Activation of ribosomal and messenger RNA synthesis in excised Jerusalem artichoke tuber slices

RNA synthesis was studied in Jerusalem artichoke (Helianthus tuberosus L.) tuber slices immediately following excision and during the early period of aging in water. Incorporation of [³H]adenosine into RNA was detected as early as 20 min after excision. Measurement of the specific activities of RNA (cpm/g) and of ATP showed that RNA synthesis proceeded at a constant rate for the first several hours of aging and then increased moderately. [³H]adenosine was incorporated into polysomes throughout the aging period examined. Sucrose gradient fractionation of EDTA-dissociated polysomes showed that during the first 2 h of aging most of this incorporation was not into ribosome subunits but into presumed mRNA. Autoradiographic analysis of [³H]adenosine labelled nuclei showed that this was caused, at least in part, by a delay in the onset of rRNA synthesis synthesized during this time chromatographed as poly(A)-RNA on oligo(dT)-cellulose, indicating that a large part of the mRNA was not polyadenylated.     

Utilization of stored messenger RNA during early aging of Jerusalem artichoke tuber slices

Using dissociation in 0.8 M KCl, it was established that in freshly excised Jerusalem artichoke (Helianthus tuberosus L.) tuber slices less than 8% of the ribosomes were in polysomes. The first hour of aging in water was the period of most rapid polysome accumulation; over 32% of the ribosomes carried nascent polypeptide chains at the end of this time. Thereafter polysome accumulation continued to increase, but more gradually. While synthesis of high-molecular-weight RNA (presumed mRNA) was inhibited more than 95% by -amanitin during the first hour of aging, the inhibitor had no effect on polysome formation. As determined by [³H]polyuridylic acid hybridization, unaged cells contained polyadenylated RNA with a size range of 6–30S. The amount of polyadenylated RNA did not change during the first hour of aging. In control cells in water the in-vivo rate of protein synthesis increased exponentially during the first 4 h of aging without a comparable increase in polysomes. In -amanitintreated tissues a similar increase in protein synthesis was not observed despite the presence of near control levels of polysomes. It is suggested that early polysome formation depends on stored mRNA. Inhibition of mRNA synthesis by -amanitin prevents the normal development of an enhanced rate of protein synthesis which is not directly related to numbers of ribosomes in polysomes.Abbreviations Poly(A) polyadenylic acid - Poly(A)+RNA polyadenylated RNA - Poly(U) polyuridylic acid - TCA trichloroacetic acid     

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices

Summary A biochemical and cytochemical study has been made of the distribution of -glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of -glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. -glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM -glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. -glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of -glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.     

Effect of actinomycin D on the formation of enzymes in Jerusalem artichoke tuber slices

Summary The formation of both peroxidase and phenol oxidase was induced by culturing slices of Jerusalem artichoke tubers. Actinomycin D inhibited the formation of enzymes only when added before the termination of the lag phase (10 h after slicing). The data suggest that 4 to 6 h after slicing, tissues produce a mRNA which does not become fully operative until a translation mechanism has been activated.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.     

Solubilization of Jerusalem artichoke tuber chromatin by 2,4-dichlorophenoxyacetic acid

Chromatin from the tuber of the Jerusalem artichoke (Helianthus tuberosus) was solubilized in 2,4-dichlorophenoxyacetic acid (2,4-D) solution (100 mM) at pH 7.0. This solubilization was much affected by the pH; below 6.0 less chromatin was solubilized. The elution pattern of the products on gel filtration with Sepharose 4B showed that the solubilization was caused by the dissociation of the DNA and associated proteins. The pattern of polyacrylamide gel electrophoresis of histones extracted from the chromatin solubilized by 2,4-D was quite different from those of histones extracted from the original chromatin or from NaCl (2.0M) solubilized chromatin. The F1 and F3 fractions seemed to be little affected by 2,4-D, but the F2a1, F2a2 and F2b fractions were greatly decreased. In addition, the ratios of histones and non-histone proteins to DNA changed considerably in 2,4-D solubilized chromatin in an inverse manner. None of these changes were observed with NaCl. which suggests that the behaviour of 2,4-D for the solubilization of chromatin differs substantially from that of NaCl.     

Auxin-induced growth of tuber tissue of Jerusalem artichoke VI. Biochemical changes in chromatin during aging and cell expansion

Possible mechanisms by which auxin and gibberellic acid stimulateRNA synthesis were examined, using slices excised from cold-storedtubers of Jerusalem artichoke. The ratio of DNA in nucleo non-histone to the total DNA in chromatinincreased during the aging process. On the other hand, cellexpansion did not involve this kind of change in chromosomalcomponents. Gibberellic acid and 2,4-D showed no significanteffects on the ratio of DNA in nucleo non-histone to the totalDNA in chromatin. Melting points of DNA and nucleohistone did not differ significantlyaccording to their sources, i.e. unaged, aged and growing tissues. (Received November 13, 1970; )     

Photosynthetic characterization of Jerusalem artichoke during leaf expansion

Gas exchange, chlorophyll a fluorescence and modulated 820 nm reflection were investigated to explore the development of photosynthesis in Jerusalem artichoke (Helianthus tuberosus L.) leaves from initiation to full expansion. During leaf expansion, photosynthetic rate (Pn) increased and reached the maximal level when leaves were fully expanded. The same change pattern was also found in the stomatal conductance and chlorophyll content. Lower Pn could not be ascribed to the higher stomatal resistance in developing leaves, as intercellular CO₂ concentration was not significantly lower in these leaves. Lower Pn partly resulted from the lower actual photochemical efficiency of PSII in developing leaves, as more excited energy was dissipated through non-photochemical quenching. The development of primary photochemical reaction and electron transport in the donor side of PSII was completed in the initiating leaves. However, the development of electron transport in the acceptor side of PSII was not accomplished until leaves were fully expanded, indicated by the change in probability that an electron moves further than primary quinone (ψo). PSI activity changed in parallel with ψo suggesting that PSI cooperated well with PSII during leaf expansion. It should be stressed that the development of carbon fixation process was later than primary photochemical reaction but earlier than photosynthetic electron transport during leaf expansion. The later development of photosynthetic electron transport may reduce the production of reactive oxygen species from Mehler reaction, particularly under low carbon fixation.     

Inhibition of auxin-induced cell elongation by galactose

Galactose at concentrations higher than 3 m M inhibited specifically auxin-induced elongation of oat, wheat and rice coleoptile segments but not of pea and mung bean stem segments. Glucose, arabinose, rhamnose, xylose, mannose and glucosamine did not inhibit auxin-induced elongation of coleoptile segments. Galactose inhibited auxin-induced but not hydrogen ion-induced growth.     

The effect of inhibitors of protein and nucleic acid synthesis on nucleolar size and enzyme induction in Jerusalem artichoke tuber slices

Summary The effects of various inhibitors of protein and nucleic acid synthesis on the development of invertase activity and increased nucleolar volume in discs excised from tubers of artichoke tissue are compared. The results are discussed with reference to the current theories relevant to the action of the inhibitors and to the nature of the increase in nucleolar volume.     

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, on 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Induction of DNA synthesis and callus formation from tuber tissue of Jerusalem artichoke by 2,4-dichlorophenoxyacetic acid

Cellus induction was observed from Jerusalem artichoke tubertissue on a synthetic medium containing 2,4-D at 10^–6,10^–5 (optimum conc.) and 10^–4 M. The first DNA synthesis(thymidine incorporation) was observed only at 2,4-D concentrationsof 10^–5 to 10^–4M. In 10^–5 M 2,4-D treatedtissue, DNA synthesis increased after a 20 hr lag and reacheda maximum at 36 hr, after which it decreased. Actinomycin Dand 8-aza-guanine; inhibitors of RNA synthesis, inhibited DNAsynthesis completely. 2,4-D caused the characteristic changesin RNA and protein syntheses. In comparison with the control,RNA and protein syntheses were first repressed then inducedbefore the peak of DNA synthesis. Treatment with cycloheximide(10^–4M) for one hour before inoculation inhibited proteinsynthesis completely for 12 hr; consequently DNA synthesis wasalso delayed. The results suggest that RNA and protein synthesesneeded for callus induction are regulated by 2,4-D in the firstDNA synthesis. (Received July 19, 1973; )     

The correlation of phenylalanine ammonia-lyase and cinnamic acid-hydroxylase activity changes in Jerusalem artichoke tuber tissues

Summary Phenylalanine ammonia-lyase (PAL) and cinnamic acid-hydroxylase (CAH) exhibit parallel activity changes in tissues of Jerusalem artichoke (Helianthus tuberosus L.) tubers during ageing and in vitro culture. The activity of both enzymes appears in the tissue only after slicing and can be stimulated by manganese. Inhibition of CAH, by the deprivation of the substrate O₂, leads to cinnamic acid accumulation and PAL inhibition, a process prevented by cycloheximide. It is propounded that the link between PAL and CAH activities is the result of changes of the cinnamic acid pool size produced by a change in the activity of CAH.     

The induction of phosphatase activity in thin slices of Jerusalem artichoke tissue by treatment with indole acetic acid

Summary Prolonged washing of thin slices of Jerusalem artichoke (Helianthus tuberosus) did not result in any apparent increase in the activity of the phosphatase enzymes, although the washing process is known to stimulate the activity of many other enzymes. However, treatment of the tissue in either 3×10^–5M 2,4-dichlorophenoxyacetic acid or 10^–4M indole acetic acid resulted in a 3-fold increase in the phosphatase activity. Significant stimulations of activity were detectable one hour after placing the tissue in the auxin. Treatment of the tissue in either kinetin or gibberellic acid failed to stimulate the activity of the enzyme. The enhancement of phosphatase activity caused by auxins could not be prevented by adding cycloheximide to the treatment solution an it is concluded that the stimulation occurred as the result of the activation of enzyme already present in the tissue rather than by the de novo synthesis of new enzyme protein.     

氮磷互作对菊芋块茎产量和品质的影响

为探明肥料互作对菊芋产量及品质的影响,以“南芋1号”为试验材料,通过正交田间试验,对氮(N)、磷(P)及其交互作用对菊芋块茎产量和品质的影响进行了研究.结果表明:N、P对菊芋块茎产量、干物质含量,总糖、还原糖和菊糖含量具有极显著影响(P＜0.01);N、P交互作用对菊芋块茎产量、干物质和还原糖含量有显著影响(P＜0.05),对块茎总糖和菊糖有极显著影响(P＜0.01);当磷肥为135 kg·hm-2时,与氮肥存在交互效应;当氮肥低于180 kg·hm-2时,为正交互效应,表现出协同促进作用,氮肥高于180 kg·hm-2时,为负交互效应,表现出拮抗作用;当氮肥为180 kg·hm-2、磷肥为135 kg·hm-2时,氮磷正交互效应增强,氮磷表现出协同促进作用,菊芋块茎产量最高,且菊芋块茎的干物质含量、总糖、还原糖和菊糖含量达到最高值,即菊芋块茎能达到最高产量和最优品质.综上,在山东莱州地区菊芋大田种植的基础施肥量宜为纯氮为180 kg·hm-2、P2O5为135 kg·hm-2.     

Cytological studies on the inhibition by 5-fluorouracil of ribosome synthesis and growth in jerusalem artichoke tuber slices

Summary Rapid auxin-induced cell expansion in artichoke tuber slices is obtained by aerating the slices in water (aging) prior to auxin treatment. 5-fluorouracil (5-FU), an inhibitor of ribosomal RNA synthesis in plant cells, markedly inhibits auxin-induced growth only if present in the pre-growth aging period. Autoradiographic studies show that 5-FU given in the aging and/or growth periods reduces the incorporation of RNA precursors into the cytoplasm. Pulse-chase experiments suggest that the reduced cytoplasmic incorporation is in large part due to decreased stability of ribosomal rNA, as nucleolar and chromatin label are only slightly depressed at the end of the pulse. Though the nucleoli continue to incorporate RNA precursors following 5-FU treatment, they lack a distinct granular zone, and appear as homogeneous fibrillar structures under the electron microscope. 5-FU has a parallel inhibitory effect on growth and protein synthesis as shown by ³H-leucine studies during the growth period. Electron-microscope studies show that treatment with 5-FU causes decreased numbers of ribosomes and rough endoplasmic reticulum. The results suggest that the ribosomes and rough endoplasmic reticulum formed during aging are important in obtaining subsequent rapid auxin-induced expansion. The new ribosomes serve in part to replace pre-existing ribosomes present at the time of excision, which from electron microscopic evidence from 5-FU treated tissue, appear to slowly disappear.     

Fructose 2,6-bisphosphate in relation with the resumption of metabolic activity in slices of Jerusalem artichoke tubers

When slices of Jerusalem artichoke tubers were incubated at 25°C, their concentration in fructose 2,6-bisphosphate increased up to 250-fold within 2 h. Fructose 2,6-bisphosphate was also formed, although at a slower rate, in slices incubated at 0°C. Its formation could not be explained by an increase in the concentration of fructose 6-phosphate or of ATP either by an activation of phosphofructo-2-kinase. Pyrophosphate—fructose-6-phosphate 1-phosphotransferase was the only enzyme present in a tuber extract which was found to be sensitive to fructose 2,6-bisphosphate. An improved procedure for the assay of fructose 2,6-bisphosphate is also reported.