Photoregulation of morphogenesis of tobacco plants transformed with the gene of human interlenkin-18

The effects of blue light (BL), green light (GL), and red light (RL) on morphogenesis photoregulation of transgenic tobacco (Nicotiana tabacum L.) plants containing a copy of human interleukin-18 gene were studied. Wild-type and transgenic (Il1 No.87–1 and Il18 No.7–11) lines and derived calluses were used. Photomorphogenesis of transgenic lines under GL and RL differed substantially from that of initial line at early stages of morphogenesis; this was expressed in hypocotyl lengthening under GL and cotyledon enhanced expansion under RL. Growth responses to light quality were related to changes in the levels of zeatin, IAA, and ABA. Thus, hypocotyl lengthening and increased cotyledon area under GL occurred at the elevated level of IAA and reduced level of ABA. Growth of callus cells in transgenic lines during zero passage differed from wild-type calluses only in darkness. The data obtained indicate that tobacco plant transformation with the human interleukin-18 gene changed functioning of the photoregulation systems at early developmental stages. This phenomenon is evidently explained by the pleiotropic effects of the gene inserted.     

Effects of Light Spectral Quality on Morphogenesis and Source–Sink Relations in Radish Plants

The accumulation of dry matter and the content of major phytohormones in the aboveground and underground plant parts, as well as light curves and the diurnal course of photosynthesis in the leaves were studied in radish (Raphanus sativusL.) plants of different ages that were grown under red (RL) or blue (BL) light. As seen from the rapid increase in plant biomass, the development of storage organs (hypocotyl or tap root) started on the 14th day after the emergence of seedling of the BL plants and on the 21st day for the RL plants. Conversely, RL stimulated biomass accumulation in the aboveground parts (petioles and stems) already in the early stages of plant development. Light spectral quality only slightly affected the activity and the diurnal course of photosynthesis. The GA content was ten times higher in the aboveground parts of the RL plants than those of the BL plants. The hypocotyl of the BL plants contained much higher amounts of cytokinins and IAA than that of the RL plants. The specific responses of the source–sink relations to the light quality were related to the distribution of various phytohormones between the aboveground and underground parts of the plants: RL increased the content of gibberellins (GA) in the aboveground parts of plants, thus increasing their sink activity, whereas BL stimulated the synthesis of cytokinins and IAA in the hypocotyl and enhanced its development. Light quality-specific morphogenetic responses were reversed when plants were treated with exogenous GA or paclobutrazol, an inhibitor of GA synthesis. The treatment of the BL plants with exogenous GA stimulated petiole and hypocotyl elongation and induced stem formation. The treatment of the BL plants with paclobutrazol led to shortened petioles, the flattening of the storage organ, and the disappearance of the stem.     

Tuber morphology and starch accumulation are independent phenomena: Evidence from ipt-transgenic potato lines

Tuber formation and carbohydrate metabolism in potatoes were studied using transgenic potato plants carrying the Agrobacterium tumefaciens ipt gene, involved in cytokinin biosynthesis. Three independent transformants, viz. clones 1, 11 and 13, whose cytokinin and auxin content had previously been shown to be different from each other and from the wild-type, were analysed in vitro. Clones 11 and 13 showed a higher ability to form stolons and tubers, as evident from: (1) stolon development in whole plants grown under non-inductive conditions, (2) total number and weight of tubers formed by cuttings of this clone in darkness, (3) tubers appeared earlier than tubers of wild-type plants and at a lower sucrose concentration in the medium. Clone 1 did not form stolons or tubers under any conditions tested, but rather formed short shoots. A series of metabolic changes, known to be characteristic for tubers, were analysed in leaves, stems and developing buds. It was found that the short type of shoots, formed by clone 1, had metabolic characteristics very similar to tubers formed in wild-type or clones 11 and 13, including glucose, fructose, sucrose, and starch levels, and activities of invertase, sucrose synthase and fructokinase. It is concluded that the regulation of the stolon swelling and of carbohydrate metabolism, normally occurring simultaneously, can be uncoupled, and are thus, at least partly independent phenomena. The present data obtained with a high-cytokinin line indicate that cytokinins (probably in concert with auxins) might be mainly involved in the regulation of tuber morphology.     

Stimulatory effect of cytokinins and interaction with IAA on the release of lateral buds of pea plants from apical dominance

Lateral buds of pea plants can be released from apical dominance and even be transformed into dominant shoots when repeatedly treated with synthetic exogenous cytokinins (CKs). The mechanism of the effect of CKs, however, is not clear. The results in this work showed that the stimulatory effects of CKs on the growth of lateral buds and the increase in their fresh weights in pea plants depended on the structure and concentration of the CKs used. The effect of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) was stronger than that of 6-benzylaminopurine (6-BA). Indoleacetic acid (IAA) concentration in shoot, IAA export out of the treated apex and basipetal transport in stems were markedly increased after the application of CPPU or 6-BA to the apex or the second node of pea plant. This increase was positively correlated with the increased concentration of the applied CKs. These results suggest that the increased IAA synthesis and export induced by CKs application might be responsible for the growth of lateral shoots in intact pea plants.     

Morphogenesis of potato plants in vitro. I. Effect of light quality and hormones

Stem cuttings of potato plants (Solanum tuberosum L., cv. Miranda) were cultured in vitro on MS medium with sucrose either without or with addition of indole-3-acetic acid (IAA) or kinetin (K) under red light (R) or blue light (B). Plants on medium without hormones under R were thin, long, with very small leaves, and produced no or only a few microtubers (after longer-lasting cultivations). In B, plants remained short, thick, with large, wellde-veloped leaves and produced a significant amount of microtubers. Darkening of both roots and shoots strongly promoted tuber formation; the tubers were formed on the darkened part of the plant. IAA had no pronounced effect on plant development in B except for slight lengthening of the stem, and, in longer cultivations, slightly enhanced tuber formation as well. In R, IAA brought about several significant effects: stem reduction and induction of tuber formation being the most significant. Kinetin in R increased tuber formation slightly. In B, kinetin not only strongly stimulated tuber formation, but also increased the total fresh weight and root (+ stolons)/shoot ratio. Results are discussed with regard to the possible role of auxins and/or cytokinins in mediating the morphogenetic effects of light.     

Effect of phytohormones synthesized by rhizosphere bacteria on plants

New strains of rhizosphere microorganisms Azotobacter chroococcum Az d10, Bacillus megaterium Pl-04, and Bacillus mucilaginosus B-1574 were found to be able to synthesize cytokinins (CKs) and indolylacetic acid (IAA). Three forms of CKs—dihydrozeatin riboside, isopentenyl adenosine, and trans-zeatin riboside—were identified, whose ratio was different in the three bacterial cultures. Inoculation of cucumber (Cucumis sativus L.) plants increased the content of CKs and IAA in them by 35.6 and 21.3%, respectively, and also stimulated seed germination and increased the growth rate, the biomass of shoots, the number of lateral roots, and the root hair area, which ensured better plant nutrition. The IAA/CKs ratio shifted during bacterization towards CKs due to increase in the content of riboside forms, which apparently caused growth stimulation.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Involvement of auxin and CKs in boron deficiency induced changes in apical dominance of pea plants (Pisum sativum L.)

It has previously been shown that boron (B) deficiency inhibits growth of the plant apex, which consequently results in a relatively weak apical dominance, and a subsequent sprouting of lateral buds. Auxin and cytokinins (CKs) are the two most important phytohormones involved in the regulation of apical dominance. In this study, the possible involvement of these two hormones in B-deficiency-induced changes in apical dominance was investigated by applying B or the synthetic CK CPPU to the shoot apex of pea plants grown in nutrient solution without B supply. Export of IAA out of the shoot apex, as well as the level of IAA, Z/ZR and isopentenyl-adenine/isopentenyl-adenosine (i-Ade/i-Ado) in the shoot apex were assayed. In addition, polar IAA transport capacity was measured in two internodes of different ages using 3H-IAA. In B-deficient plants, both the level of auxin and CKs were reduced, and the export of auxin from the shoot apex was considerably decreased relative to plants well supplied with B. Application of B to the shoot apex restored the endogenous Z/ZR and IAA level to control levels and increased the export of IAA from the shoot apex, as well as the 3H-IAA transport capacity in the newly developed internodes. Further, B application to the shoot apex inhibited lateral bud growth and stimulated lateral root formation, presumably by stimulated polar IAA transport. Applying CPPU to the shoot apex, a treatment that stimulates IAA export under adequate B supply, considerably reduced the endogenous Z/ZR concentration in the shoot apex, but had no stimulatory effect on IAA concentration and transport in B-deficient plants. A similar situation appeared to exist in lateral buds of B-deficient plants as, in contrast to plants well supplied with B, application of CKs to these plants did not stimulate lateral bud growth. In contrast to the changes of Z/ZR levels in the shoot apex, which occurred after application of B or CPPU, the levels of i-Ade/i-Ado stayed more or less constant. These results suggest that there is a complex interaction between B supply and plant hormones, with a B-deficiency-induced inhibition of IAA export from the shoot apex as one of the earliest measurable events.     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Alteration of Hormonal Levels in a Rootless Epiphytic Bromeliad in Different Phenological Phases

Major changes in indole-3-acetic acid (IAA) and cytokinin (CK) levels occur at different phenological phases of Tillandsia recurvata shoots. This epiphytic rootless bromeliad was chosen as suitable material for hormonal analysis because CK synthesis is restricted to the shoots, thus avoiding problems in the interpretation of results caused by translocation and interconversion of CK forms between roots and leaves encountered in plants with both organs. Young plants of T. recurvata have weak apical dominance because side shoots appeared early in development, and branch growth was correlated with a strong increase in the level of zeatin. The flowering phase was characterized by a significant increase in free base CKs, zeatin, and isopentenyladenine compared with the levels found in adult vegetative shoots. In contrast, both free-base CKs declined in the fruiting phenological phase, and the IAA level increased dramatically. It was concluded that in phases characterized by intense organ formation, such as in the juvenile and flowering stages, there was an enhancement of CK content, mainly caused by zeatin, leading to a lower IAA/CK ratio. Higher ratios were correlated with phases that showed no organogenesis, such as adult and fruiting phenologies. Received April, 15, 1999; accepted September 7, 1999     

Influence of Spectral Quality of Light on Pollen Tube Elongation in Arachis hypogaea

The effect of light of different wave lengths (red RL, blueBL, yellow YL, green GL and far-red FR) on pollen tube elongationhas been studied in Arachis hypogaea. Short exposure of pollento RL caused enhanced tube elongation and this effect was reducedby FR and BL. The effects of RL and FR were mutually reversible.Similarly, the inhibitory effect of blue light could be overcomeby IAA and riboflavin separately whereas acetylcholine and GA₃mimicked the RL effect. The activities of peroxidase (PO) andmalate dehydrogenase (MDH) were increased by RL and IAA whereasBL increased the activity of IAA-oxidase. The regulatory roleof light of different spectral composition in controlling pollentube elongation is discussed. Arachis hypogaea, pollen tube elongation, light     

Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

The <Emphasis Type="Italic">trans</Emphasis> and <Emphasis Type="Italic">cis</Emphasis> zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.     

Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants,and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.