Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3′(2′) monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M_r of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2′,3′-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5′ purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate.     

Phenylpropanoids as a Protectant of Aluminum Toxicity in Cultured Tobacco Cells

Aluminum (Al) enhances ferrous ion [Fe(II)]-mediated peroxidationof lipids, which is lethal to normal tobacco cells, but notto phosphate (P_i)-starved cells ( –P cells). We foundthat tobacco cells accumulated phenylpropanoid compounds includingchlorogenic acid (CGA) and caffeic acid (CA) during P_i starvation.The accumulation was inhibited by 2-aminoindan-2-phosphonicacid (AIP), a specific inhibitor of L-phenylalanine ammonialyase (PAL). CGA, CA and also an extract containing the phenylpropanoidcompounds from –P cells protected normal cells ( +P cells)efficiently from both lipid peroxidation and the loss of viabilitycaused by the combined application of Al and Fe(II), indicatingthat the phenylpropanoids acted as antioxidant molecules. –Pcells exhibited approximately 25-fold higher specific activityof PAL than +P cells. The content of the phenylpropanoids andthe activity of PAL were not affected by the combined treatmentwith Al and Fe(II) in either +P cells or –P cells. Theseresults suggest that an increase in PAL activity during P_i starvationenhances the accumulation of phenylpropanoids, and that thephenylpropanoids protect tobacco cells from cytotoxic lipidperoxidation caused by the combination of Al and Fe(II) ⁴CREST, Japan Science and Technology Corporation (JST).     

Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation

We report the isolation of five cDNA clones whose corresponding mRNAs accumulate in cultured soybean cells (Glycine max cv Mandarin) during cytokinin or auxin starvation. The levels of three of these mRNAs decrease rapidly after addition of 5 micromolar zeatin to cytokinin-starved cells or after addition of 10 micromolar α-naphthaleneacetic acid to auxin-starved cells. These mRNAs also exhibit various patterns of accumulation in the tissues of intact soybean plants. Partial nucleotide sequence analysis demonstrates that one of the cDNAs in the collection, called SAM46, is 46% identical at the amino acid level to the iron superoxide dismutase gene of Escherichia coli. Expression of this cDNA in Escherichia coli cells results in detectable iron superoxide dismutase activity, confirming the identity of the cDNA.     

Changes Related to Salt Tolerance in Thylakoid Membranes of Photoautotrophically Cultured Green Tobacco Cells

A line of cultured tobacco cells (Nicotiana tabacum cv. SamsunNN) was established that was able to grow photoautotrophicallyin a medium that contained 0.2 M NaCl or in a medium withoutNaCl. Thylakoid membranes of the NaCl-adapted cells had higheroxygen-evolving activities, on the basis of chlorophyll, thanthose of unadapted cells. Furthermore, the oxygen-evolving activitiesof thylakoid membranes from NaCl-adapted cells were more tolerantto high concentrations of NaCl than those from unadapted cells. Glycinebetaine at 1 M protected the oxygen-evolving activityof thylakoid membranes from unadapted cells but not that fromadapted cells. Examination of the dissociation of 23-kDa and33-kDa polypeptides from the water-splitting complex of photosystemII at high concentrations of NaCl indicated that the affinitywith which the 23-kDa polypeptide was bound to thylakoid membranesof salt-adapted cells had been altered. (Received March 22, 1993; Accepted November 15, 1993)     

Changes in Endogenous Gibberellin Contents during Growth of Cultured Tobacco Cells

Changes in the contents of endogenous gibberellins (GAs) wereexamined in three kinds of cultured tobacco cells; a crown gallcell and two cultured cells derived from normal tissue of Nicotianatabacum. The relative amounts of the GAs were analyzed by systematicchromatographic purifications followed by GC-SIM. In all thecell lines examined, the content of GAj was the highest duringthe logarithmic phase of growth, indicating that GA₁ has a physiologicalrole in the growth of dedifferentiated cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received April 11, 1984; Accepted July 10, 1984)     

Effect of Inorganic Phosphate on the Extracellular Acid Phosphatase Activity of Tobacco Cells Cultured in vitro

Acid phosphatase activity in culture medium of tobacco cells growing in suspension increased with the age of the culture from which the medium was obtained. The increase in the activity was accelerated by omitting inorganic phosphate from nutrient medium, and it was depressed by addition of inorganic phosphate or cycloheximide. Amylase and β-galactosidase activities were not induced by the omission of inorganic phosphate. It was concluded that derepression of acid phosphatase synthesis was involved in the increase in the extracellular acid phosphatase activity upon inorganic phosphate depletion.     

Autophagy in Tobacco Suspension-Cultured Cells in Response to Sucrose Starvation

The response of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated. When the cells that were grown in Murashige-Skoog medium containing 3% (w/v) sucrose were transferred to the same medium without sucrose, 30 to 45% of the intracellular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were degraded nonselectively. With the same treatment, protease activity in the cell, which was measured at pH 5.0 using fluorescein thiocarbamoyl-casein as a substrate, increased 3- to 7-fold after 1 d. When the cysteine protease inhibitor (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane (10 [mu]M) was present in the starvation medium, both the protein degradation and the increase in the protease activity were effectively inhibited. Light microscopy analysis showed that many small spherical bodies accumulated in the perinuclear region of the cytosol 8 h after the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 to 6 [mu]m in diameter that contained several particles. Quinacrine stained these vesicles and the central vacuole; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1-naphthylphosphate and [beta]-glycerophosphate as substrates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions.     

Long-Term Phosphate Starvation and Respiratory Metabolism in Suspension-Cultured Catharanthus roseus Cells

In order to clarify the metabolic adaptation of respiratorypathways in plants to limited levels of P_i, the effects of long-termstarvation of P_i on the activities of various enzymes relatedto respiratory metabolism were examined in suspension-culturedCatharanthus roseus cells. When the activities were expressedas units per g fresh weight, only those of phosphoenolpyruvate-hydrolyzing(PEP-hydrolyzing) enzyme (which may possibly be equivalent tothe acid phosphatase activity derived from vacuoles) and PEPcarboxylase were higher in the Pⁱ-starved cells than in controlcells. Activities of other enzymes in the Pⁱ-starved cells werelower than or similar to those of the control cells. Time-coursestudies indicated that PEP-hydrolyzing activity was inducibleby starvation of Pⁱ. However, in contrast to the results reportedby Duff et al. [(1989a) Plant Physiol. 90: 1275.], fluctuationsin the activity of PP¹:fructose-6-phosphate 1-phosphotransferaseduring starvation of Pⁱ were similar to those in levels of phosphofructokinaseand 6-phosphogluconate dehydrogenase. These data suggest thatthe concept of the phosphate starvation-inducible ‘bypasses’,which are engineered via the coarse control (i.e., induction)of specified enzymes and were proposed initially by Duff etal. in Brassica nigra cells, is not directly applicable to Catharanthusroseus cells in suspension. Tracer experiments using [U-¹⁴C]glutamineindicated that a significant proportion of respiratory substratescould be supplied from the enlarged pool of amino acids duringstarvation of Pⁱ. These assumptions are supported by the observedfluctuations in levels of free amino acids and of protein inP¹-fed and P¹-deficient Catharanthus roseus cells. ¹Part 41 in the series ‘Metabolic Regulation in PlantCell Cultrue’ ²Present Address: Morinaga Mild Industry, 5-1-83, Higashihara,Zamma-shi, Kanagawa, 228 Japan     

Dark Formation of Chlorophyll in Cultured Tobacco Cells

Dark accumulation of chlorophyll was demonstrated in calluscells of Nicotiana glutinosa. The chlorophyll content of dark-growncallus cells was dependent on the agar concentration in theculture medium: the content was significantly higher at lowerconcentrations of agar. Some properties of the dark-formed chlorophyllare described. (Received June 25, 1983; Accepted December 2, 1983)     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)     

An Electrophysiological Study of Photomixotrophically Cultured Tobacco Cells

Membrane potential properties of photomixotrophically culturedgreen tobacco cells with chloroplasts were studied in comparisonwith white tobacco cells without chloroplasts. In the dark therewas almost no difference in their membrane potential properties.In the light some of the green cells showed a light-inducedpotential change (LPC), but other green cells did not, nor didthe white cells. Our results indicate that the green cells arecomposed of two kinds of cells, one that shows the LPC and onethat does not. (Received October 5, 1983; Accepted May 10, 1984)     

Tissue-Specific Variation in the Cytokinin Habituation of Cultured Tobacco Cells

Cells of tobacco pith parenchyma sometimes lose their requirement for an exogenous supply of a cell division factor usually supplied as the synthetic cytokinin, kinetin. This change in phenotype, known as cytokinin habituation, is inherited by individual cells and appears to result from epigenetic changes rather than from rare, random, genetic mutations. We have found that tissues from different regions of the tobacco plant exhibit different states of habituation in culture. Pith tissues, as reported earlier, are usually cytokinin requiring and rapidly shift to the habituated state in culture. Leaf tissues are very slightly habituated and require kinetin for optimal rates of growth. Tissues from the stem-cortex are initially habituated. Both the leaf and cortex phenotypes are inherited by individual cells and persist for many cell generations in culture. These results show that certain tissue-specific phenotypes persist in culture and provide evidence that a process akin to habituation leading to different stable states of cytokinin requirement occurs in normal development.     

A Ferredoxin-Like Electron Carrier from Non-Green Cultured Tobacco Cells

The electron carrier effective in nitrite reduction in proplastidsof cultured tobacco cells has been purified by DEAE-celluloseand Sephadex G-100 chromatography. Its electron carrying activityin the nitrite reduction system with dithionite showed that355 nmol NO₂^– reduced mg^–1 protein min^–1.The electron carrier had absorption maxima at 419, 459 and 469nm, and the absorbance peak at 419 nm was decreased 56% on reduction.The reduced form of the electron carrier showed an electronparamagnetic resonance signal with g=1.93. Thus, this electroncarrier is a kind of ferredoxin. It did not, however, show electroncarrying activity in the NADP-photoreduction system of chloroplasts.Its molecular weight was calculated as 19,500 by Sephadex G-100chromatography. ¹Present address: Second Department of Anatomy, Fukushima MedicalCollege, Sugitsuma-cho, Fukushima 960, Japan. (Received April 11, 1983; Accepted February 6, 1984)     

Ion Composition of Tobacco Cells Cultured under Sulfur Deficiency

In both photoheterotrophic and heterotrophic tobacco cells areduced supply of sulfate in the medium did not alter the ratebut the duration of exponential growth. The higher the sulfatesupply in the medium the longer exponential growth proceeded.However, the ion composition of photoheterotrophic and heterotrophiccells was affected by sulfur deficiency in completely differentways. The dynamics in the K⁺-, Na⁺-, Mg²*-, nitrate-, phosphate-,and malate-con-tents of photoheterotrophic cells during growthwere not at all, or only slightly changed, when the sulfatesupply in the medium was reduced from 1.8mM to 1.2 mM, 0.6 mM,or 0.3mM. In heterotrophic tobacco suspensions, however, severesulfur deficiency caused K⁺, Na⁺, Mg²⁺, and malate to accumulateand nitrate to begin to accumulate earlier inside the cells.Addition of sulfate after 4 days to heterotrophic suspensionsgrown under sulfur-limiting conditions prevented the accumulationof these cations and anions. During the initial period of growthalso phosphate accumulated inside heterotrophic tobacco cellsto amounts found to be the higher the smaller the sulfate-contentof the media. Apparently, in photoheterotrophic tobacco cellsthe ion composition can homeostatically be regulated independentfrom the cells' sulfate supply, whereas the ion compositionof heterotrophic tobacco cells appears to be highly dependenton the sulfate supply of the cells. ⁴Present address: Fraunhofer Institut für AtmosphärischeUmwéltforschung, Kreuzeckbahnstr. 19, D-8100 Garmisch-Partenkirchen, F.R.G. (Received August 30, 1988; Accepted January 18, 1989)     

Ultraviolet Radiation-stimulated Efflux of 86-Rubidium from Cultured Tobacco Cells

Ultraviolet (254 nm) radiation stimulated the efflux of ⁸⁶Rb⁺ from liquid-cultured tobacco (Nicotiana tabacum) cells; it did not stimulate the movement of mannitol or 2-deoxyglucose. These results indicate that the efflux of ⁸⁶Rb⁺ is not to a generalized disruption of membrane structure.     

Size and Structure of Replicating Mitochondrial DNA in Cultured Tobacco Cells

The BY-2 tobacco cell line was used to study the size and structure of replicating mitochondrial DNA (mtDNA). Approximately 70 to 90% of the newly synthesized mtDNA did not migrate during pulsed-field gel electrophoresis. Moving pictures of the fluorescently labeled molecules showed that most of the immobile well-bound DNA was in structures larger than the size of the BY-2 mitochondrial genome of ~270 kb. Most of the structures appeared as complex forms with multiple DNA fibers. The sizes of the circular molecules that were also observed ranged continuously from ~20 to 560 kb without prominent size classes. Pulse-chase and mung bean nuclease experiments showed that the well-bound DNA contained single-stranded regions and was converted to linear molecules of between 50 and 150 kb. MtDNA replication in plants may be initiated by recombination events that create branched structures of multigenomic concatemers that are then processed to 50- to 150-kb subgenomic fragments.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

烟草培养细胞中钙离子,钙调素与细胞质流的关系

烟草愈伤组织的培养细胞中,当钙离子载体将Ca~(2 )导入细胞时,细胞质流停止.CaM拮抗剂试验表明,高钙使细胞质流停止的效应可能与CaM无关,除W7外的多种CaM拮抗剂都明显而且可逆地抑制细胞质流。酶联免疫吸附分析(ELISA)检出培养细胞中存在有CaM。间接酶标免疫组织化学分析进一步证明CaM存在于胞质条纹中。     

Resistance Mechanism of Cultured Plant Cells to Tobacco Mosaic Virus

In somaclonal tissues obtained from systemically TMV-infected tobacco plants, a relation between changes of TMV amounts and the callus growth was examined. The culture medium was suitable for maintaining a constant concentration of TMV as well as active callus growth. By using the shake-culture method, somaclonal tissues were separated into two classes on the basis of callus sizes. In large callus tissues, TMV amounts were constant during subculturing but the tissues did not either grow or release the newly divided cells after the last subculture. On the other hand, smaller callus tissues grew markedly and the TMV amounts were conspicuously lowered. After shake-subculture of smaller tissues, they were successfully regenerated to plantlets. None of the plantlets expressed any mosaic symptoms, while plantlets from the original somaclones showed severe mosaic symptoms of TMV in leaflets. Thus, the present report describes the successful production of virus-free plantlets from infected somaclonal callus cultures.