plants mobilized iron from the free space iron pool of another plant in the same vessel.     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

Uptake of glutamine by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up [¹⁴C]glutamine at an initial rate of about 10 micromoles·gram⁻¹·hour⁻¹ in the standard assay conditions (pH 5, 30°C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent K_m of about 2 millimolar and a V_max of about 25 micromoles·gram⁻¹·hour⁻¹.

The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine.

Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella.

     

Effect of l-aminocyclopropane-l-carboxylic acid,silver nitrate,and norbornadiene on plant regeneration from maize callus cultures

The effect of the ethylene antagonists norbornadiene and silver nitrate and the ethylene precursor l-aminocyclopropane-l-carboxylic acid (ACC) on Zea mays plant regeneration was studied. A 12-fold increase in plant regeneration, as measured by number of plants obtained per gram fresh weight from callus cultures of maize inbreds Pa91 and H99, was obtained by 250 M norbornadiene and 100 M silver nitrate treatments. An increase in amout of nonregenerable tissue and a 68% decrease in plant regeneration were associated with callus treated with 1 mM ACC. Ethylene emanation from 1 mM ACC treated callus reached a maximum of 170 nl g^–1 h^–1 after 3 days compared to 7 nl g^–1 h^–1 for the control. The free proline content was up to 80% lower in 1 mM ACC treated callus grown for 30 days on medium with or without 12 mM proline, respectively, as compared to each control. These studies indicate that ethylene action inhibitors such as norbornadiene and silver nitrate can be used to increase plant regeneration efficiency from maize callus cultures.Abbreviations ACC l-aminocyclopropane-l-carboxylic acid - gfw gram fresh weight     

Accumulation of Putrescine during Chilling Injury of Fruits

Putrescine (Put) increased 68% in lemon (Citrus limon (L.) Burm. f. cv Bearss) flavedo, 39% in grapefruit (C. paradisi Macf. cv Marsh) flavedo, 49% in grapefruit juice, and 149% in pepper (Capsicum annuum L. cv Early Calwonder) pericarp when fruits were stored at chilling temperatures. In lemon flavedo, the coefficient of correlation (r²) between Put concentration with severity of chilling was 0.90 and Put levels almost doubled; the injury index going from 1 to 2 units. Pepper pericarp, which was the most chilling-sensitive tissue tested (injury index going from 1 to 3.8 units), showed the greatest difference in Put accumulation (166 to 413 nanomoles per gram fresh weight) between storage temperatures of 7.2 and 1°C. The least difference (338 to 470 nanomoles per gram fresh weight) was found in grapefruit flavedo between storage temperatures of 15.5 and 4.4°C; the injury index going from 1 to 1.3 units.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Further studies on the growth and differentiation of single,isolated cells of tobacco in vitro

Summary Single cells of hybrid tobacco callus, isolated from germinating seeds, were grown individually in microchambers in the absence of any other cells, in fresh, liquid, coconut milk-medium. These produced a conlony of 50–75 cells in 10–15 days. In all instances the plane of the first cell division, and in most cases also that of the second division, were at right angles to the long axis of the cell. There was more variation in the size, shape and pattern of the second and subsequent divisions in single cells isolated from fresh stem pith and seed callus than those from old stem callus. Upon transfer from the microchambers to agar medium the colony of cells gave rise to a huge mass of callus tissue in about 3 months. In no instance did the cell colonies or any stages preceding them resemble or simulate any stages of normal embryogeny of tabacco. Single-cell clones obtained by this method from seed callus or fresh pith callus produced shoots with leaves and roots in synthetic medium with various indoleacetic acid-kinetin combinations. Normal plants established from the above cultures in the greenhouse flowered in due course of time. This method offers the possibility of producing a very large number of clones or identical plants in species where vegetative propagation is not otherwise possible, apart from its use in studies on the genetics and morphogenesis in higher plants.     

Endosperm cell division in maize kernels cultured at three levels of water potential

The influence of osmoticum treatments on early kernel development of maize (Zea mays L.) was studied using an in vitro culture method. Kernels with subtending cob sections were placed in culture at 5 days after pollination. Sucrose (0.29, 0.44, or 0.58 molar) and sorbitol (0, 0.15, or 0.29 molar) were used to obtain six media with water potentials of −1.1, −1.6, or −2.0 megapascals. Kernel water potential declined in correspondence with the water potential of the medium; however, fresh weight growth was not significantly inhibited from 5 to 12 days after pollination. In stress treatments with media water potentials of −1.6 or −2.0 megapascals, endosperm tissue accumulated water and solutes from 10 and 12 days after pollination at a rate similar to or greater than that of the control (−1.1 megapascals). In contrast, endosperm cell division was inhibited in all treatments relative to control. At 10 days after pollination, endosperm sucrose concentration was greater in two of the −2.0 megapascal treatments with 0.44 or 0.58 molar media sucrose compared to control kernels cultured in 0.29 molar sucrose at −1.1 megapascals. Significant increases in abscisic acid content per gram of fresh weight were detected in two −2.0 megapascal treatments (0.29 molar sucrose plus 0.29 molar sorbitol and 0.58 molar sucrose) at 10 days after pollination. We conclude that in cultured maize kernels, endosperm cell division was more responsive than fresh weight accumulation to low water potential treatments. Data were consistent with mechanisms involving abscisic acid or lowered tissue water potential, or an interaction of the two factors.