Isolation and some physiological properties of natural plant growth inhibitors

Using paper chromatography and conventional methods of isolation, natural growth inhibitors were isolated from green leaves of different plants (Brassica oleracea, Zea mays, Pisum sativum andSalix rubra). All isolated inhibitors were found to be phenolic compounds and the chemical structure of most of them was determined; only the final structure of theBrassica inhibitor has not yet been ascertained. 500 mg of natural inhibitor ofPisum sativum was isolated from 1500 g of leaves and was identified as quercetin-glucosil-p-coumarate (QGC), described earlier byFuruya, Galston andStowe (1961). The structure of the natural inhibitor ofZea mays (4 mg from 100 g of leaves) was identical with p-coumaric acid and the chemical nature of the plant growth inhibitor fromSalix rubra (700 mg from 1,5 kg of leaves and young bark) was that of 2-chalconaringenin-glucoside or isosalipurposide, described earlier byCharaux andRabaté (1931) andHarborne (1966). All isolated substances had inhibiting properties in the straight growth test of wheat coleoptile sections and decreased the growth of isolated stem sections prepared from plants—donors of inhibitors. Thus, maximum growth inhibition (LG max) was attained, if wheat coleoptile sections were incubated with:Brassica inhibitor in the concentration of 0·5 mg/ml, withPisum inhibitor (QGC) in the concentration of 16 mg/ml, withZea inhibitor (p-coumaric acid)—0·35 mg/ml and with Salix inhibitor (isosalipurposide) in the concentration of 0·5 mg/ml. In small concentrations no mentioned substances were able to enhance the growth as actively as indolic auxins (on 250–300%); only slight growth activation in biotests was sometimes observed for low concentrations. Inhibition in p-coumaric acid was much more active in a free form than in the bound form as an acyl-rest of QGC. As a rule, the wheat coleoptile test was much more sensitive (3–5 times) to the plant growth inhibitors, than tests prepared from tissue and organs of plants—donors. The retardation activity of plant growth inhibitors is not correlated with their molecular weight. Dormin (or±abscissin II) was also tested on wheat coleoptile sections. In neither of the applied concentrations (10-0·05 μg/l range) was dormin able to depress straight growth of wheat coleoptile sections, but even in a 1·7 μg/l concentration it inhibited the IAA-activated growth of sections. However, additional experiments showed that dormin in higher concentrations (40 μg/l and more was able even to depress endogenous straight growth of wheat coleoptise sections. The differences between the properties of natural phenolic growth inhibitors and dormin were discussed.     

Contribution to the study of seasonal dynamics of endogenous stimulators and inhibitors in peach trees

For three consecutive years the content of natural stimulators and inhibitors was observed in leaves and shoots of peach trees. Research was directed to the question of development of flower buds. The substances under study were isolated from the acidic fraction of ether extracts by means of paper chromatography and their concentration was determined by biological test. The activities of substances which either stimulate or inhibit the growth of oat coleoptiles, were added up. The curve expressing the content of stimulators in shoots in relation to the fresh weight showed its maximum in the period of full growth of shoots (15 June–15 July according to the fluctuating vegetative conditions) and it showed a decreasing tendency at the end of the season. The decline of the curve showing the content of inhibitors is of similar direction but less steep. The trend of substances under study is the same in leaves as in shoots, but their quantity is lower. Stimulation and inhibition effects in individual sampling intervals were added up to make a common curve which seems to express the combined action of stimulators and inhibitors in the plant, which determines its growth pattern.     

Contribution to the study of seasonal dynamics of endogenous stimulators and inhlbitors in peach trees

For three consecutive years the content of natural stimulators and inhibitors was observed in leaves and shoots of peach trees. Research was directed to the question of development of flower buds. The substances under study were isolated from the acidic fraction of ether extracts by means of paper chromatography and their concentration was determined by biological test. The activities of substances which either stimulate or inhibit the growth of oat coleoptiles, were added up. The curve expressing the content of stimulators in shoots in relation to the fresh weight showed its maximum in the period of full growth of shoots (15 June–15 July according to the fluctuating vegetative conditions) and it showed a decreasing tendency at the end of the season. The decline of the curve showing the content of inhibitors is of similar direction but less steep. The trend of substances under study is the same in leaves as in shoots, but their quantity is lower. Stimulation and inhibition effects in individual sampling intervals were added up to make a common curve which seems to express the combined action of stimulators and inhibitors in the plant, which determines its growth pattern.     

Efect of decapitation on the level of endogenous stimulators and inhibitors and some morphogenetic phenomena in peach trees

In experiments from 1966–1968 a comparison was performed concerning the level of endogenous stimulators and inhibitors in leaves and shoots of peaches decapitated in three different times and in controls. For the determination of these substances a biological test was employed after separation of the acid fraction of ethyl ether extracts by means of paper chromatography. Mathematical addition of total stimulations and inhibitions gave a new value, called “growth disposition” by the authors. Its graphical demonstration gives a simple curve which documents the growth rhythm of control plants as well as the response of plants to the time-regulated decapitation. In the case of shoots this response has always been positive,i.e. at all periods of sampling for analyses the growth disposition with decapitated plants was higher in comparison with controls.     

The Growth Substances separated from Plant Extracts by Chromatography. I

Methods for the chromatographic separation on paper of indolecompounds and for the direct biological assay of the chrornatograinsusing the Avena coleoptile straight-growth method are described.Reagents for the detection of the indole-3-carboxylic acids,indole-3-acetonitrile, and gramirte as coloured spots on chromatogramsare compared and the areas of such spots are shown to be proportionalto the logarithms of the quantities of substance present. The procedure of chromatography described is shown not to involvea loss of indole-3-acetic acid activity if chromatography isdone in darkness and chrornatograms are not stored in lightand air. Methods are described for the extraction of growth aubstancesfrom plant materials, the purification and chromatography, onpaper, of the extracts and the bioassay of the chromatogramsusing Avena coleoptile sections. The ether extracts, containing acidic substances, of etiolatedbroad bean and pea shoots and roots, etiolated sunflower shoots,maize roots, and potato etiolated shoots and tuber have beenchromatographed and the chromatograms bioassayed. On all chromatogramsthree areas active in Avena coleoptile section growth are found.One area of growth promotion is shown due to indole-3-aceticacid [IAA]. Another area of growth promotion and, one of growthinhibition are due to unknown substances, which are named accelerator () and inhibitor ß (ß) respectively. On chromatograms of potato tuber a fourth growth-promoting area,in addition to those described above, is detected and is shownto be probably due to indole-3-acetonitrile [IAN]. IAN or indole-3-pyruvicacid may occpr together with IAA on chrormatograms of extractsof immature maize kernels and cauliflower head respectively. On cabbage extract chromatograms the growth-promoting activitycorresponding in position with IAA is shown to be due to IAAand to IAA alone. In etiolated broad bean shoots IAA is the predominating growthsubstance in the stem and ß predominates in the firstlateral bud. The latter is suggested as an explanation of apicaldominance, and the predominance of ß in potato tuberskin is suggested as an explanation of dormancy in tubers. In the broad bean root the acidic growth-substance patterns,for the whole root and for the sections 0–2 cm. and 2–4cm. from the tip, are the same. The acidic growth substances extractable from broad bean shootsare the same whether the plant material is boiled or frozenbefore extraction.     

Gibberellins in Diffusates from Shoots of Apple Trees

Excised shoot apices, leaves and internodes from shoots of apple trees (Malus×domestica) give off gibberellins by diffusion on agar. A methanol extract of the agar was prepared, the extract separated on thin layer plates, and the gibberellin activity estimated by means of Rumex and lettuce hypocotyl bioassays. The largest amounts of gibberellin are found in diffusates from the shoot apex, the two upper leaves and the two upper internodes. Several promotive fractions are found on the chromatograms as well as growth inhibitors. Removal of young leaves retards elongation of the internodes. Probably gibberellins produced in young leaves exercise some control over this process. The growth regulators Alar and CCC also retard internode elongation. Diffusates from shoots treated with these substances were also examined. Preliminary results suggest that the amount of diffusible gibberellins from treated shoots is not reduced.     

ON THE RELATION BETWEEN GROWTH AND RESPIRATION IN THE AVENA COLEOPTILE

1. The growth of Avena coleoptile sections in sucrose and auxin solutions is inhibited by various substances which are known to act as dehydrogenase inhibitors. 2. Iodoacetate, which is particularly active in this connection, inhibits all growth at a concentration of 5 x 10^–5 M, but produces only a slight inhibition of oxygen uptake. 3. The growth inhibition by iodoacetate is completely removed by malate and fumarate, and to a lesser extent by succinate and pyruvate. 4. These acids themselves increase the effect of auxin on growth and also increase the respiration of the coleoptile sections, but only if auxin is present. 5. When sections have been soaked in malate or fumarate, the addition of auxin considerably increases the total respiration. Further, the concentration range over which this increase takes place parallels that active in promoting growth. 6. The four-carbon acids provide a respiratory system which is part of the chain of growth processes, and which is in some way catalyzed by auxin. It represents a small but variable fraction of the total respiration.     

Effects of Short-term Red Radiation and Choline Compounds on Cytokinin Content, Chlorophyll Accumulation and Photomorphogenesis in Wheat Seedlings

Effects of choline compounds (2-chloroethyltrimethylammonium chloride and 2-ethyltrimethylammonium chloride) as well as red radiation (R) pulse on the dynamics of cytokinin changes, growth and chlorophyll (a + b) accumulation were studied during the growth and greening of etiolated wheat seedlings (Triticum aestivum L., var. Mironovskaya-808). The seedlings were grown for 120 h in the dark and then exposed for 72 h to white light. Pre-treatment of caryopses with cholines and pre-irradiation of etiolated seedlings with R inhibited elongation of both coleoptile and first leaf; but the same factors accelerated these growth responses when seedlings were exposed to white light. Chlorophyll (Chl) accumulation and the first leaf appearance from coleoptile were accelerated by the pre-treatments as well. Far-red radiation (FR) reversed all effects of R but choline pre-treatment eliminated partly R/FR photoreversibility. Two compounds with high cytokinin activity (tested on a fresh weight basis by the bioassay with Amaranthus caudatus L.) were found in shoots and first leaves. One of them had R_f, UV absorbance spectrum and the biological activity similar to N⁶-(Δ²-isopentenyl)adenosine. Another cytokinin-like substance was not identified with the used standards. Stimulation of greening by R pulse and cholines was accompanied with accelerated accumulation of both cytokinin-like substances. We conclude that the influence of R and cholines on the concentration of substances with cytokinin activities detected in the leaves might be involved in the stimulation of Chl accumulation.     

Sources of Free IAA in the Mesocotyl of Etiolated Maize Seedlings

Sources of free indole-3-acetic acid (IAA) for the mesocotyl of intact etiolized maize ((Zea mays L.) seedlings are evaluated. The coleoptile unit, which includes the primary leaves and the coleoptilar node, is the main source of free IAA for the mesocotyl. The seed and the roots are not immediate sources of IAA supply. Dependence of the apical growing region of the mesocotyl on the coleoptile unit as a source of free IAA is almost total. One-half or more of the supply of IAA comes from the coleoptile tip, the rest mainly from the primary leaves. Removal of the coleoptile tip results in inhibition of mesocotyl elongation. The hypothesis that growth of the mesocotyl is regulated by auxin supplied by the coleoptile is supported. Conjugated forms of IAA appear to play little part in regulating the levels of free IAA in the shoot.     

The occurrence of auxins in the old leaves of apple trees

A relatively high auxin level was found in old leaves from the lower part of long shoots of young apple trees using the straight growth bioassay on wheat coleoptile segments. The auxin level remains almost constant during summer and autumn.     

Involvement of plant growth substances in the alteration of leaf gas exchange of flooded tomato plants

Ethylene, abscisic acid, and cytokinins were tested for their ability to either induce or prevent the changes which occur in gas exchange characteristics of tomato (Lycopersicon esculentum Mill. cv. Rheinlands Ruhm) leaves during short-term soil flooding. Ethylene, which increases in the shoots of flooded plants, had no effect on stomatal conductance or photosynthetic capacity of drained plants. Abscisic acid, which also accumulates in the shoots of flooded plants, could reproduce the stomatal behavior of flooded plants when sprayed on the leaves of drained plants. However, photosynthetic capacity of drained plants was unaffected by abscisic acid sprays. Cytokinin export from the roots to the shoots declines in flooded plants. Spray applications of benzyladenine increased stomatal conductance in both flooded and drained plants. In addition, the decline in photosynthetic capacity during flooding was largely prevented by supplementary cytokinin applications. The possible involvement of these growth substances in modifying leaf gas exchange during flooding is discussed.     

SEEDLING GROWTH STUDIES OF EARLY-RIPENING PEACHES. I. INTERRELATIONSHIP BETWEEN EMBRYO MATURITY,GROWTH SUBSTANCES AND SEEDLING GROWTH

Weaver , Gerald M., and L. Fredric Hough . (Rutgers, The State Univ., New Brunswick, N. J.) Seedling growth studies of early-ripening peaches. I. Interrelationship between embryo maturity, growth substances and seedling growth. Amer. Jour. Bot. 46(10): 718–724. Illus. 1959.—Peach fruits of ‘Raritan Rose’ were harvested 75, 83, 91, 99 and 107 days after full bloom. Embryos were excised, and cultured on artificial media. Growth responses following 8 wk. cold treatment varied considerably with embryo maturity. No correlation existed between seedling survival and the initial responses of shoots. Root growth of 83-day embryos exceeded that of all other stages of maturity. This was reflected in maximum seedling survival and vigor. Initial shoot growth was greatest from embryos harvested at 91 days and at maturity (107 days), but all shoots developed into rosettes which may expand slightly or develop normally from axillary buds. Bioassays of growth substances suggest a strong correlation between early shoot development and auxin concentration. Root growth-responses appeared to be correlated with a promotor-inhibitor balance. The results of wheat coleoptile assays of embryo extracts chromatographed in an isopropanol: ammonia: water solvent (8:1:1) suggest the presence of three growth promotors, in greatest concentration at Rf 0.70, but also at Rf values 0.10 and 0.30. Two growth inhibitors were present in embryo tissue at Rf values 0.27 and 0.96, the former decreasing in concentration with increasing embryo maturity.     

Growth Regulators in Populus tremula

Shoots grown out from aspen root sections in light or in darkness were extracted and the content of growth-regulating substances in various fractions was determined with the Avena coleoptile straight-growth assay. The most obvious difference was obtained for the inhibitor β fraction. This was about ten times more active in the light-grown than in the dark-grown shoots. Some indications of more growth inhibitory material in ether-insoluble fractions of light-grown plants were also obtained. The stimulation obtained at the Rf of indol-3-yl-acetic acid was somewhat lower in the etiolated shoots than in the light-grown ones.     

Effect of umbelliferone,as rooting stimulator,on endogenous growth substances in bean cuttings

Endogenous growth substances were estimated byAvena coleoptile test in hypocotyls of cuttings ofPhaseolus vulgaris which were treated with Umbelliferone, a coumarin which has been found to have a stimulating effect on rooting. A similar growth promoting zone which corresponded chromatographically to indole-3-acetic acid, was found in both treated and control cuttings. A growth-inhibiting zone detected in control extracts was absent in UM treated cuttings. The results are discussed in relation to Umbelliferone effect on rooting stimulation.     

How do plant growth substances work?

Abstract. Despite intensive research effort, the role of growth substances in the life of the intact growing plant is far from clear. Two reasons are suggested which may account for the lack of progress. The first is the failure to recognize the unique aspects of plant development. These which are expressed by the regenerative, organizational and developmental plasticity of the meristem probably result from the possession of growth substances. The second is the concept of growth substances as hormones. This represents the main conceptual thrust of research and is considered critically, starting with the historical system, the coleoptile and dealing with other major growth substance systems in turn. It is concluded that a hormonal concept which includes control by changes in growth substance concentration fails to explain the developmental phenomena under examination. A role for growth substances as integrating agents is suggested and the notion of quantitative tissue sensitivity variation is developed to explain the major growth patterns of developing shoots.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: II. Auxin- and Abscisic Acid-like Activity

The wilty tomato mutant, flacca, and the control variety, Rheinlands Ruhm, were compared with regard to the endogenous activity and concentration of auxin- and abscisic acid-like substances during ontogeny. The mutant wilts fast under water deficit because of inability to close its stomata. Symptoms characteristic of excessive auxin are evident in the developing mutant. Among these symptoms are branch and leaf epinasty, excessive rooting along the stem, and increased apical dominance. By using a leucine-incorporation assay, spray of whole plants with 2,4-dichlorophenoxyacetic acid, and wheat coleoptile bioassay, indications were found of an excess of activity and concentration of auxin-like substances in shoots of young and mature mutant plants. The wheat coleoptile bioassay also revealed a much lower amount of substances with abscisic acid-like activity in the mutant compared with the normal plant. In contrast to the appearance during ontogeny of morphological symptoms characteristic of auxin excess in the mutant, the absolute amount of auxin-like substances and their activity in incorporation of leucine decreased with age. A parallel decrease of the concentration and activity of auxin-like compounds was also found in the normal plant. The concentration of abscisic acid-like substances increased with age in both genotypes. The disagreement between the increasing morphological symptoms and the decrease of auxin-like activity and concentration is discussed, together with the possibility of a causal relationship between auxin-and abscisic acid-like activity and stomatal behavior.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

Effects of shoot position on shoot and leaf morphology of Avicennia marina in the hyperarid Red Sea coastal region of Egypt

We examined the effects of shoot position on shoot growth and morphology of Avicennia marina (Forssk.) Vierh. in the Red Sea coastal region of Egypt. To determine differences in morphological characteristics, we collected shoots from the upper and lower canopies of A. marina individuals in the wild and compared the morphological characteristics of these shoots. The study plot was established in an A. marina mangrove forest. Heights and diameters of individual trunks (n = 14) in the plot were measured at ground level. Then, five shoots with young but fully expanded leaves were collected from the upper and lower canopies of the individuals. We measured shoot length, and dry weight and also area, dry weight, thickness, and Soil Plant Analysis Development (SPAD) value of collected leaves. Our measurements showed that leaf area, dry weight, specific leaf area, and SPAD value of leaves from the upper canopy were smaller than those of lower-canopy leaves in most individuals. From the differences in traits between upper and lower leaves, we concluded that leaves in the upper canopy are typically adapted to high light levels, whereas leaves in the lower canopy exhibit adaptations to low light conditions. In addition, soil-water salinity at the study site was far higher than the optimum salinity for A. marina. Hence, it is also suggested the salinity level at this site may have influenced the reduced leaf size in the upper canopy.     

Acorn production is linked to secondary growth but not to declining carbohydrate concentrations in current-year shoots of two oak species

In trees, reproduction constitutes an important resource investment which may compete with growth for resources. However, detailed analyses on how growth and fruit production interact at the shoot level are scarce. Primary canopy growth depends on the development of current-year shoots and their secondary growth might also influence the number and size of fruits supported by them. We hypothesise that an enhanced thickening of current-year shoots is linked positively to acorn production in oaks. We analysed the effect of acorn production on shoot growth of two co-occurring Mediterranean oak species with contrasting leaf habit (Quercus ilex, Quercus faginea). Length and cross-sectional area of current-year shoots, apical bud mass, number of leaves and acorns, xylem and conductive area, number of vessels of acorn-bearing and non-bearing shoots were measured in summer and autumn. Nitrogen and carbohydrates analyses were also performed in stems and leaves of both shoot types. Stem cross-sectional area increased in acorn-bearing shoots when compared with non-bearing shoots for both species and such surplus secondary growth was observed since summer. In bearing shoots, the total transversal area occupied by vessels decreased significantly from basal to apical positions along the stem as did the xylem area and the number of vessels. Leaves of bearing shoots showed lower nitrogen concentration than those of non-bearing shoots. Carbohydrate concentrations did not differ in stems and leaves as a function of the presence of acorns. Such results suggest that carbohydrates may preferentially be allocated towards reproductive shoots, possibly through enhanced secondary growth, satisfying all their carbon demands for growth and reproduction. Our findings indicate that acorn production in the two studied oaks depends on shoot secondary growth.