Stabilization of Oat Leaf Protoplasts through Polyamine-mediated Inhibition of Senescence

Protoplasts isolated from Avena sativa L. leaves undergo progressive senescence when incubated aseptically in 0.6 m mannitol with or without added nutrients. This senescence is manifested by morphological deterioration and ultimate lysis of protoplasts, by a decrease in incorporation of [(3)H]uridine and [(3)H]leucine into macromolecules, and by a sharp increase in ribonuclease activity.The presence in the incubation medium of l-arginine, l-lysine, certain polyamines related to these amino acids (cadaverine, putrescine, spermidine), Ca(2+), or streptomycin stabilizes the protoplasts. Protoplasts incubated with 10 mml-arginine or l-lysine show an initial inhibition of [(3)H]uridine incorporation, but with time, incorporation is restored to levels greater than in control protoplasts. The rise in ribonuclease activity of protoplasts is completely inhibited if the protoplasts are incubated with 10 mml-arginine. Greater incorporation of [(3)H]uridine into RNA of aging protoplasts is also maintained by appropriate concentration of cadaverine, putrescine, spermidine, Ca(2+), or streptomycin in the incubation medium; the same concentrations of these substances stabilize the protoplasts against additional lysis.     

Dual Mechanisms in Polyamine-mediated Control of Ribonuclease Activity in Oat Leaf Protoplasts

Dibasic amino acids and polyamines added to oat (Avena sativa L.) leaf protoplast isolation media decrease the RNase activity of extracted protoplasts relative to controls. This effect, which is manifested even when the added polyamine is removed by exhaustive dialysis prior to assay, is due to a prevention of the rise in RNase activity which usually follows protoplast isolation. Polyamines, but not dibasic amino acids, also decrease RNase activity in vitro. This in vitro effect seems to result from electrovalent attachment of the polyamine to the RNA, because the greater the net positive charge on the polyamine, the greater is its inhibitory effect in vitro. The activity of dibasic amino acids when added during protoplast isolation probably results from their conversion to polyamines.     

Synthesis of DNA during Mitosis

Growing roots of Zea mays in tritiated thymidine for brief periodsconfirms that G₁ may be eliminated from the mitotic cycle andDNA synthesis may be advanced into telophase but no further,in the fastest dividing cells of the cap initials, but not inthe stele.     

Polyamine Binding to Proteins in Oat and Petunia Protoplasts

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozenthawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.     

Electrorotation of Oat Protoplasts before and after Fusion

This paper describes an experimental chamber suitable for inducingcell fusion by an electric field and for measuring the rotationalbehaviour of single protoplasts and fusion products in high-frequencyrotating fields. Intact protoplasts from Avena sativa L. leavesrotate before, during and after fusion, as demonstrated by therotation spectra of cells. The electrorotation technique allowselectrical properties of fused cells to be examined within asecond after applying the fusion pulse. (Received June 23, 1986; Accepted June 19, 1987)     

Regulation of Amino Acid Uptake into Oat Mesophyll Cells: A Comparison between Protoplasts and Leaf Segments

The uptake of -aminoisobutyric acid (AIB) into protoplasts andinto 1 cm sections of leaves from 7 d old light-grown oats (Avenasativa L. cv. ‘Garry’) was studied. Both protoplastsand leaf sections with cuticle and epidermis removed accumulatedAIB against a concentration gradient although the rate of uptakeinto protoplasts was one-third to one-sixth that into leaf sections.AIB uptake into both protoplasts and leaf cells in situ wasstimulated by ‘aging,’ and low pH, and inhibitedby osmotic shock, respiratory poisons, and KCl concentrationsabove 1 mM. It was concluded that the rate of uptake of AIBand its accumulation ratio could be accounted for by the energyinherent in the proton-motive force, the proton-motive forcebeing the sum of the pH gradient and potential difference acrossthe plasma membrane. The similarities between oat mesophyllprotoplasts and leaf cells in situ suggest that these protoplastsare suitable material for the study of certain membrane-regulatedevents.     

Polyamine-induced hydrolysis of apurinic sites in DNA and nucleosomes.

The ability of different polyamines to catalyze hydrolysis of phosphodiester linkages in apurinic and apyrimidinic (AP) sites has been investigated in supercoiled, relaxed and denatured DNA, and also in core and chromatosome particles. The rate constants for the hydrolysis in the DNAs have been determined. In general the order of effectiveness of the polyamines were: spermine greater than spermidine greater than putrescine greater than cadaverine. A 9 fold difference in rate constants was found between spermine and cadaverine. No difference in the rate of hydrolysis was seen between AP-sites in supercoiled and relaxed DNAs, whereas the rate for the single-stranded DNA and DNA in core and chromatosome particles was only half of that in the double-stranded DNA. All AP-sites in both free DNA and DNA-histone particles were hydrolyzed in the presence of polyamines. For all polyamines, with the exception of spermine, increasing concentration of both Mg++ and salts such as KCl both led to a large decrease in the rate of polyamine-induced hydrolysis of AP-sites. The rate of hydrolysis increased markedly with increasing pH in the pH range pH 6 - pH 11.     

DNA Synthesis, Mitosis and Ploidy of Dividing Cells in Early Crown Gall

DNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with ³H-thymidine in slides in which mitoses had been counted and analyzed before autoradiography. The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two. The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.     

Swelling of Etiolated Oat Protoplasts Induced by cAMP and Red Light

Etiolated oat protoplasts were treated with dibutyryl cAMP tostudy possible function of cAMP in the development by measuringthe protoplast swelling. The mean diameter of protoplasts inthe absence of any chemical treatment was 33.58±1.26(SE) µm, which increased to 36.96±0.86 µmin the presence of 100 µM dibutyryl cAMP. Prostacyclin,a potent activator of adenyl cyclase, also showed a significantswelling effect (diameter 38.01±0.98 µm). Red lightalso elicited the swelling of protoplasts (40.26±0.8µm). ¹Present address: Department of Biology, Pusan National University,Pusan 607, Korea. ²Present address: Department of Horticulture, Cheju NationalUniversity, Cheju 590, Korea. ³Present address: Department of Biological Sciences, Texas TechUniversity, Lubbock, TX 79409, U.S.A. (Received June 29, 1985; Accepted November 18, 1985)     

Osmoregulation by Oat Coleoptile Protoplasts (Effect of Auxin)

The effect of auxin on the physiology of protoplasts from growing oat (Avena sativa L.) coleoptiles was investigated. Protoplasts, isolated iso-osmotically from peeled oat coleoptile segments, were found to swell steadily over many hours. Incubated in 1 mM CaCl2, 10 mM KCl, 10 mM 2-(morpholino)ethanesulfonic acid/1,3-bis-[tris(hydroxymethyl)methylamino]propane, pH 6.5, and mannitol to 300 milliosmolal, protoplasts swelled 28.9% [plus or minus] 2.0 (standard error) after 6 h. Addition of 10 [mu]M indoleacetic acid (IAA) increased swelling to 41.1% [plus or minus] 2.1 (standard error) after 6 h. Swelling (in the absence of IAA) was partially dependent on K+ in the bath medium, whereas auxin-induced swelling was entirely dependent on K+. Replacement of mannitol in the bath by Glc increased swelling (in the absence of IAA) and eliminated auxin-induced swelling. Swelling with or without IAA was inhibited by osmotic shock and was completely reversed by 0.1 mM NaN3. Sodium orthovanadate, applied at 0.5 mM, only gradually inhibited swelling under various conditions but was most effective with protoplasts prepared from tissue preincubated in vanadate. Our data are interpreted to suggest that IAA increases the conductance of the plasma membrane to K+.     

Malate and Dihydroxyacetone Phosphate-dependent Nitrate Reduction in Spinach Leaf Protoplasts

Isolated spinach (Spinacia oleracea L. var. Bloomsdale) leaf protoplasts reduced nitrate at rates of 9 micromoles per milligram chlorophyll per hour in light with a 3- to 4-fold stimulation in the presence of HCO₃⁻. A similar stimulation of nitrate reduction in the absence of CO₂ fixation was obtained by the addition of malate, oxaloacetate (OAA), phospho-3-glyceric acid (PGA), or dihydroxyacetone phosphate (DHAP). Stimulation by malate and DHAP was light-independent, while the PGA and OAA effect was light-dependent. Nitrate reduction was found to be coupled to the cytoplasmic oxidation of DHAP or malate. The PGA/DHAP and OAA/malate shuttle across the chloroplast envelope has been demonstrated to support CO₂ fixation and/or nitrate reduction. The leaf protoplasts readily assimilated nitrate into amino-N in a stoichiometric relationship.     

Osmotically-Induced Surface Area Expansion in Oat Protoplasts Depends on the Transmembrane Potential

The transmembrane potential of mesophyll cell protoplasts fromAvena sativa was altered in two different ways: first by raisingthe external KCI concentration with and without the additionof valinomycin and, secondly, by using the proton carrier CCCPand varying the external pH. The percentage of unlysed protoplastswas determined after decreasing the osmotic pressure from 1.2to 0.7 MPa in order to characterize their ability to enlargetheir surface area. Lysis strongly increased when the KCI concentrationwas raised from 0 to 90 mol m^–3. This effect did not dependon the presence of valinomycin. Using CCCP, lysis increasedsimilarly if the external pH was lowered from 7.2 to 5.5. Inthe presence of CCCP the influence of the external KCI concentrationdisappeared completely. This led to the conclusion that depolarizationinhibited surface area expansion. Since the expansion is thoughtto be based on exocytotic-like membrane incorporation, theseresults suggest that exocytosis is controlled, at least in part,by the transmembrane potential. Key words: Oat protoplasts, osmotic expansion, transmembrane potential     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

DNA Synthesis in Tobacco Mosaic Virus-Infected Nicotiana tabacum Protoplasts and Regeneration of Persistently Infected Calli

Tobacco mosaic virus infection of Nicotiana tabacum mesophyll protoplasts did not affect the pattern of chloroplast or total cellular DNA synthesis for at least 120 h when compared with that of mock-infected cells. Calli derived from infected protoplasts often showed large amounts of tobacco mosaic virus RNA and coat protein.     

燕麦叶片衰老与活性氧代谢的关系

燕麦连体叶片与高体叶片衰老中,过氧化氢酶和超氧物歧化酶(SOD)活性下降,脂类过氧化产物丙二醛(MDA)迅速积累,组织自动氧化速率显著加快。植物激素BA,GA_3,2,4—D及光、亚胺环己酮(CH),EDTA处理均不同程度地延缓离体叶片的衰老过程,同时抑制过氧化氢酶和SOD活性下降,阻止MDA的积累和组织自动氧化速率的提高.推测叶片衰老中活性氧起着重要的作用。     

Light-Stimulated Changes in the Acidity of Suspensions of Oat Protoplasts: Dependence upon Photosynthesis

Light induced an alkalinization and stimulated a subsequent acidification of the medium surrounding oat (Avena sativa L. cv Garry) leaf protoplasts. Blue light was less effective than would be predicted from photosynthetic action spectra. Nonetheless, 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented alkalinization and reduced acidification to the dark rate for protoplast suspensions exposed to all light regimes tested.

Alkalinization increased in parallel with initial rates of O₂ evolution as the quantum flux density of white light was raised to 75 microeinsteins per square meter per second. Alkalinization was accompanied by a decrease in the CO₂ content of the medium; therefore, it was attributed to photosynthetically induced CO₂ uptake. The effect of CO₂ depletion on the acidity of the medium appeared to be mainly restricted to the first 15 minutes of exposure to light. Consequently, subsequent pH changes primarily reflected a constant net proton efflux. Acidification occurred in the dark, but rates of acidification increased in response to increased light approximately in parallel with changes in a concomitant net O₂ efflux. The results indicated that protoplasts could acidify the medium in response to nonphotosynthetic activity, but that photosynthesis mediated light stimulation of acidification.

     

土壤水分胁迫对燕麦叶片渗透调节物质含量的影响

以旱棚内盆栽的'内农大莜一号'燕麦品种为材料,测定了其不同水分胁迫下各生育期叶片的脯氨酸、可溶性糖含量和细胞膜相对透性,分析土壤含水量对燕麦叶片渗透调节物质的影响,以明确燕麦不同生育时期的抗旱特性.结果表明:(1)随着土壤相对含水率的下降,燕麦各生育期叶片脯氨酸含量、可溶性糖含量、细胞膜相对透性均呈上升趋势.(2)随着生育期的推进,燕麦叶片脯氨酸含量在30%和45%土壤含水量下呈持续上升趋势,而在含水量 60%、75%、90%处理下则于生育前期上升,灌浆期略有下降;随着生育期的延续,可溶性糖的含量呈先升后降的抛物线型变化,且其最大值随水分胁迫强度增加而提前,含水量 30%和45%处理的最大值均出现在拔节期,含水量60%和75%处理则分别出现在孕穗期和开花期;随着生育期的延续,各处理燕麦叶片的细胞膜相对透性均呈持续上升趋势.可见,水分胁迫能诱导燕麦叶片渗透调节物质的积累,且增幅随着胁迫强度的增加而上升,从而使燕麦表现出较强的抗旱性.     

Isolation of Leaf Protoplasts: Macromolecule Uptake and Growth Substance Response

A simple method, using mixtures of commercially available pectinasesand cellulases, is described for the isolation of very largenumbers of tobacco leaf protoplasts. These protoplasts havebeen cultured for several days in suitable media. Pinocytosis was shown to occur when protoplasts were incubatedwith ferritin. These isolated protoplasts respond dramatically,by progressively bursting, when incubated with growth substancesat physiological levels. There was no detectable response togrowth substances such as indol-3yl-acetic acid when the anti-auxintranscinnamic acid was also present, indicating that these responsesare specifically auxin-induced.