Isolation, Culture, and Plant Regeneration of Protoplasts of Two Shrubby Oxalis Species from South America

Protoplasts were successfully isolated from internodal callustissues of both Oxalis glaucifolia and O. rhombeo-ovata whenthey were digested in a solution containing 0.1% (w/v) MacerozymeR-10, 0.5% (w/v) cellulase Onozuka R-10 and 0.3 mmol m^–3sucrose. Protoplasts proliferated to give cell colonies on Gamborget al.'s B5 medium supplemented with 0.3 mmol m^–3 mannitol,0.5 mg dm^–32, 4-D, and 2.0 mg dm^–3 kinetin. Calluswas produced upon transfer of cell colonies to Murashige andSkoog medium containing 2.0 mg dm^–3 l-naphthaleneaceticacid (NAA) and 0.1 mg dm^–3 kinetin for O. glaucifolia,or with 5.0 mg dm^–3 NAA and 0.5 mg dm^–3 6-benzylaminopurine,for O. rhombeo-ovata. Plants were regenerated from O. glaucifoliaprotoplasts on a medium containing 0.1 mg dm–3 NAA, 1.0mg dm^–3 kinetin and 1.0 mg dm^–3 gibberellic acid,but only vascular nodules were differentiated by O. rhombeo-ovataprotoplast-derived calli. Key words: Tissue culture, protoplasts, plant regeneration, Oxalis spp     

Germination of Sporangia of Phytophthora palmivora (Butl.) Butl.

Sporangia of Phytophthora palmivora germinated by either forminggerm tubes or producing zoospores. Two distinct modes of germ-tubedevelopment have been described. Sporangia in distilled waterformed zoospores at 10–34°C with an optimum at 22°Cbut germinated by means of germ tubes at 30 and 34°C only.Zoospore formation was inhibited to varying degrees by cocoapod extract, I.0 per cent (w/v) peptone and yeast extract, 100–500µg1^-1 thiamine, and by very low concentrations of severalamino acids, carbohydrates, and inorganic salts. Germ-tube formation was encouraged at 22°C by 1'0 per cent(w/v) peptone and yeast extract, by cocoa pod extract and exudate,10mM CaCl2, 1–10 mM MgSO₄. 7H₂O, 0.5 per cent (w/v) fructose,galactose, glucose, lactose, maltose, and sucrose, by 100 ppmarginine, aspartic acid, glutarnic acid, glycine, leucine, andtryptophane, and by 100–500 µg 1^-1 thiamine.     

Comparative Studies of Leaf Tissue 4: III. EFFECTS OF WALL-DEGRADING ENZYMES AND OSMOTIC STRESS

Commercially available cell wall-degrading enzymes frequentlyused for protoplast isolation inhibited CO₂ fixation and photosyntheticO₂ evolution, and stimulated dark respiration by leaf tissueand isolated mesophyll protoplasts of Nicotiana tabacum L. andAntirrhinum majus L. They also depolarized the membrane potentialof cells of leaf tissue, inhibited uptake of ⁸⁶Rb by tobaccoleaf tissue and isolated mesophyll protoplasts, and stimulated³⁶CI uptake by tobacco leaf tissue. Where studied, these effectswere found to be reversible. The depolarization effect on Antirrhinumleaf cells occurred even when the enzyme preparations had beendenatured, dialysed, or desalted, and the effect was greatestin those fractions of the enzyme preparation which showed thehighest cellulase activity. Plasmolysis of tobacco leaf tissue inhibited photosyntheticO₂ evolution, CO₂ fixation, and ⁸⁶Rb uptake to levels belowthose exhibited by isolated protoplasts in media of the samecomposition and osmolarity. The implications of these resultsfor work with leaf tissue and isolated protoplasts are discussed.     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Activity of Gibberellins A1 to A0 in the Avena First-leaf Bioassay, and Location after Chromatography

Activity curves are determined for gibberellins A₁ to A₀ bythe Avena first-leaf bioassay method. Gibberellins A¹, A₄ andA₅ can be detected at 10^-11 or 10^-10 g/ml and give optimum activityof approximately 230 per cent elongation (water controls = 100per cent). Gibberellins A²A³, and A⁹ can be detected at 10^-3g/mland give optimum activity of approximately 200 per cent. GibberellinsA₆ and A₇ can be detected at 10^-5g/ml; GA₇ gives optimum activityof around 190 per cent. All the gibberellins except GA₈ canbe detected by this bioassay method after chromatography inn-butanol: 1.5 N ammonia (3: 1) and benzene: acetic acid: water(4: 2: 1) when applied to the paper at concentrations from O.Ito µg. The sensitivity of the method is compared withthat of other gibberellin bioassay methods.     

A Reanalysis of Particle Motion in Sieve Tubes of Heracleum

From an analysis of the Brownian motion of particles in sieveelements of Heracleum mantegazzianum and Heracleum sphondylium,Barclay and Johnson have suggested that the in situ viscosityof sieve tube sap is four to six times higher than has previouslybeen assumed. In particular, they obtained a value for the sapviscosity of about 10^–2 Pa s, which compares with a valueof 2 x 10^–3 Pa s for a 20 per cent (w/v) sucrose solution.The present paper describes a reanalysis of their data. It isargued that Barclay & Johnson underestimated the Brownianmotion of sieve element particles and so overestimated the sapviscosity. An exact correction was not possible, but it is concludedthat the in situ viscosity of Heracleum sieve tube sap mustbe less than 3 x 10^–3 Pa s, which corresponds to a sucroseconcentration of less than 29 per cent. Hence it may not beunreasonable to suppose that the viscosity of sieve tube sapis determined primarily by the concentration of sucrose, ashas been assumed in theoretical analyses of the Munch hypothesis.It is also concluded that the sieve tubes studied by Barclayand Johnson were not functional, in the sense that they didnot exhibit an axial bulk flow of sap. Heracleum, sieve tubes, Brownian motion, viscosity, Munch hypothesis     

Metabolism of Pyrimidines in Protoplasts from Cultured Catharanthus roseus Cells

The metabolism of [2-¹⁴C]thymine, [2-¹⁴C]thymidine, [2-¹⁴C]uraciland [¹⁴C]uridine was investigated in protoplasts obtained fromsuspension cultures of Catharanthus roseus. Most of the exogenouslysupplied thymine, thymidine and uracil was degraded, and salvageof these pyrimidines accounted for 5–36 per cent of thetotal amount of ¹⁴C-labelled precursors which was metabolized.However, more than 80 per cent of the labelled uridine was utilizedfor the biosynthesis of nucleotides and nucleic acids, and therest was degraded. In contrast to the results from protoplastsof sugar cane cells in suspension culture, the data indicatethat protoplasts possess a pathway for the degradation of pyrimidines,and that the overall metabolism of these pyrimidines in protoplastsis very similar to the metabolism in the intact cells. Catharanthus roseus, madagascar periwinkle, protoplasts, pyrimidine metabolism     

Flavanoids in Pollen and Stigma of Brassica oleracea and Their Effects on Pollen Germination In Vitro

Brassica oleracea pollen was applied to a basic medium of 1.5per cent agar and 15 per cent sucrose to which flavanoids wereadded at three concentrations. Two types of agar were used;with agar 1, quercetin at a concentration of 0.5 x 10^–3per cent gave an increase in percentage germinating grains.With agar 2, an increase in germination occurred with kaempferoland naringin at concentrations of 0.5 x 10^–3 and 0.5 x10^–1 per cent respectively. Increase in pollen tube lengthoccurred with agar 2 and quercetin at a concentration of 0.5x 10^–3 per cent. The stigma tissue of B. oleracea contains at least three andthe pollen at least one glycoside of quercetin. The sugars inthe glycosides were not identified. Pollen germination and pollentube extension were not stimulated exclusively by the flavanoidspresent in the stigma. The flavanoid composition of the stigmadid not vary amongst five different S-allele genotypes, indicatingthat flavanoids are probably not directly involved in the incompatibilityreaction of B. oleracea.     

Growth of Plants in Different Oxygen Concentrations

Dwarf french beans (Phaseolus vulgaris var. Canadian Wonder)were grown in chambers at 25?C with the roots aerated at 20per cent oxygen and tops variously maintained at: T₁ O₂ 0.21;CO₂ 270?10^–6: T₂; O₂ 0.05, CO₂, CO₂ 270?10^–6: T₃;O₂ 0.21; CO₂ 550?10^–6. Experiment 1 (T₁ and T₂) lasted2 weeks: Experiment 2 (T₁ T₂ and T₃) only one week. Hourly estimatesof CO₂ uptake were made by gas analysis and weekly estimatesof fresh weight, dry matter in tops and roots, and leaf area,by sampling. Light intensity was 80 W m^–2 of photosyntheticallyactive radiation. An attempt was made to explain the results in terms of a simplelight absorption model such that where dV/dt is the rate of CO₂ uptake per plant, ßis the photosynthetic efficiency, I₀ is the incident light intensity,f is the fraction of incident light absorbed by unit leaf layerand L is the leaf area index. The analysis showed that ß(T₂)was at least double ß(T₁), whilst f(T₂) was smallerthan f(T₁) at a given leaf area. The results also required thatthroughout the period of the experiment, fL(T₁)=fL(T₂) at anygiven time, i.e. the treatment with the larger leaf area (T₂)has the smaller value of f, and therefore intercepts less lightper unit leaf area. This could be advantageous for plant growth,but requires further experiments. The photosynthetic rates per unit leaf are about 40 per centgreater in T₂ than T₁. Over the relatively short period of the experiment the resultsare adequately described by U=btⁿ, where U is the accumulatedcarbon dioxide uptake, b is related to the photosynthetic efficiency(different for the differing treatments), and n is a constant(similar for all treatments). This relationship with time isbelieved to be a relationship with accumulated radiation, forthe light was constant throughout the experiments. Comparisons of carbon fixed (measured gas uptake) and dry matteraccumulation (sampling) show great scatter with an average valueof 0.43. The first week's results were generally smaller thanthis value and the second week's greater. Energy fixation as a fraction of photosynthetically active radiationon the ground area covered by the plants ranged from 3.5 to10 per cent. The results from treatment T₃ were similar to T₂ suggestingthat increasing CO₂ concentration decreases the growth inhibitionat 21 per cent O₂.     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

Release of Guttation Fluid from Passive Hydathodes of Intact Barley Plants. II. The Effects of Abscisic Acid and Cytokinins

Guttation was used as a non-destructive way to study the flowof water and mineral ions from the roots and compared with parallelmeasurements of root exudation. Guttation of the leaves of barley seedlings depends on age andon the culture solution. Best rates of guttation were obtainedwith the primary leaves of 6- to 7-day-old seedlings grown onfull mineral nutrient solution. The growing leaf tissue becomessaturated with K⁺ below 1.5 mM K⁺ in the medium, whereas K⁺concentration in the guttated fluid still increases furtheras K⁺ concentration in the medium is raised. At 3 mM K⁺ averagevalues of guttation were 1.4–2.4 mm³ h^–1 per plantwith a K⁺ concentration of 10–20 mM; for exuding plantsthe flow was 4.2–7.6 mm³ h^–1 per plant and K⁺ concentration35–55 mM. Abscisic acid (ABA) at 10^–6 to 10^–4 M 0–2h after addition to the root medium increased volume flow ofguttation and exudation and the amount of K⁺ exported. Threeh after addition of ABA both volume and amount of K⁺ were reduced.There was an ABA-dependent increase in water permeability (Lp)of exuding roots shortly after ABA addition. Later Lp was decreasedby 35 per cent and salt export by 60 per cent suggesting aneffect of ABA on salt transport to the xylem apart from itseffect on Lp. Benzyladenine (5 x 10^–8 to 10^–5 M)and kinetin (5 x 10^–6 M) progressively reduced volumeflow and K⁺ export in guttation and exudation and reduced Lp. Guttation showed a qualitatively similar response to phytohormonesas found here and elsewhere using exuding roots. Hordeum vulgare L., barley, guttation, abscisic acid, cytokinins, benzyl adenine, kinetin     

Response of Self-thinning to Artificially Reduced Levels of Leaf Area in Monocultures of Trifolium pratense

Greenhouse grown monocultures of Trifolium pratense L, werepartially defoliated to test the hypothesis that the constantof proportionality (K) in the self-thinning rule is a functionof leaf area. This constant equates mean weight (w) and density(p) in self-thinning populations by the equation Pruning leaflets did not affect the pattern or occurrence ofdensity-related mortality but, as predicted, did affect K, whichwas reduced by 25 per cent as leaf area was decreased from anaverage of 7.3 to 3.9 m²m^–2. For both self-thinning andnon-self-thinning populations, leaf area was substituted forK in eqn (1) to give Multiple linear regression showed that this expression was significantfor all three defoliation treatments. Regressions with treedata grouped by genera were also significant and indicate thateqn (2) may be a more general expression of the relation betweenmean weight and density in even-aged monocultures. The self-thinningrule may be a special case of eqn (2) which expresses itselfwhen leaf area attains some upper limit. Trifolium pratense, red clover, leaf area, self-thinning, defoliation     

Microbiological Synthesis of Fat: THE EFFECTS OF NITROGEN LEVEL ON THE METABOLISM OF PENICILLIUM LILACINUM THOM IN A SUCROSE MEDIUM

1. A study has been made of the relationships between the synthesesof carbohydrate, protein, and fat by Penicillium lilacinum Thomin presence of different amounts of sodium nitrate us a definedsucrose salts medium.
2. Under the defined experimental conditionsincreases in the concentrationof NO^–₂ in the medium werefollowed by increases in therates at which nitrogen and sugarwere taken up by the fungus,in the quantities assimilated,and in total and protein nitrogenin the felt. These conditionsprevailed so long as unassimilatedsugar was available.
3. Mediaof lower NO^–₃ concentration (for example, 0·32or 0·64 per cent. (w/v) NaNO^–₂;) yielded feltsricher in carbohydrate than were those grown in media of higherNO^–₂; content (0·96 or 1·28 per cent. (w/v)NaNO₃ The carbohydrate content of the felts increased graduallyuntil the sugar in the medium was exhausted; carbohydrate contentthen decreased.
4. Media of lower NO^–₃; concentration weremore conduciveto fat synthesis than those of higher NO^–₃;content.

     

Somatic Embryogenesis in Sugarcane (Saccharum officinarum L.): Growth and Plant Regeneration from Embryogenic Cell Suspension Cultures

Embryogenic cell suspension cultures were established from calliderived from young leaves of sugarcane (Saccharum officinarumL.) by placing them in liquid medium containing 5 per cent coconutwater (CW), 2–3 mg 1^–1 2, 4-D and 500 mg 1^–1casein hydrolysate (CH). The cultures were maintained by transferring2.5–5.0 ml of the suspension to 35 ml of fresh mediumevery 4–5 days. Organized structures resembling the earlystages of embryogeny were formed when 2, 4-D in the medium waslowered (0.1–1.0 mg 1^–1) but these did not developbeyond the globular or early scutellar stages. High levels ofsucrose (6–10 per cent) promoted the formation of proembryoids.Plating of the suspension on MS agar medium supplemented with0.25–2.0 mg 1^–1 2, 4-D, 5 per cent CW, 500 mg 1^–1CH, with or without activated charcoal, resulted in the formationof embryogenic calli. A large number of embryoids were formedin media containing lower 2, 4-D concentrations. Transfer ofembryoids to half-strength MS medium with 6 per cent sucroseestablished plantlets which were successfully transferred tosoil. Saccharum officinarumL, sugarcane, suspension culture, embryogenesis, regeneration     

Isoforms of NADP-dependent Malic Enzyme in Tissues of the Greening Maize Leaf

Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoformsof NADP-dependent malic enzyme in tissues of the greening maizeleaf.—J. exp. Bot. 39: 1109–1119. The compartmentation of the isoforms of NADP-dependent malicenzyme (E.C. 1.1.1.40 [EC] ) has been studied in cell-free extractsand in enzymatically-isolated protoplasts of mesophyll tissue(MT) and bundle sheath (BS) strands of greening maize leaves.The etiolated leaf of 10-d-old seedlings contains a cytosolicisozyme with a pl of 5.4 ?0.1 at low specific activity (s.a,45 ? 3 nmol min^–1 mg^–1 protein), found both in MTand BS. The green leaf on the other hand contains the dominantBS chloroplast isozyme with pl 4.6 ? 0.2 at a s.a, of 370 ?40 nmol min^–1 mg^–1 protein (3.2 ? 0.5 µmolmin^–1 mg^–1 chl) and a minor, previously undescribedisoform with pl 6.5 ? 0.1 also localized in the BS at a s.a.of 38 ? 6 nmol min^–1 mg^–1 protein. Green MT protoplastshave only traces of pl 4.6 isozyme. After illumination of dark-grown seedlings, the total leaf activityshows a rapid increase (1.5-fold within 2 h), attributed mainlyto the pl 5.4 isozyme of MT protoplasts and BS strands. Thisis followed by a large increase of enzyme activity due to thecontinued rise of pl 5.4 isozyme for about 24 h and, after aninitial lag of a few hours, to the accumulation of pl 4.6 isozyme.After 18 h illumination, pl 4.6 and 5.4 isozyme activities tendto decline in the MT whereas they are still increasing in theBS, particularly the former. This pl 4.6 species has becomethe major one by 48 h illumination. The final pattern of greenleaves is established around 96 h light, when the chloroplastisozyme has attained its maximum level, the pl 5.4 isozyme ofBS strands has been superceded by the pl 6.5 species (also supposedto be cytosolic) and MT protoplasts retain little residual activity.Some metabolic implications of the changing pattern of NADP-dependentmalic isozymes during maize leaf greening are discussed. Key words: C₄, isozymes, malic enzyme, photodifferentiation, Zea mays     

Respiratory Loss of Recently Assimilated Carbon in Wheat

A series of experiments was undertaken to assess the amountof respiration associated with the growth of wheat at differentstages. Plants (or in some cases just the flag leaf) were labelledwith ¹⁴CO₂ and the amount of ¹⁴CO₂ respired during the subsequent48 or 72 h was measured. The evolution of ¹⁴C, expressed asa percentage of the amount initially assimilated (referred toas the R/A value) was used as a measure of the overall efficiencyof dry matter production. Respiratory ¹⁴CO₂ evolution from labelledplants was most rapid in the first 12 h after labelling, thereafterdeclining rapidly. Evolution was also more rapid following labellingsat the end of the light period (dusk) than at the beginningof it (dawn). The R/A values were greatest (42 and 50 per centrespectively for dawn- and dusk-labelled plants) for young plantsand least (13 and 28 per cent respectively) for plants duringmid grain filling. When flag leaves, as distinct from wholeplants, were labelled, R/A values were lower still (9 and 21per cent respectively), indicating that flag leaf assimilatewas used efficiently in grain production. The calculated minimum R/A for the formation of grain material(10 per cent protein, 90 per cent starch) was 6.2 per cent.That the experimentally determined values were greater thanthis is attributed to the turnover of carbon in enzymes, toother maintenance processes, and possibly to the operation ofthe pentose phosphate pathway of glucose oxidation. R/A valueswere lower in those plants labelled at the beginning than thoseat the end of the photoperiod. This was considered to be a consequenceof refixation of respiratory ¹⁴CO₂ during the light. The higherR/A values found for young plants were considered to be a consequenceof the greater percentage of ¹⁴C translocated to the roots (rootsbeing unable to refix respired CO₂) and of greater turnoverof enzymes associated with more active metabolism. Triticum, wheat, respiration, carbon assimilation, carbon loss, grain-filling     

Nuclear and Cell Sizes in Different Regions of Root Meristems of Zea mays L.

Nuclear volumes and cell areas were determined for seven regionsof the meristem of roots of Zea mays. Roots were fixed in 10per cent neutral buffered formalin, in 3 per cent glutaraldehydeor in acetic acid/alcohol; they were prepared as sections oralls were teased apart. Mean volumes of interphase nuclei weresimilar in all regions of the root except the vascular tissueof the stele. Mean nuclear volumes and the overall range ofvolumes were similar in sub-populations of cells with differentproportions of G¹, S and G² cells, e.g. in row I of root capinitials, whose cells lack a G¹ phase, and in quiescent centrecells, which are mainly in G¹. Nuclear volume does not appearto be closely correlated with DNA content. Nuclear volumes covereda 6 to 12-fold range within a meristem and even within specificregions, in which cells are part of the same cell lineages,there was a 4- to 9-fold range. Nuclear volumes were comparedin sister cells in rows I and II of the root cap initials. In10 per cent of the pairs, sister nuclei had identical volumes;the other pain had different volumes and mean difference was68 µm³. Mechanisms by which this variability could begenerated are discussed, particularly asymmetry, at mitoses,of factors that regulate nuclear growth. Zea mays L., nuclear volume, cell size, root mcristem, DNA content, mitosis     

Plant regeneration of mesophyll protoplasts from a cytoplasmic albino mutant ofLycopersicon esculentum cv. Large Red Cherry

Mesophyll protoplasts from in vitro grown plants of a cytoplasmic albino mutant ofLycopersicon esculentum cv. Large Red Cherry were isolated with yields between 0.4 to 4.4 × 10⁶ protoplasts per gram leaf tissue. Success in the culture of these protoplasts was dependent on embedding of the protoplasts in 100 µ1 agarose droplets 0.6% (w/v). A plating efficiency of 4.0% was obtained when the protoplasts were cultured in TM-2 medium with sucrose concentrations of 8.7 to 9.6% (w/v) resulting in an osmotic pressure of 432 to 469 mOsmol kg^-1. After 14 days of protoplast culture, microcalli with a diameter of 3 mm were observed. After 3 weeks, macrocalli were obtained which were transferred to regeneration medium. Regeneration of shoot primordia, with a frequency of 19%, was obtained on TM-4 medium supplemented with 1% (w/v) sucrose. The first shoot primordia were visible 10 weeks after protoplast plating. For development of the shoot primordia into shoots it was necessary to increase the sucrose concentration to 6% (w/v). Eight out of eleven regenerants were diploid (2n = 2x = 24); the other three were tetraploid. Efficient regeneration of mesophyll albino protoplasts from tomato opens the way to select at the cellular level for the chloroplast transfers.     

No Light Activation and High Malate Sensitivity of Phosphoenolpyruvate Carboxylase in Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts (GCP) were prepared from leaves of Commelinacommunis L. and phosphoenolpyruvate carboxylase (PEPc) activityrecorded after injection of the protoplasts directly into theassay medium. The GCP were lysed immediately by the presenceof Triton X-100 and a lowered osmotic concentration in the assaycuvette enabling PEPc activity to be measured with ‘nascent’enzyme. There was no light activation of the enzyme with K_mPEP (about 3.7 mol m^–3) and Vmax being similar in light-ordark-treated protoplasts. Illumination of the GCP in the presenceof CO₂-free air and KCI, a treatment which is known to swellGCP, did not change the kinetics. PEPc activity at saturating PEP was very sensitive to malateinhibition, 20 mmol m^–3 (the I₅₀ value) inhibiting activityby about 50%. Inhibition was similar in light- or dark-treatedprotoplasts. Malate inhibition was, however, much less (I₅₀= 500 mmol m^–3) if the enzyme source was a protoplastextract kept in the absence of glycerol. Inclusion of 20% glycerolin the extraction medium maintained the enzyme in the malate-sensitiveform as occurred in the in vivo assays. The high apparent K_mPEP and the high sensitivity to malate inhibition of GCP PEPcare features unlike those observed with PEPc from leaf tissuesof C₄ and CAM plants and from GCP extracts. PEPc activity increased slightly in the presence of KCI in theassay medium up to about 10 mol m^–3 and thereafter activityslowly declined as KCI concentrations increased further. Key words: Guard cell protoplasts, phosphoenolpyruvate carboxylase     

Isolation and Biochemical Characteristics of Protoplasts of Jojoba (Simmondsia chinensis (Link) Schneider)

Two types of protoplasts were isolated from leaves of shootsor callus of subcultures of jojoba (Simmondsia chinensis (Link)Schneider). Protoplasts from leaves were rich in chloro-plastsand were about half the volume of protoplasts from callus. Theviability of preparations as determined by the Evans blue techniquewas 80%. From cell cycle analysis by flow cytometry of nuclei,leaf protoplasts were uniformly in a non-proliferating phase(G0-G1), while callus protoplasts presented many phases of thecell cycle. Protoplasts from calli had only half the Chi ofthose from leaves. Yet Chi a/b ratio, as well as protein andtotal lipid content per cell, were similar in both types ofprotoplasts. A major drop in polar lipids, chiefly in mono-and digalactosyldiacylglycerol, and a parallel increase in neutrallipids occurred during protoplast isolation. The 18:2/18:3 ratiodecreased in neutral lipids concomitant with an increase intriglycerides rich in linolenic acid. Our results suggest atriggering of lipolytic acylhydrolases during the protoplastisolation, as reported for other species. Plasmolysis of thecells with high osmolarity medium and long incubation timeswere required to get a good yield of jojoba protoplasts. Inthe course of this procedure water-deficit stress takes place.A parallel with lipid changes occurring under this type of stressis discussed. (Received April 22, 1991; Accepted July 3, 1991)