Ecophysiology of cuticular transpiration: comparative investigation of cuticular water permeability of plant species from different habitats

Water permeabilities of astomatous, isolated cuticular membranes (CM) of 24 different plants species were measured. Permeances varied from 1.7×10^–11 m·s^–1 (Vanilla planifolia leaf) up to 2.1×10^–9 m·s^–1 (Malus cf. domestica fruit) among different plant species, thus covering a range of over 2 orders of magnitude. Ranking of species according to permeances resulted in four distinct groups. The first group, of species with the lowest cuticular transpiration rates, included evergreen species growing in warm dry tropical climates (e.g. Vanilla planifolia and Monstera deliciosa leaves). The second class, with slightly higher water permeabilities, included evergreen species with typical scleromorphic leaf properties, adapted to a typical mediterranean type of climate with a dry period during the year (e.g. Citrus limon and Olea europaea leaves). The third group of species, where the highest leaf cuticular transpiration rates were observed, included deciduous species normally growing in a tempeate climate (e.g. Juglans regia and Forsythia suspensa leaves). Fruit cuticular membranes (CM) made up the fourth group (e.g. Capsicum annuum and Malus cf. domestica fruits), with even higher permeances than leaves of species from group 3. Thus, it appears that the plant species investigated show ecophysiological adaptations to the climatic demands of their natural habitats in cuticular water permeability.     

The effect of the environment on the permeability and composition of Citrus leaf cuticles

The constituents of the soluble cuticular lipids (SCL) of the leaf blades of Citrus aurantium L. were identified by gas chromatography-mass spectrometry and quantified. Major components were 1-alkanols (C₂₄ to C₄₀), n-alkyl esters (C₃₆ to C₅₆), n-alkanoic acids (C₂₈ to C₃₄), n-alkanes (C₂₂ to C₄₀) and triterpenones, while n-alkanals (C₂₉ to C₃₈), sterols, and alkyl benzenes (molecular weights 260, 274 and 288) made minor contributions. Leaf age and side significantly affected the quantitative composition of SCL. Increased day temperature during the development of leaves led to decreased amounts per unit area of n-alkanes, 1-alkanols, n-alkanoic acids and n-alkyl esters while increased night temperatures resulted in increased amounts of n-alkanes n-alkanoic acids and 1-alkanols. Relative humidity had no effect on the amounts or composition of SCL. The permeability of cuticular membranes to water (described in part I of this paper) and the composition of SCL were not related. A model for the molecular structure of the transport-limiting barrier of plant cuticles and for the transport of water across it is proposed.Abbreviations CM cuticular membrane - GC gas chromatogra-phy - MS mass spectroscopy - TLC thin-layer (planar) chromatography - SCL soluble cuticular lipids The authors are indebted to Dr. R. Winkler and H. Krause, Laboratorium für Strukturchemie des Fachbereichs Chemie, Biologie und Geowissenschaften, Technische Universität München, FRG, for performing the GC-MS analyses and their valuable help in the identification of SCL constituents. This work has been supported by the Deutsche Forschungsgemeinschaft and the Bayerische Staatsministerium für Wissenschaft und Kunst.     

Periclinal penetration of potassium permanganate into mature cuticular membranes ofAgave andClivia leaves: new implications for plant cuticle development

Staining cuticular membranes ofAgave americana andClivia miniata en bloc with potassium permanganate results in a strong contrast in the interior cuticular layer while the exterior part remains unstained. This is not caused by a selective chemical reaction with the interior part but by the unidirectional penetration of the reagent from the interior side, the outside being protected by the cuticle proper. In transverse cryosections of the cuticular membrane, permanganate penetrates nearly as easily into the exterior cuticular layer as into the interior one giving the same contrast. However, compared with the periclinal penetration into the cuticle proper this penetration is accelerated five-to tenfold by the polysaccharide network within the cuticular layer which serves as a distribution-channel system. Periclinal penetration into the cuticle proper occurs independently in each cutin penetration unit included between two obvious lucent lamellae and further divided into subunits.     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

Water permeability of plant cuticles

Using the system vapor/membrane/liquid, permeability coefficients of cuticular transpiration (P _ct) were determined as functions of water activity in the vapor (a _wv). Enzymatically isolated cuticular membranes (CM) of Citrus aurantium L. and nonisolated CM of onion bulb scales and eggplant fruits were investigated. P _ct of Citrus and eggplant CM decreased with decreasing a _wv, while permeability coefficients of CM of onion were independent of a _wv. Extraction of soluble cuticular lipids (SCL) from the CM of Citrus increased permeability coefficients by a factor of approximately 500. This extraction had no effect on the dependence of P _ct on a _wv.Treating cuticular membranes as a resistance network consisting of SCL and the polymer matrix, it is shown that the permeability of onion CM is determined by the resistance of the SCL arranged in series with the polymer matrix. In this type of CM liquid and vapor are separated by a continuous, nonporous layer of SCL, and the driving force of transpiration is the gradient of partial pressure of water vapor across the SCL layer. In the CM of Citrus and eggplant, the SCL layer is traversed by polar pores that swell or shrink depending on a _wv. However, liquid continuity is maintained across these membranes down to a _wv=0.22, the lowest value used. In this type of membrane the driving force of transpiration is the water potential gradient across the membrane.Abbreviations CM cuticular membrane - MX polymer matrix - SCL soluble cuticular lipids - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES (N-morpholino)ethane sulfonic acid - SADH succinic acid 2,2-dimethyl hydrazide     

AgCl precipitates in isolated cuticular membranes reduce rates of cuticular transpiration

Counter diffusion of chloride, applied as NaCl at the inner side of isolated cuticles, and silver, applied as AgNO₃ at the outer side, lead to the formation of insoluble AgCl precipitates in isolated cuticles. AgCl precipitates could be visualized by light and scanning electron microscopy. The presence of AgCl precipitates in isolated cuticles was verified by energy dispersive X-ray analysis. It is argued that insoluble AgCl precipitates formed in polar pores of cuticles and as a consequence, cuticular transpiration of 13 out of 15 investigated species was significantly reduced up to three-fold. Water as a small and uncharged but polar molecule penetrates cuticles via two parallel paths: a lipophilic path, formed by lipophilic cutin and wax domains, and a aqueous pathe, formed by polar pores. Thus, permeances P (m s⁻¹) of water, which is composed of the two quantities P _Lipid and P _Pore, decreased, since water transport across polar pores was affected by AgCl precipitates. Cuticles with initially high rates of cuticular transpiration were generally more sensitive towards AgCl precipitates compared to cuticles with initially low rates of transpiration. Results presented here, significantly improves the current model of the structure of the cuticular transpiration barrier, since the pronounced heterogeneity of the cuticular transport barrier, composed of lipophilic as well as polar paths of diffusion, has to be taken into account in future.     

Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.     

The permeability of the epidermal cell plasma membrane of barley leaves to abscisic acid

Uptake of ³H-labelled (±)-abscisic acid (ABA) into isolated barley (Hordeum vulgare L.) epidermal cell protoplasts (ECP) was followed over a range of pH values and ABA concentrations. The present results show that ABA uptake is not always linearly correlated with the external concentration of undissociated ABA (ABAH). At pH 7.25, ABA uptake exhibited saturation kinetics with an apparent K _m value of 75 mmol·m^–3 to tal ABA. This saturable transport component was inhibited by pretreating the protoplasts with 1 mol·m^–3 p-chloromercuribenzenesulfonic acid at pH 8.0, conditions that minimized the uptake of this acid sulfhydryl reagent. Moreover, the rate of (±)-[^3]HABA uptake was reduced by addition of 0.1 mol·m^–3 (±)-ABA to 41%, whereas the same concentration of (±)-ABA was approximately half as effective (46% of the inhibitory effect). Thus, it was concluded that only (±)-ABA competes for an ABA carrier that is located in the epidermal cell plasma membrane. The permeability of the epidermal cell plasma membrane was studied by performing a Collander analysis. At pH 6 the overall plasma-membrane permeability of epidermal cells was similar to that of guard cells but was about two times higher than that of mesophyll cells.Abbreviations ABA abscisic acid - ABA^– anion of ABA - ABAH undissociated ABA - 2,4-D 2,4-dichlorophenoxyacetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - ECP deepidermal cell protoplast - K_r partition coefficient - M_r relative molecular mass - NEM N-ethylmaleimide - PCMBS p-chloromercuriben zenesulfonic acid - P_s permeability coefficient We are grateful to Barbara Dierich for expert technical assistance, to Prof. H. Gimmler (Lehrstuhl für Botanik I, Universität Würzburg, FRG) for helpful discussions and to the Deutsche Forschungsgemeinschaft (SFB 251, TP 3) for financial support.     

Fine structure of plant cuticles in relation to water permeability: The fine structure of the cuticle of Clivia miniata reg. leaves

The fine structure of the upper cuticular membrane (CM) of Clivia miniata leaves was investigated using electron microscopy. The CM is made up of a thin (130 nm) lamellated cuticle proper (CP) and a thick (up to 7 m over periclinal walls) cuticular layer (CL) of marbled appearance. Evidence is presented to show that the electron lucent lamellae of the CP do not simply represent layers of soluble cuticular lipids (SCL). Instead, the lamellation is probably due to layers of cutin differing in polarity. It is argued that the SCL in the Cp are the main barrier to water. Thickening of the CM during leaf development takes place by interposition of cutin between the CM and the cellin wall. The cutin of young, expanding leaves has a high affinity for KMnO₄ and is therefore relatively polar. As leaves mature, the external CL underneath the CP becomes non-polar, as only little contrast can be obtained with permanganate as the post fixative.Abbreviations CM cuticular membrane - CP cuticle proper - CL cuticular layer - SCL soluble cuticular lipids (cuticular waxes)     

Gas permeability of plant cuticles

Cuticles from the adaxial surface of Citrus aurantium L. leaves and from the pericarp of Lycopersicon esculentum L. and Capsicum annuum L. were isolated enzymatically and their oxygen permeability was determined. Isolated cuticles were mounted between a gaseous and an aqueous compartment with the physiological outer side of the membrane facing the gaseous compartment. Permeability for oxygen was characterized by permeability (P) and diffusion (D) coefficients. P and D were independent of the driving force (gradient of oxygen concentration) across the cuticle, thus, Henry's law was obeyed. P values for the diffusion of oxygen varied between 3·10^-7 (Citrus), 1.4·10^-6 (Capsicum), and 1.1·10^-6 (Lycopersicon) m·s^-1. Extraction of soluble lipids from the cuticles increased the permeability. By treating the cutin matrix and the soluble lipids as resistances in series, it could be demonstrated that the soluble lipids were the main resistance for oxygen permeability in Citrus cuticles. However, in Lycopersicon and Capsicum, both the cutin matrix and the soluble lipids determined the total resistance. P values were not affected by either the proton concentration (pH 3–9) or the cations (Na⁺, Ca²⁺) present at the morphological inner side of the cuticles. It is concluded that the water content of cuticles does not affect the permeability properties for oxygen. Partition coefficients indicated a high solubility of oxygen in the cuticle of Citrus. The data suggest a solubility process in the cuticle of Citrus with respect to oxygen permeation.Abbreviations CM cuticular membrane - MX cutin polymer matrix - SCL soluble cuticular lipids     

Ultrastructure and histochemistry of Citrus limon (L.) stigma

Citrus limon has a wet stigma which can be divided in two zones: a glandular superficial one formed by papillae, and a non-glandular one formed by parenchymatic cells. The stigmatic exudate is produced by the papillae after the latter have reached their ultimate size. The papillae of the mature pistil are of varying size and composition. Both the unicellular and multicellular ones are present. The cells at the base of the papillae are rich in cytoplasm, whereas the tip cells are vacuolated. Histochemical analysis has shown that the exudate of Citrus is composed of lipids, polysaccharides, and proteins. Our results indicate that the lipidic component is produced and secreted first, followed by production and secretion of the polysaccharidic component. The lipidic component of the exudate is produced in the basal papillae cells and accumulates as droplets in dilated parts of the smooth endoplasmic reticulum (SER). Subsequently the lipid droplets are transported to the plasma membrane, and transferred by the latter into the cell walls. Then the exudate component is accumulated in the intercellular spaces and in the middle lamellar regions of the walls. Subsequently, the polysaccharidic component of the exudate is produced and secreted by the tip cells of the papillae.Abbreviations RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Respiratory and cuticular water loss in insects with continuous gas exchange: comparison across five ant species

Respiratory water loss (RWL) in insects showing continuous emission of CO(2) is poorly studied because few methodologies can measure it. Comparisons of RWL between insects showing continuous and discontinuous gas exchange cycles (DGC) are therefore difficult. We used two recently developed methodologies (the hyperoxic switch and correlation between water-loss and CO(2) emission rates) to compare cuticular permeabilities and rates of RWL in five species of ants, the Argentine ant (Linepithema humile) and four common native ant competitors. Our results showed that RWL in groups of ants with moderate levels of activity and continuous gas exchange were similar across the two measurement methods, and were similar to published values on insects showing the DGC. Furthermore, ants exposed to anoxia increased their total water loss rates by 50-150%. These results suggest that spiracular control under continuous gas exchange can be as effective as the DGC in reducing RWL. Finally, the mesic-adapted Argentine ant showed significantly higher rates of water loss and cuticular permeability compared to four ant species native to dry environments. Physiological limitations may therefore be responsible for restricting the distribution of this invasive species in seasonally dry environments.     

Action of ultraviolet radiation (UV-B) upon cuticular waxes in some crop plants

The surface structure and composition of surface lipids were examined in leaves of barley, bean, and cucumber seedlings grown in a growth chamber under white light and low levels of ultraviolet (UV-B; 280–320 nm) radiation. The cuticular wax of cucumber cotyledons and bean leaves appeared as a thin homogeneous layer, whereas on barley leaves crystal-like structures could be observed under these irradiation conditions. Principally, the amount of cuticular wax found in barley leaves was five times greater than in bean or cucumber leaves. The prediominant wax components were primary alcohols in barley, primary alcohols and monoesters in bean, and alkanes in cucumber cotyledons. Irradiation with enhanced UV-B levels caused an increase of total wax by about 25% in all plant species investigated. Aldehydes, detected as a minor constituent of cucumber and barley wax, increased twofold. Distribution patterns of the homologs within some wax classes were different at low and enhanced UV-B levels. In general, the distribution of the homologs was shifted to shorter acyl chain lengths in wax of leaves exposed to enhanced UV-B levels. This was most apparent in cucumber wax, less in bean or barley wax. The UV-B-caused effects upon cucumber wax were mainly due to a response by the adaxial surface of the leaf.Abbreviation UV-B Ultraviolet radiation (280–320 nm)     

Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 g · ml^–1 of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53–57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.Abbreviations kDa kilodalton - PEM proembryogenic masses - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis We thank Dr. S. Satoh (Institute of Biological Sciences, Tsukuba, Japan) for sending protein samples of the purified 57-kDa glycoprotein. This research was supported by a grant from the Charles H. Revson Foundation for Basic Research in the Life Sciences of the Israel Academy of Sciences. R.F. is a recipient of the Jack and Florence Goodman Career Development Chair.     

Protoplast-fusion-mediated transfer of organelles from Microcitrus into Citrus and regeneration of novel alloplasmic trees

Summary Iodoacetate-treated Citrus protoplasts from embryogenic nucellar calli of Sour orange (C. aurantium) or from Rough lemon (C. jambhiri) were fused with -irradiated protoplasts from a related genus, Microcitrus. The fused protoplasts were cultured to obtain colonies and micro-calli. Micro-calli derived from these two fusion combinations were isolated, propagated and differentiated into embryos, which subsequently regenerated trees having the morphology of Sour orange or Rough lemon. These intergeneric fusions resulted in mitochondria with novel DNA, indicating recombination between the chondriomes of Citrus and Microcitrus. Chloroplast DNA analyses of fusion-derived embryos indicated that they contained the chloroplasts of either fusion-partner or a mix of these chloroplasts. Later plastome analyses of leaves from fully differentiated plants showed that cybrids having Rough lemon morphology had either Rough lemon or Microcitrus chloroplast DNA, indicating complete sorting out of chloroplasts. Likewise, sorting out of Microcitrus chloroplasts was detected in a cybrid plant having Sour orange morphology.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250 Israel. No. 2663-E, 1989 series     

Comparative water relations of adult and juvenile tortoise beetles: differences among sympatric species

Relative abundance of two sympatric tortoise beetles varies between drought and 'wet' years. Differing abilities to conserve water may influence beetle survival in changing environments. Cuticular permeability (CP), percentage of total body water (%TBW), rate of water loss and percentage of body lipid content were determined for five juvenile stages and female and male adults of two sympatric species of chrysomelid beetles, the golden tortoise beetle, Charidotella bicolor (F.) and the mottled tortoise beetle, Deloyala guttata (Olivier). There were significant differences in %TBW and lipid content among juvenile stages. Second instars had the greatest difference in CP (37.98 and 11.13 microgcm(-2)h(-1)mmHg(-1) for golden and mottled tortoise beetles, respectively). Mottled tortoise beetles had lower CP and greater %TBW compared with golden tortoise beetles, suggesting that they can conserve a greater amount of water and may tolerate drier environmental conditions. This study suggests that juvenile response to environmental water stress may differentially affect the survival of early instars and thus affect the relative abundance of adult beetles in the field. This is supported by the low relative abundance of golden tortoise beetle larvae in a drought year and the higher abundance in two 'wet' years.     

A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves

Transpiration of cuticular membranes isolated from the lower stomatous surface of Hedera helix (ivy) leaves was measured using a novel approach which allowed a distinction to be made between gas phase diffusion (through stomatal pores) and solid phase diffusion (transport through the polymer matrix membrane and cuticular waxes) of water molecules. This approach is based on the principle that the diffusivity of water vapour in the gas phase can be manipulated by using different gases (helium, nitrogen, or carbon dioxide) while diffusivity of water in the solid phase is not affected. This approach allowed the flow of water across stomatal pores ('stomatal transpiration') to be calculated separately from the flow across the cuticle (cuticular transpiration) on the stomatous leaf surface. As expected, water flux across the cuticle isolated from the astomatous leaf surface was not affected by the gas composition since there are no gas-filled pores. Resistance to flux of water through the solid cuticle on the stomatous leaf surface was about 11 times lower than cuticular resistance on the astomatous leaf surface, indicating pronounced differences in barrier properties between cuticles isolated from both leaf surfaces. In order to check whether this difference in resistance was due to different barrier properties of cuticular waxes on both leaf sides, mobility of 14C-labelled 2,4-dichlorophenoxy-butyric acid 14C-2,4-DB) in reconstituted cuticular wax isolated from both leaf surfaces was measured separately. However, mobility of 14C-2,4-DB in reconstituted wax isolated from the lower leaf surface was 2.6 times lower compared with the upper leaf side. The significantly higher permeability of the ivy cuticle on the lower stomatous leaf surface compared with the astomatous surface might result from lateral heterogeneity in permeability of the cuticle covering normal epidermal cells compared with the cuticle covering the stomatal cell surface.     

The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

Factors affecting the binding between fusicoccin and plasma membranes from maize roots

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (K_D); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [³H]dihydroFC and the competition between [³H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H. The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.