Substances with cytokinin activity extracted from growing apices of apple-tree shoots

The presence of native cytokinins was studied in growing apices of apple-tree annual shoots. During June the presence of two compounds showing cytokinin activity in biotest was observed and their R_F value was determined in three chromatographic systems.     

Effects of flooding the root system of sunflower plants on the cytokin in content in the xylem sap

The severe chlorosis observed in the lower most of flooded sunflower plants (Helianthus annuus L. cv. Tall Single) may lie initiated by a reduction in the import of cytokinins by the stoot from the flooded root system. Experiment indicates that during 12 hours following the release of flooding, plants previously flooded for 72 hours or less recover their ability to exude sap when the root systems are aerated, and the root systems synthesize and export amino-acids to the shoot. Plants flooded for longer periods lose these abilities. The metabolic activity of the root apices declines parallel with the decline in eytnkinin concentration in the sap with increase in flooding time up to 72 hours. Flooding for 96 hours drastically reduce all four parameters of root activity. After flooding for this period there was a large increase in the number of blackened and tetrazolium-negative root apices which were in all probability dead. The correlation between the metabolic activity of the root apices and the total cytokinin content of the sap supports tbe view that root apices may be sites of cytokinin synthesis.     

Substances with cytokinin activity in apple shoots during the vegetative season

The content of naturally occurring free substances with cytokinin activity was investigated in growing apices of apple shoots during the vegetative period. In the end of May one substance with cytokinin activity was found. During the first period of growth activity we found three such substances with more hydrophobic behaviour and in the culmination of the second period of growth activity we found other three or four substances displaying on the chromatogram more hydrophylic behaviour.     

Polyamine biosynthesis and titer during various developmental stages of Phaseolus vulgaris

The activities of arginine decarboxylase (ADC; EC 4.1.1.19) and ornithine decarboxylase (ODC; EC 4.1.1.17) as well as polyamine content were examined in Phaseolus vulgaris L. cv. Taylor's Horticultural before and during anthesis, during fruit development and throughout vegetative growth. The specific activities of polyamine biosynthetic enzymes were highest in all rapidly growing tissues, e.g., root apices, hypocotyls, young internodes, young leaves, flower buds, young pods and pericarps. They were lowest in mature, non-growing tissues. Similarly, the content of the major polyamines (putrescine, spermidine, spermine) is highest in rapidly growing tissues, and lowest in mature tissue. These correlations reinforce the growing connection between polyamines and rates of cell division and metabolic activity during both vegetative and reproductive development.     

Specific arrangements of cortical microtubules are correlated with the architecture ofmeristems in shoot apices of angiosperms and gymnosperms

Arrangements of corticalmicrotubules (MTs) as seen in median longitudinal cryosections of shoot apices of several angiosperms and gymnosperms were studied by indirect immunofluorescence microscopy.Bryophyllum, Clethra, Helianthus, Houttuynia, Vinca (angiosperms), andPinus, Cedrus, Cedrus andGinkgo (gymnosperms) were examined. In all angiosperm apices collected during the growing season, MTs were mainly arranged anticlinally in the tunica, randomly in the corpus, and transversely in the rib meristem. This pattern of arrangements of MTs was further confirmed by electron microscopy inBryophyllum apices. In the apices of winter shoots MTs in the rib meristem were arranged randomly, indicating a seasonal change with respect to their arrangment. In all examined gymnosperm apices, populations of superficial cells showed both random and anticlinal arrangements of MTs, in contrast to those of angiosperm apices that consistently show anticlinally arranged MTs. In the shoot apices of both angiosperms and gymnosperms, cortical MTs were arranged perpendicularly to the directions of cell expansion. The significance of MTs in the maintrnance of the different architectures of shoot apices in angiosperms and gymnosperms is discussed.     

Gibberellin Induced Changes in Diffusible Auxins from Savoy Cabbage

Diffusates from apices of young plants of savoy cabbage treated with gibberellie acid (GA) and apices of control plants have been examined with respect to their content of Indole auxins. Three indole Compounds were detected and identified on the basis of their chromatographic characteristics in several systems. These compounds were: glucoubrassicin, indole-3-acetic acid (IAA) and indole-3-acetonitrile (IAN). An effect of GA on the total auxin activity of the diffusate was noted 90 hours after treatment, while an increase in stem height occurred 48 hours later. This increase in auxin effect of the entire diffusates was shown bv chromogenic development and bioassay of chromatograms of diffusates to be a result of an increase in level of the IAA content. A concomitant decrease in I the glucobrassicin content was indicated. Since GA was found to have no effect on the enzymatic conversion of tryptophan or tryptamine to IAA, it is proposed that the effect of GA is on the conversion of glucobrassicin to IAA.     

The Relationship Between Flowering Induction and the Level of Adenosine Triphosphate in Pharbitis nil

A study has been made on the changes of ATP and protein content in cotyledons and apices of Pharbitis nil after flowering induction. Protein content of the cotyledons which have just got through the induction is 68% higher than that of the control, but the difference trends to disappear there after. The. difference of protein content between the induced and uninduced apices is not so obvious in the first three days after induction, but quite evident on the fourth day (30% higher in the induced apices) suggesting that there is some relationship between protein metabolism and flowering induction both in the cotyledons and in the apices. Just after the seedlings have been induced, ATP content of the cotyledons is getting much (134%) higher than that of the control and the tendency is retained towards the fourth day after induction. Generally ATP content in apices is one order of magnitude higher than that in cotyledons. Although ATP content in the apices is only slightly higher than that of the control soon after induction, it gains quite a lot in the second day until the fifth day the end of our experiment. In the third day after induction ATP level in the apices reaehs to the maximum (20.6×10-2 μmol/g, apices) which is 37% higher than that of the control. The results show that flowering induction is bound to be followed by increase of proteins and ATP both in apices and in cotyledoms. It also. shows both formation of the stimulus in induced cotyledons and evocation in the apices might be all concerned in expression of some genes and synthesis of new RNA and protein. According to the maximum peak of ATP in the apices and cotyledons appeared in 3rd to 4th day after induction, it seems that the inductive effect both in the cotyledons and apices might continue for some time under the following uninduced condition.     

Molecular cloning and expression analysis of spermidine synthase gene during sex reversal induced by Ethrel in cucumber (Cucumis sativus L.)

A full-length cDNA encoding spermidine synthase CsSPDS of 317 amino acids was isolated from cucumber. CsSPDS showed high similarity to SPDSs of other species, with the greatest similarity to SPDS of Coffea arabica (identity, 87%). Northern blot analysis revealed that CsSPDS was expressed predominantly in rapid growing tissues, such as roots, young leaves and flower buds. The response of CsSPDS to the sex reversal of three-leaf stage androecious cucumber seedlings by Ethrel treatment was also examined. Our results showed that the CsSPDS was up-regulated in shoot apex during sex reversal process, which correlated with increases of spermidine synthase activity and free spermidine (Spd) content, and a decrease of putrescine (Put) content in shoot apices.     

Quantitative analysis of Schizophyllum commune metalloprotease ScPrB activity in SDS-gelatin PAGE reveals differential mycelial localization of nitrogen limitation-induced autolysis

The basidiomycete Schizophyllum commune produces a variety of proteolytic enzymes. A number of these, detected in native gelatin-containing polyacrylamide gels, have their activities increased during nitrogen-limited growth of the mycelium. ScPrB, a metallo-endoprotease, appears to have the greatest nitrogen stress-induced increase in activity of all of these enzymes. Quantifying ScPrB has proven difficult because no artificial chromogenic substrate has been found. In addition, it is poorly resolved from another highly active protease, ScPrA, in native gelatin-containing gels. We have developed a method using SDS gelatin-containing polyacrylamide gels for resolving ScPrB from ScPrA and for quantifying its activity by densitometry. This method was used to assess the intramycelial location of ScPrB induction after the transfer of exponentially growing colonies to nitrogen deprivation conditions. By all analyses (proportional, normalized to fresh weight, and normalized to protein), the increase in ScPrB activity was found to occur exclusively in midsections of the growing mycelium, whereas ScPrB activity was found to be decreased or unchanged in the centers of colonies and in colony margins. This implies that proteolysis mediated by ScPrB may supply translocatable amino acids only from the region directly behind the growing hyphal apices.     

EFFECT OF GROWTH RATE,MORPHOGENIC ACTIVITY,AND PHYLOGENY ON SHOOT APICAL ULTRASTRUCTURE IN OPUNTIA POLYACANTHA (CACTACEAE)

Dormant short shoot apices of Opuntia polyacantha were cultured under three conditions: cytokinin and high sucrose to stimulate the formation and rapid growth of a leafy long shoot; cytokinin and no sucrose (slow growth of a leafy long shoot); gibberellic acid and high sucrose (rapid growth of a spiny short shoot). These meristems, and also dormant (uncultured) ones, were analyzed by stereological, ultrastructural techniques. By comparing meristems growing with cytokinin but with or without sucrose, correlations between metabolic rate and apical ultrastructure were studied; comparison of leaf-producing and spine-producing meristems permitted examination of correlations with morphogenic role; comparison with published data for four other species permitted study of phylogenetic effects, and comparison with dormant apices revealed information about meristem activation. Ultrastructure varied according to each condition: metabolic rate, morphogenic activity and species can be distinguished by quantitative methods. Apical ultrastructure is most strongly correlated with rate of growth such that apices of differing species resemble each other if growing at similar rates, whereas apices of a single species differ markedly if growing at differing rates or if performing different morphogenic activities. Hyaloplasm is an excellent indicator of metabolic rate; mitochondria, nuclei, and vacuoles are not.     

Formation of Polyamines in the Rumen of Goats During Growth

Ornithine decarboxylase (E.G. 4.1.1.17) and S-adenosylmethionine decarboxylase (E.G. 4.1.1.50) and their products putrescine, spermidine and spermine were estimated in the rumen liquid from 3 groups of growing kids and 23 adult goats. Polyamines were also estimated in the feedstuff used. Marked differences in polyamine synthesis in rumen liquid were observed between the different groups of kids. Two groups of kids growing up together with adult goats had at an age of 2–4 months a peak of a few days duration in enzyme activity as well as in polyamine concentration. In these groups ornithine decarboxylase activity reached maximal values of 158±79 s (n = 4) and 100 (66–117) (n = 3) nmol[14CO2]/ml rumen liquid/h at an age of 120 and 77 days, respectively. The corresponding activity in rumen liquid from kids who were isolated from other animals was only about 1/10 of this value. By comparison ornithine decarboxylase activity in adult goats was 30.7±20 (n = 43) nmol[14CO]/ml/h. In rumen liquid from kids grown up together with adults, concentrations of the polyamines reached maximum at about the same time as ornithine decarboxylase activity. The mean maximal concentration of putrescine in the 2 groups was about 350 and 500 nmol/ml, while the corresponding value for spermidine was about 200 nmol/ml in both groups. Relatively constant and high concentration of polyamines were present in the feedstuff used. However, in growing kids the ruminai putrescine and spermidine concentration at times far exceeded those that could be accounted for by the estimated intake of polyamines by the food. The results therefore strongly indicate that polyamines are formed in considerable amounts in rumen content of kids during the phase of rapid growth. Results from a few experiments with calves also indicate that this may be true for cattle. polyamines; putrescine; spermidine; spermine; ornithine-decarboxylase; rumen liquid.     

Histochemical study of vegetative and floral bud formation in tobacco stem expiants

Primary explants from the inflorescence stem of tobacco and primary explants from the stem of vegetative plants, cultivatedin vitro under the same conditions, display different morphogenetic ability. The former give rise mostly to floral buds, whereas the latter exclusively to vegetative ones. Histological and histochemical analyses of both original andin vitro cultivated explants were made. They showed differences in chlorophyll content and alcohol dehydrogenase (AD) activity of the original explants reflecting their different metabolic status. Bud primordia were initiated in the superficial meristematic layer derived from epidermal tissues. Floral or prefloral apices were characterized by a strong AD activity in all cells of the meristem, while in vegetative apices AD activity was restricted to their uppermost parts. A high rate of procambium differentiation connected with leaf primordia formation was typical of vegetative buds. A higher concentration of glucose (5 %) enhanced cell division in explants, which is also correlated with a higher AD activity. The significance of vascular tissues for differentiation of vegetative buds is discussed. Presented at the International Symposium “Plant Growth Regulators” held on June 18-22, 1984 at Liblice, Czechoslovakia.     

Changes in Gene Expression from Diploid to Autotetraploid Status of Lycopersicon esculentum

The cytokinin activity of the root exudate, the leaves, and the apices of vegetative and flowering white lupin plants (Lupinus albus L.) was investigated. The level of cytokinin activity in the root exudate decreased over the 11-week experimental period. Four peaks of cytokinin activity were recorded in the root exudate of 8-week-old plants after fractionation on Sephadex LH-20. Two of these peaks co-eluted with zeatin and zeatin riboside. It is suggested that the remaining peaks represent nucleotide and glucoside cytokinins. The cytokinin levels in extracts of the mature leaves fluctuated slightly over the experimental period. Three peaks of activity co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in extracts of mature leaves of 8-week-old plants. In the apices cytokinin activity could only be detected in the inflorescences of flowering plants. It would appear that cytokinins produced by the roots accumulate in the fully expanded mature leaves, but are utilized in the rapidly growing apical region and in young expanding leaves.     

Apical meristems of tomato roots and their modifications induced by arbuscular mycorrhizal and soilborne pathogenic fungi

Functional morphological patterns in root apices of tomato ( Lycopersicon esculentum ) dependent on growth, ageing and infection by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and/or by the soilborne pathogenic fungus Phytophthora nicotianae var parasitica ( P. parasitica ) were studied. Uninfected root apices were characterized by closed, tri-layered meristems with nonreticulate nuclei; however, some apices of each treatment lost their meristematic nature, stopped growing and differentiated, becoming 'parenchymatized'. The pathogenic fungus reduced the apex diameter and the number of mitotically active and viable apices inducing plasmolysis, cell and nucleus degeneration, and necrosis. The AM fungus, on the other hand, produced an increase in apex size and reduced the percentage of necrosis both in uninfected roots and in roots infected by P. parasitica . Thus, the AM fungus protected the apices from the pathogenic infection, allowing normal root growth. Furthermore, larger apices, which produce thicker roots, might indirectly contribute to plant protection. Increased volumes of colonizable tissues favour the spreading of the symbiont, and P. parasitica hyphae are always excluded from arbuscule-containing cells.     

Increased Pentose Phosphate Pathway Activity Linked to Floral Induction in Apices of Spinacia oleracea during Short Days

A quantitative cytochemical study of glucose-6-phosphate dehydrogenaseactivity as a marker of the pentose phosphate pathway (PPP)was made on shoot apices of Spinacia oleracea kept either continuouslyin short days for up to eight weeks or transferred for a singleperiod of 20 h to continuous light after between three and eightweeks in short days. By the time the apices kept in continuousshort days showed morphological changes relating to the floralstate, the PPP activity was already elevated. From four weeksonwards, the apices were more readily induced to the floralstate as evidenced by the increased PPP activity. In addition,the level of PPP activity achieved in the short-day apices asthey progressed to the floral state was as great as that observedin the apices induced to flower by a 20-h day. Spinacea oleracea, pentose phosphate pathway, shoot apex, cytochemistry, glucose-6-phosphate dehydrogenase, floral induction     

A promoter directing epidermal expression in transgenic alfalfa

We describe the isolation, cloning and expression of a 2.8 kb promoter fragment of the Blec4 gene from pea (Pisum sativum cv. Alaska) and demonstrate that it is capable of directing the expression of the -glucuronidase coding region to the developing epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa (Medicago sativa cv. RegenSY). The Blec4 promoter represents a useful tool with which to target the expression of foreign genes to the epidermal layer of actively growing shoots. The activity of the Blec4 promoter in the epidermis of the shoot apex makes it particularly suitable for genetically engineering defense against insects and diseases that attack the growing shoot apex.     

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.     

The water status of the roots of soil-grown maize in relation to the maturity of their xylem

The long delayed maturation of the late metaxylem of maize ( Zea mays ) roots imposes a high-resistance barrier between the immature apices and the negative water potential of the leaves. These apices (20+ cm) bear strongly adhering soil sheaths to within 0.5 to 2 cm of the distal end. It was hypothesized that the sheathed immature apices should show less response to transpiration stress than bare regions. Measurements were made of the relative water content (RWC) of the sheathed and bare zones of the axile roots, both at different ages of the plant, and early and late in the day's transpiration. Sheathed roots maintained a steady RWC of about 83% irrespective of age or transpiration. Bare roots had RWCs of about 63% in the morning, but this fell to 55% in the afternoon. The first-order branches on the bare roots in the morning had still lower values of RWC, near 50%. Plots of RWC against water potential were indistinguishable for the three root types. It is concluded that the immature apices are indeed relatively isolated from the fluctuating tensions in the stem xylem, and that these tensions reduce the water content of bare roots and their branches to low values.     

Micronutrient content does not reflect the status of health in the aquatic carnivorous plant <Emphasis Type="Italic">Aldrovanda vesiculosa</Emphasis>

Tissue micronutrient content (B, Fe, Mn, Zn, Cu, Mo, Co) was compared in healthy and ill shoots of a rootless aquatic carnivorous plant Aldrovanda vesiculosa growing in an outdoor culture, in plants in the field, in different accessions from around the world, and in shoots and turions. On the basis of microelemental analyses, the observed disorder of shoot apices cannot be explained as deficiency of any of the above micronutrients.     

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca²⁺-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.