Autoradiographic and microspectrophotometric studies of DNA synthesis in excised tobacco pith tissue

Summary Pith tissue was cultured on modified White’s nutrient medium supplemented, except for controls, with 2 mg/l of IAA and/or 0,5 mg/l of kinetin. For autoradiographs sections were used from tissue grown on medium containing tritiated thymidine. Nuclear DNA contents (Feulgen) were measured by the microspectrophotometric two-wavelengths method. No fading of Feulgen dye in nuclei was found in 11 weeks, in contrast to considerable fading observed in earlier work when a different batch of basic fuchsin had been employed. Counts of radioactive nuclei in autoradiographs agreed well with microspectrophotometric results on the occurrance of DNA synthesis. In control cultures, with or without tritiated thymidine, DNA doubling took place in about 20% of the nuclei during the first two days but in few, if any, thereafter. It was confirmed that kinetin, as well as IAA, increases the frequency of nuclei undergoing DNA synthesis. However, IAA, in contrast with kinetin, still induced considerable DNA doubling after two days. Continued cell reproduction was maintained only in the presence of both substances. This work has been supported in part by research grants toK. Patau from the US Public Health Service (grant No. C-3313) and the American Cancer Society; and by grants toF. Skoog from the American Cancer Society and the Research Committee of the University of Wisconsin Graduate School with funds from the Wisconsin Alumni Research Foundation.     

Involvement of ethylene in growth induction of stationary tobacco pith tissue in vitro

The effects of ethylene on growth initiation of tobacco pith tissue in vitro were investigated. Pith explants were incubated on a double inorganic modified White’s media containing 0.2 mg l⁻¹ kinetin with and without indole-3-acetic acid (IAA) and the ethylene synthesis inhibitor aminooxyacetic acid (AOA). The burst of wound ethylene had no effect on growth initiation, was not affected by the AOA, and decreased to its minimum level during the initial 24 h in culture. Tissue growth was initiated after 72 h and continued on IAA-containing media only. A marked increase in ethylene evolution occurred only in tissues subjected to an IAA-containing medium prior to growth initiation. AOA inhibited this ethylene synthesis and the following growth of the tissues. The initial water uptake by the pith explants occurring even in the absence of IAA was also inhibited by AOA. The metabolic indicators for growth initiation such as enhanced respiration, increased activity of nitrate reductase, and initiation of cathodic isoperoxidases were all inhibited by AOA. It was concluded that the primer function of IAA in growth initiation is via inducing the biosynthesis of a marked ethylene signal, which in the absence of which active growth will not occur. The inhibiting effect of AOA is continuous and a transfer of the pith explants to fresh IAA-containing media did not result in a new ethylene burst nor tissue growth induction. The morphological changes in the tissues and cells during the initial stages of their development on the different media are demonstrated.     

Cytokinin effects on growth of quiescent tobacco pith cells

Excised pith tissue from Nicotiana glauca or the tumor-prone hybrid N. glauca × N. langsdorffii has no growth requirement for exogenous cytokinins. Addition of kinetin to cultures of these lines results in growth inhibition at a kinetin concentration 1000-fold lower than the optimal level for kinetin-requiring lines. Cytological comparison of the kinetin-inhibited 2N hybrid and glauca tissues with pith from the kinetin-requiring N. tabacum var. Wisconsin 38 suggests that the nature of the cytokinin action is similar in both situations and that the primary function of cytokinin, when it stimulates growth, may be to curtail cell expansion, thereby facilitating a balance of cell expansion and division requisite for maximal growth.     

Repression of isoperoxidase formation in excised tobacco pith by exogenous, auxin-controlled RNA

Previous work has shown that tobacco pith tissue contains two constitutive isoperoxidases migrating toward the anode at pH 9·0. Within 24 hr of aseptic culture on basal medium, such tissue develops five new isoperoxidases, three cathodic and two anodic. The appearance of the new isoperoxidases involves de novo protein formation; it is inhibited by anaerobic conditions, by such inhibitors as Actinomycin D, and by the plant hormone indole-3-acetic acid (IAA). We now find that phenol RNA extracted from parent pith and injected or vacuum infiltrated into cultured pith explants prevents the appearance of the new isozymes; RNA from cultured pith has no such effect. Hydrolysis with 0·3 N KOH, ribonuclease or proteolytic enzymes partially destroys this activity, while treatment with both ribonuclease and proteolytic enzymes completely destroys it. Fractionation of the RNA indicates that part of the repressor activity is associated with an mRNA-like fraction.     

Responses of intact and excised young bean plants to fusicoccin

The effect of fusicoccin (FC) on the growth of epicotyls and leaves of Phaseolus vulgaris L. intact and excised seedlings has been examined, and several unexpected responses were observed. FC was added either to small wounds on one side of the epicotyl of 10 day old red-light grown seedlings or to the base of shoots excised at the base of the hypocotyl. Plants were kept in either dim red light (4 mol m^-2sec^-1) or bright white light (175 mol m^-2sec^-1) during the FC treatment. FC added to the base of the shoots was found to inhibit leaf expansion in either light condition. At the same time stem elongation was enhanced. The active concentration range was 10^-7–10^-5M. The basal fed FC caused a rapid and severe bending of the epicotyl starting at the base of the elongation zone. The direction of curvature was random, not related to the plane of the cotyledons or the direction of the gravity vector. Application of FC to one side of the epicotyl caused a similar but smaller bending away from the treated side. The bending occurred at either end of the elongation zone in accordance with site of FC application above or anywhere below it along the epicotyl and hypocotyl. It is concluded that the curvature of the epicotyl induced by FC fed in the transpiration stream may either be due to a differential loss of the capacity of cells at the base of the elongation zone to grow in response to FC or as a result of elongation of the first responsive cells encountered by the FC.     

Structural physiological, and biochemical gradients in tobacco pith tissue

Explants of tobacco pith taken at various distances from the apex of a mature stem show a sharp gradient in growth potential in vitro; growth is highest in the extreme apical and basal explants, and is minimal in explants removed ca. 75 cm from the apex. Calluses produced by the vigorously growing basal explants are harder and more compact than those produced from more apical explants. The gradient in growth potential is directly correlated with gradients in RNA, protein of cell sap and soluble N per unit fresh weight, but is inversely correlated with peroxidase activity. Cell size increases from apex to base of plants.

The peroxidase activity of pith explants is electrophoretically resolvable into 2 isoperoxidases, moving anodically at pH 9.0. During in vitro culture, this activity rises, due to the formation of several new isozymes moving toward the cathode. The appearance of these isozymes occurs most rapidly in apical and extreme basal explants.

     

Comparative studies on tobacco pith and sweet potato root isoperoxidases in relation to injury, indoleacetic Acid, and ethylene effects

Sweet potato (Ipomoea batatas) root parenchyma and tobacco (Nicotiana tabacum) stem pith, both known to increase peroxidase activity after excision, differed from each other in their isoperoxidase patterns and in the isoperoxidase responses to injury and exogenous ethylene.     

Membrane proteins of chloroplasts of intact and TMV-infected tobacco plants

The effects of viral infection on the membrane proteins from tobacco plant chloroplasts differing in their stability for TMV infection were studied. It was shown that the changes in the chloroplasts of labile and resistant tobacco varieties are oppositely directed. The data from amino-acid analysis, SH-group determination and infrared spectra of the membrane proteins of the resistant variety are indicative of conformational changes caused by disruption of the hydrogen bonds, which stabilize the protein, by changes in aggregability, etc. due to infection and metabolic disturbances in the infected cell. The conformational changes in the chloroplasts of the stable variety are adaptive and affect the biological activity, enzymatic and immunological properties and energy metabolism of the chloroplasts.     

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O (2) (.-) ) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O (2) (.-) generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)-respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O (2) (.-) in intact and excised roots; ECPOX was an important component of them in both, together with NOX-RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane-redox systems, with different terminal oxidases, activated by different effectors or stresses.     

The relation of DNA synthesis and mitosis in tobacco pith tissue cultured in vitro

Summary DNA synthesis and mitosis were initiated in cultured tobacco pith tissue by means of IAA and kinetin. DNA classes were determined by microspectrophotometric measurements (Feulgen); autoradiographs (tritiated thymidine) served to ascertain whether or not nuclei had undergone DNA synthesis during culture.All mitoses in new cells (resulting from divisions in culture) were diploid and had been preceded by DNA synthesis in culture.Whereas many of the old cells (which had not previously divided in culture) found in diploid or polyploid mitosis had undergone DNA synthesis during culture, others had not. Such non-radioactive mitoses still occurred after 16 days.In view of this, a 4 C nucleus in differentiated tissue should be considered as potentially both diploid and tetraploid, for it appears impossible to predict whether it would, upon restoration of conditions conducive to DNA synthesis and mitosis, enter a diploid mitosis or, after undergoing DNA synthesis, a tetraploid one.A high nuclear DNA content seems to have a much more inhibiting effect on the onset of DNA doubling than on that of mitosis.Somatic polyploidization is understood as the result of two DNA doublings between which mitosis was omitted, or aborted, or in effect undone by a failure of cytokinesis leading to fusion during a later mitosis.This work has been supported by research grants to K. Patau from the U.S. Public Health Service (grant No. C-3313) and the American Cancer Society.     

Effect of carbon dioxide and light on ethylene production in intact sunflower plants

High CO₂ concentration (0.5%) increased the rate of ethylene production, measured in a continuous flow system, in intact sunflower (Helianthus annuus L.) plants. However, the rate of ethylene production subsided to near control levels after approximately 24 hours. The effect of high CO₂ could only be observed in light. Although high CO₂ concentration had no effect on the rate of ethylene production in darkness, prolonged exposure (approximately 16 hours) of plants to high CO₂ in the dark prevented the increase in ethylene production when the plants were exposed to light and high CO₂.     

Effects of carbon dioxide on ethylene production and action in intact sunflower plants

A continuous flow system was used to study the interactions between carbon dioxide and ethylene in intact sunflower (Helianthus annuus L.) plants. An increase in the concentration of carbon dioxide above the ambient level (0.033%) in the atmosphere surrounding the plants increased the rate of ethylene production, and a decrease in carbon dioxide concentration resulted in a decrease in the rate of ethylene production. The change in the rate of ethylene production was evident within the first 15 minutes of the carbon dioxide treatment. Continuous treatment with carbon dioxide was required to maintain increased rate of ethylene production. The rate of carbon dioxide fixation increased in response to high carbon dioxide treatment up to 1.0%. Further increases in carbon dioxide concentration had no additional effect on carbon dioxide fixation. Carbon dioxide concentrations higher than 0.11% induced hyponasty of the leaves whereas treatment with 1 microliter per liter ethylene induced epinasty of the leaves.     

Isoelectric patterns of peroxidase isoenzymes from tobacco tissue cultures

Summary Peroxidases from tobacco tissue cultures have been separated by thin-layer isoelectric focusing into 12–14 isoenzymes, which have been divided into three groups according to differences in isoelectric points. The isoelectric patterns of callus tissues with and without buds have been compared with those of leaves and stems developed in vitro. Qualitatively, there was a basic similarity of the isoelectric patterns, the same isoenzymes being present in all samples. Distinct quantitative differences in the content and substrate specificity were noted for some of the isoenzymes.     

Factors influencing the incidence of habituation for cytokinin of tobacco pith tissue in culture

Pith tissue of Nicotiana tabacum L. cv. Havana 425 exhibits a gradient in its tendency to habituate for cytokinin on an auxin-containing medium at 35° C, about 10° C above the standard culture temperature. Explants of pith from below the 8th to 11th internode, counting from the bottom of the plant, rarely habituate for cytokinin; explants from above this threshold habituate rapidly. The explants must also be above a critical size, about 20–30 mg, to habituate. There was a pronounced interaction between size and position effects; the threshold position for cytokinin habituation shifted upward with decreasing explant size.     

Aged preparations are electrophysiologically more representative of intact tissue than are freshly excised preparations

Studies using excised tissues usually suffer from a discrepancy in the results between freshly excised preparations and aged ones (i.e. preparations which are allowed to rest and-or regenerate for about a day between excision and experimentation). This is especially true for measurements of the membrane potential (V_m) and its changes upon exposure to light in slices of extensor tissue of the laminar pulvini of Phaseolus coccineus L. Measurements of the V _m of extensor cells in situ reveal virtually identifical results to those obtained from aged excised preparations (more negative resting V _m; dramatic, light-induced transients of V _m), whereas freshly excised preparations display a small and light-insensitive V _m. It is concluded, therefore, that for the given example aged preparations are more suitable than fresh ones for investigating the properties of intact tissues.Symbol V_m membrane potential