Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Promotion by gibberellic Acid of polyamine biosynthesis in internodes of light-grown dwarf peas

When gibberellic acid (GA₃; 5-35 micrograms per milliliter) is sprayed on 9-day-old light-grown dwarf Progress pea (Pisum sativum) seedlings, it causes a marked increase in the activity of arginine decarboxylase (ADC; EC 4.1.1.9) in the fourth internodes. The titer of putrescine and spermidine, polyamines produced indirectly as a result of ADC action, also rises markedly, paralleling the effect of GA₃ on internode growth. Ammonium (5-hydroxycarvacryl) trimethyl chloride piperidine carboxylate (AMO-1618; 100-200 micrograms per milliliter) causes changes in the reverse direction for enzyme activity, polyamine content, and growth. GA₃ also reverses the red-light-induced inhibition of ADC activity in etiolated Alaska pea epicotyls; this is additional evidence for gibberellin-light interaction in the control of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17), an alternate source of putrescine arising from arginine, is not increased by GA₃ or by AMO-1618.     

Apparent endogenous circadian rhythm inent-kaurene biosynthesis

Net synthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid was assayed in cell-free enzyme extracts prepared from Alaska pea (Pisum sativum L.) seedlings throughout 44 h of a regimen consisting of a 16-h day and an 8-h night. Activities generally followed an upward trend during the dark period and a downward trend during the photoperiod. Activity was also assayed in enzyme extracts prepared at intervals during a 12-h photoperiod and a following, continuous 36-h dark period after entrainment of plants to a regimen of 12-h days and 12-h nights.Ent-kaurene synthesis activity again followed an upward trend in enzyme extracts prepared during what would have been the entrainment dark period, and a downward trend during the entrainment photoperiod. The apparent endogenous rhythm ofent-kaurene biosynthesis may have implications for the regulation of gibberellin biosynthesis.     

Gibberellin-antagonizing protein in dwarf peas

Summary Seedlings of dwarf pea grown under red light for 9 d were homogenized in 0.1 M phosphate buffer (pH 6.8) and a water-soluble extract was obtained by centrifugation, dialysis and lyophilization. The extract contained a proteinaceous substance (or substances) which interfered with the GA₃ response of dwarf peas, probably due to complex formation with the hormone.     

Effect of inhibitors of polyamine biosynthesis on gibberellin-induced internode growth in light-grown dwarf peas

When gibberellic acid (GA3) is sprayed on 9-day-old light-brown dwarf Progress pea (Pisum sativum) seedlings, arginine decarboxylase (ADC; EC 4.1.1.9) activity increases within 3 h and peaks at about 9 h after GA3 application. This is followed by a second lower peak at about 30 h; both peaks were higher than the corresponding peaks in the controls. In contrast, no appreciable effect of GA3 on internode length was observed until about 12 h, after which time a dramatic increase in growth rate occurred and persisted for about 12 h. Specific (DL-alpha-difluoromethylarginine) and non-specific (D-arginine and L-canavanine) inhibitors of ADC strongly inhibited ADC activity and to a lesser extent internode growth. The inhibition was reversed only slightly by the addition of polyamines. Actinomycin D and cycloheximide inhibited the rise in ADC activity induced by GA3. The half-life of the enzyme was increased by GA3 treatment. The results suggest that part of the GA3-induced increase in internode growth may result from enhanced polyamine biosynthesis through the ADC pathway. Furthermore, the GA3 induced increase in ADC activity probably requires de novo synthesis of both RNA and protein.     

Inhibition ofEnt-kaurene oxidation by cytokinins

Cytokinins, which have some structural similarities to ancymidol, a plant growth retardant, were tested for their effects on the cell-free oxidation ofent-kaurene. Results indicate that several cytokinins inhibit this reaction in microsomal extracts of liquid endosperm from immature wild cucumber seeds. N⁶-cyclohexanemethyladenine was the most active (inhibiting 50% of the controlent-kaurene oxidation at 2×10^–6 M). N⁶-isoamyladenine, N⁶-benzyladenine, N⁶-(²-isopentenyl)adenine and dihydrozeatin were active at successively higher concentrations. Zeatin, kinetin, adenine, N⁶-benzyladenosine, and N⁶-(isopentenyl)adenosine were inactive in this system.The basis for the inhibition ofent-kaurene oxidation by cytokinins may be similar to that of ancymidol: interaction with cytochrome P-450. A binding spectrum similar to that of ancymidol with cytochrome P-450 from wild cucumber endosperm microsomes was obtained with four active cytokinins. The cytokinin binding properties of this protein are currently under investigation.No metabolism of N⁶-benzyladenine could be detected under conditions in which the cytokinin inhibited the oxidation ofent-kaurene toent-kaurenol.This work was supported in part by National Science Foundation Grant PCM 7619279 and PCM 8016237, and a grant from Eli Lilly and Company.A portion of this work was presented as a poster paper at the Tenth International Conference on Plant Growth Substances, Madison, Wisconsin, USA, July 22–26, 1979.     

Regulation of growth and morphogenesis in phenotyplc and genotyplc dwarf peas

White light suppressed the stem growth and promoted leaf expansion. With increased irradiance the light effect was enhanced. The morphogenetic effects induced by light and by dwarf-mutation are similar. However, the nature of phenotyplc and genotyplc dwarfism is different. In the dwarf mutants the auxin level is not changed in contrast to the GA, ABA and QGC contents. Under high irradiance which depressed the stem growth, auxin and GA-levels were lowered while the content of QGC and of growth-inhibitor non-identical with ABA increased, but the level of ABA was not affected. The sensitivity of various pea forms to the light and to exogenous phytohormones (GA and IAA) is different. The plants with the shortest stems were more sensitive to light and GA. Data on the stem growth and rhizogenesis induced by light and by GA are presented. The metabolism of 2-¹⁴C-PCA (precursor of QGC) in tall and dwarf forms is different. The possible role of phytohormones and some phenolic compounds in the regulation of growth and morphogenesis of phenotyplc and genotyplc dwarf forms is discussed.     

Vitamin biosynthesis by germinating seeds at low temperature and in overmoistened soil]

The distribution of farnesene and farnesene hydroperoxide in apples of different varieties was studied. The relationship between the content of these compounds and the pathological browning of the apple surface, scald was followed. It was shown that 1) farnesene and farnesene hydroperoxide were concentrated in the cuticle; 2) there was no distinct correlation between farnesene concentration and scald, 3) there was a direct correlation between the concentration of farnese hydroperoxide and scald development; 4) the factors that inhibited farnesene oxidation (antioxidants, low oxygen concentration, mineral oils--farnesene absorbers) slowed down the scald development during apple storage.     

Radioactive gibberellin a(5) and its metabolism in dwarf peas

Radioactive gibberellin A₅ (³H-GA₅) was synthesized from gibberellic acid. When it was applied to dwarf peas grown in the dark, an average of 3% was converted to another acid gibberellin within 48 hours. The biological activity of the metabolite did not account for the response to applied GA₅. GA₅ is therefore assumed to be biologically active per se.³H-GA₅ did not appear to form a stable complex with a macromolecule in pea shoots. When injected into dwarf pea pods, ³H-GA₅ was readily metabolized by maturing seed to more water-soluble substances and to two other acidic compounds. This metabolism continued even throughout germination of the seed without reconversion of the metabolites to GA₅. It is concluded that “bound” GA₅ plays no part in the germination of dwarf pea seeds.     

Induction of secondary dormancy in sunflower seeds by high temperature. Possible involvement of ethylene biosynthesis

High temperature (45°C) inhibits seed germinition and seedling sunflower ( Helianthus annuus L. cv. Mirasol). Treatment of imbibed seeds at 45°C for more than 48 h induces a secondary dormancy, which is associated with progressive decrease of germination ability at optimal temperature (25°C) as well as with abnormal seedling growth. Ethylene (55μl l⁻¹) and 2-chloroethylphosphonic acid (ethephon) (2.5 m M ) improve germination of thermodormant seeds at 25°C. but the abnormal growth of the seedlings remains. O₂-enriched atmosphere and dry storage improve germination and normal seedling growth. The induction of thermodormancy in sunflower seeds seems associated with loss of their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. Possible effects of high temperature on membranes and ethylene forming enzyme (EFE) are discussed.     

Protein biosynthesis changes in Trypanosoma cruzi induced by supra-optimal temperature

Early during vertebrate infection, T. cruzi is exposed to the host blood at an elevated temperature. Bearing this in mind, the pattern of protein synthesis of two parasite forms was examined. SDS-PAGE of heated organisms showed an increase in at least four proteins (103, 92, 75 and 61 kD). The temperature effect is also manifested in cells whose RNA synthesis is reduced by actinomycin D treatment. The synthesis of the '29 degrees proteins' is inhibited at 40 degrees C in organisms growing in culture medium; when the organisms were maintained in serum, the inhibition was not observed. The inhibitory effect observed at 40 degrees C was reversed when the temperature was shifted to 29 degrees C. These proteins were synthesized for 180 min at 37 degrees C or 360 min at 40 degrees C. The increased protein synthesis manifested at 37 degrees C had decreased 45 min after the temperature was lowered to 29 degrees C. When the cells were pre-incubated at 40 degrees C and shifted to 29 degrees C, the synthesis of the heat-induced proteins proceeded for at least 180 min. This pattern of heat induction in epimastigotes and trypomastigotes is the same irrespective of whether the incubation medium is LIT (for epimastigotes), M-16 (for trypomastigotes), or when serum was used for both cell types.     

Preparation of radioactive gibberellin a(1) and its metabolism in dwarf peas

Gibberellin A₁-3,4-³H was prepared by selective catalytic reduction of gibberellic acid with a mixture of tritium and hydrogen. ³H-GA₁ was applied at physiological concentrations to dwarf peas and the metabolism of the hormone was investigated. ³H-GA₁ was converted to an acidic, biologically active compound. Radioactive but biologically inactive compounds were also found in the neutral fraction and could not be converted to acidic gibberellins by hydrolysis. No attachment of gibberellin to any macromolecular fraction was evident.     

Induction of proteins in response to low temperature in Escherichia coli.

When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10 degrees C, growth stops for several hours before a new rate of growth is established. During this growth lag the number of proteins synthesized is dramatically reduced, and at one point only about two dozen proteins are made; 13 of these are made at differential rates that are 3 to 300 times increased over the rates at 37 degrees C. The protein with the highest rate of synthesis during the lag is not detectably made at 37 degrees C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.     

Gibberellic acid-induced microtubule reorientation in dwarf peas is accompanied by rapid modification of an α-tubulin isotype

To test whether shifts induced in microtubuie orientation by gibberellic acid (GA₃) involved changes in tubulin isotypes, pea stem cells were examined when elongation had been enhanced by GA₃. The behaviour of a dwarf recessive mutation (le), with very low endogenous levels of gibberellin, was compared with the tall (Le) plant. Two hours after adding GA₃, cells were measurably longer than controls and this coincided with a net shift of microtubule orientation from longitudinal and oblique to transverse — an effect that was more pronounced in the dwarf. There were always more cells with net-transverse microtubules in GA₃-treated tissue than in controls, but as growth ceased, the major orientation of the microtubule arrays became oblique in both samples. Microtubule reorientation was rapid and was closely correlated with the growth of the cells. Although changes in orientation and isotype were monitored over a 40 h period, immunoblotting 2D gels with the well characterized antibodies YL1/2 and YOL1/34 confirmed that alterations to the α-tubulin constellation could be detected as early as the 2 h time point. Again the effect was especially pronounced in dwarf plants. In the presence of added GA₃, one α-tubulin isotype (designated α₁) retained its position in the α-tubulin constellation (as determined by total protein staining and with YOL1/34 that recognizes detyrosinated as well as tyrosinated tubulin). It was no longer recognized, however, by the anti-tyrosinated α-tubulin antibody YL1/2. This indicates that as GA₃ begins to cause a reorientation of the cortical microtubules (and to enhance the rate of cell elongation) the α₁ isotype is rapidly changed, probably by post-translational modification.     

Induction of cellular transglutaminase biosynthesis by sodium butyrate

Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9–11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.     

The importance of starch biosynthesis in the wrinkled seed shape character of peas studied by Mendel

The wrinkled-seed mutant (rr) of pea (Pisum sativum L.) arose through mutation of the gene encoding starch-branching enzyme isoform I (SBE1) by insertion of a transposon-like element into the coding sequence. Two isoforms of starch-branching enzyme have been documented in the developing pea embryo. The second isoform, SBEII, is expressed towards the later stages of embryo development while SBEI is expressed highly in the early stages. Due to mutation of SBEI the total amount of starch and the proportion of amylopectin, a branched starch polymer, are greatly reduced in the wrinkled (rr) line as compared to that in the wild-type, round (RR) line. Consequently, the level of sucrose in the rr line is nearly two fold that of the RR line. Increased sucrose concentration in the developing embryos of this mutant line causes increased uptake of water and thereby increases the cell size and fresh weight. During seed maturation in these mutant seeds a greater loss of water occurs. As a result, the wrinkled seed phenotype develops. Besides this morphological variation, the mutation also causes changes in the amount of lipid and of one storage protein, legumin. This review article discusses the role of the SBEI enzyme in causing such metabolic changes in the developing embryos with the implication that metabolism can play a central role in plant development.     

Induction of hepatitis B virus gene expression at low temperature

There is a limited understanding of the cellular regulation of HBV gene expression in differentiated hepatocytes. We previously demonstrated that HBV replication inversely correlates with cell proliferation and DNA synthesis. In this report, temperature-induced modulation of cell growth was used as a novel approach to study HBV gene expression in the absence of indirect effects from drugs or serum deprivation. We observed markedly elevated levels of hepatic HBV mRNA expression from integrated and episomal HBV DNA at 32 degrees C. Additionally, hepatoblastoma cells cultured at 32 degrees C expressed increased levels of albumin mRNA and decreased levels of c-myc mRNA, which demonstrates that liver-derived cells cultured at low temperature exhibit characteristics of functional and differentiated hepatocytes. In transiently transfected HepG2 cells cultured at 32 degrees C, the HBV enhancer 1 activated the X promoter and core/pregenomic promoter by 7.3- and 28-fold, respectively. In the absence of enhancer 1, core/pregenomic promoter activity was 2.4-fold higher than the X promoter in HepG2 cells at 32 degrees C. In contrast, enhancer 1 exclusively activated the X promoter in transfected non-liver cells at 32 degrees C. Therefore, the core/pregenomic promoter exhibits strict liver-specificity at low temperature. This work supports the hypothesis that HBV replication and gene expression are optimal in non-activated hepatocytes, and provides a novel system for delineating molecular aspects of the HBV replication process.     

Induction by nitrogen and low temperature of storage-protein synthesis in poplar trees exposed to long days

The synthesis of storage proteins in trees of poplar (Populus x canadensis Moench) could not only be induced by a shift from long-day to short-day conditions but also by either a low-temperature treatment or by nitrogen feeding under continuous long-day conditions. The synthesis of the protein did not depend on the cessation of growth and the formation of a terminal bud. The accumulation of the storage protein was in all cases preceded by a drastic increase in the level of the corresponding mRNA.Abbreviations cDNA copy DNA - kDA kilodalton