Quantized Fluxes of Chloride to the Vacuole of Nitella translucens

It has been shown previously that the transfer of tracer chloridefrom the outside solution to the vacuole of Nitella translucensis initially linear with time. In this paper the relations betweenthe initial rate of chloride transfer to the vacuole and thetotal influx to the cell are further examined. In the individualcells in each experiment the ratio (initial rate of transferto the vacuole/rate of entry to the cell), M_ov/M_T, is quantized;that is, in each experiment the ratio takes values close to, 2, 3,... etc. Formation of pinocytotic vesicles is a processwhich could be imagined to be quantized, but the fact that itis a flux ratio, rather than a flux, which is quantized suggeststhat entry to a small cytoplasmic phase, such as the endoplasmicreticulum, must precede a quantized discharge to the vacuole.It is suggested that the kinetics of tracer movement to thevacuole are consistent with transfer in small vacuoles buddedoff the endoplasmic reticulum which then fuse with the centralvacuole.     

The Electrical Potential Difference Across the Tonoplast of Root Cells

Changes in electrical potential, measured as a microelectrodewas advanced into epidermal cells and from cell to cell in rootsof Lolium multiflorum and Zea mays, are described. The recordingssuggest that the electrical potential difference between thecytoplasm and vacuole, E_vc is of the order of a few millivolts,the vacuole tending to be the more positive. E_vc appeared tobe approximately the same for epidermal, cortical, endodermal,and pericycle cells.     

Leaf Water Potential and Control of Water Loss in Droughted Sitka Spruce Seedlings

Sitka spruce seedlings were subjected to drought in experimentsin a growthroom, a greenhouse, and out of doors. The plantswere grown in a double chamber with the bulk of the roots inthe upper part where they dried out the soil when water waswithheld. A few new roots penetrated into the lower part inwhich the soil remained moist. The double chamber system enabledthe plant to attain a high water psotential by night and theshoot was only periodically under mild water stress. Measurementswere made on soil water potential (_solt), leaf water potential(₁), transpiration (E), and stomatal conductance (k_s). As soildecreased over a period of 4.5 d, E and k_s decreased progressively.The decline in E and k_s which indicated stomatal closure, occurredat a higher ₁ than has been reported for Sitka spruce. The behaviourof the stomata appeared to be modified by conditions at theroot, and it is proposed that differences in the response to₁,depend on Whether the latter is reduced by resistances in thexylem between root and leaf, as is known to occur in large treesin moist soil, or by stresses at the root itself.     

Uptake and rapid transfer of fluorescent ceramide analogues to acidosomes (late endosomes) in Paramecium.

The ciliated protozoan Paramecium incorporates sphingolipids into its cell membranes. However, it is still unclear if these sphingolipids are metabolically synthesized in the cell or if their precursors are taken up from exogenous materials. Here we studied the route of uptake of fluorescence-labeled analogues of ceramide. Fluorescent ceramide was taken up rapidly independent of phagosome formation. Cold treatment caused a decrease in uptake, while reduction in the amount of cytosolic ATP induced by NaN(3) and deoxyglucose resulted in accumulation without internalization of fluorescence at the plasma membrane. These results suggest that uptake of fluorescent ceramide occurs at the plasma membrane, that it is an ATP-dependent process, and that it is not a result of simple diffusion. At first intracellular fluorescence appeared principally in the posterior half of the cell and then spread throughout the cytosol. In particular, a high accumulation of fluorescence occurred in association with acidosomes (late endosome or multivesicular body-like vesicles) that bind to the surface of nascent and young phagosomes. Therefore, in the Paramecium cell a significant proportion of ceramide apparently enters the cell by endocytosis and is quickly relayed to acidosomes along the endocytic pathway before becoming part of the digestive vacuole (phagoacidosome) membrane.     

Anther-filament Extension in Lilium: Potassium lon Movement and Some Anatomical Features

The growth of the anther filaments in the Lilium bud is essentiallyexponential after a length of 5 mm is reached, when the celldivision rate declines and growth is by cell extension, particularlyof cells in the central part of the filament. During the periodof extension, fresh vascular tissue is formed and xylem continuityis conserved up to the time of anther dehiscence, in contrastto the Graminae where the filament extends much more rapidly.The expansion of the lily filament depends in part on the continuoustransfer of potassium ions from the receptacle throughout theperiod of growth. Although starch to sugar conversion may bean important source of osmoticum as the cells expand, regulationof K⁺ ion movement would be likely to provide a more sensitiveand faster method of regulation of cell osmoticum. Anther filament, extension growth, filament, ion movement, Lilium, lily, potassium, stamen filament, turgidity, vasculation, xylem     

Intercellular Transport in Plants: I. THE RATE OF TRANSPORT OF CHLORIDE AND THE ELECTRIC RESISTANCE

A quantitative study has been made of the intercellular movementof chloride in Chara corallina, using pairs of joined internodalcells. One cell of the pair (cell 1) was exposed to a solutioncontaining ³⁶Cl; the distribution of this tracer between thecells was determined at the end of the uptake period. Of thechloride taken up, 0.29 was transported out of cell 1 for alluptake times from 1.8 to 22 ks and 0.57 was transported to thevacuole of cell 1, in experimental series I. In series II, thefraction transported out of cell 1 was 0.43 at 22 and 43 ks,but 0.17 at 600 s. These results represent a rate of transport of 4 to 60 pmols^–1, across an intercellular wall of area 1.5 x 10^–6m²; the wall has 0.03 to 0.04 of its area occupied by plasmodesmata.The estimate of transport rate is based on an attempt to determinethe specific activity of the cytoplasm of cell 1. The electricresistance of the node was found to be 47 m m². The observed transport rate can be explained by diffusion inthe plasmodesmata, without the need to postulate active processes.Diffusion in the plasmodesmata is slower than in free solutionby an ‘impediment factor’ of 7 to 700, dependingon the assumed chloride concentration of the ground-plasm. Ifthe plasmodesmata offer the major conducting path for electriccurrent, the electric impediment factor is 390. Chloride entersthe plasmodesmata from the same small kinetic compartment whichsupplies the flux to the vacuole, or from a smallintermediatecompartment.     

Thermodynamics of Malate Transport across the Tonoplast of Leaf Cells of CAM Plants

The electrochemical potential difference for each dissociationstate of malic acid across the tonoplast of leaf cells was examinedin two CAM plants, Graptopetalum paraguayense and Kalanchoëdaigremontiana. The concentration of malic acid in each dissociationstate was estimated from an analysis of pH and concentrationsof ionic species that included calcium, malate and isocitrate.The vacuoles contained 30–40 mM isocitrate and 50–70mM calcium in G. paraguayense, and 20–30 mM isocitrateand 70–100 mM calcium in K. daigremontiana. For the calculationof the pattern of dissociation of malic acid, the formationof chelates of calcium with malate and isocitrate, which havedifferent stability constants depending on the dissociationof the acids, were also taken into consideration. The vacuolarconcentrations of the divalently dissociated form of malic acid(mal^2– were 4–7 mM and 1-3 mM in G. paraguayenseand in K. daigremontiana, respectively. To obtain informationabout the cytoplasmic concentration of malate, the apparentinhibition constant for malate of phosphoenolpyruvate carboxylasewas measured. It was about 330 µM in the dark period and60 µM in the light period. Considering an inside-positivemembrane potential, we conclude that mal^2– can be takenup passively into the vacuole during the dark period and canbe released passively from the vacuole during the light period.Two types of channel (the "SV-type" channel and a novel "MU-type"channel) which we found recently in G. paraguayense [Iwasakiet al. (1992) Plant Physiol. 98: 1494] are probably involvedin the uptake and the release of malate in the diurnal CAM rhythm.The existence of a large pH-buffering capacity due to isocitricacid in the vacuole allows the accumulation of a large amountof malic acid during the diurnal CAM rhythm. (Received February 12, 1992; Accepted July 10, 1992)     

Light-Activated H+ Transport into the Vacuole of Valonia ventricosa

Using glass capillary microelectrodes for the measurement ofpotential differences (PD) and antimony microelectrodes forthe measurement of pH, we investigated the light-induced changesof PD between the central vacuole and the external medium, ofpH in the vacuole (pH_v), as well as of pH in the external medium(pH_o) of the green marine alga Valonia ventricosa. PD in thedark was about +30 to +40 mV (vacuole positive), pH_v 6.3, andthe resistance of the protoplast (cell wall-plasmalemma-tonoplast)17.8 kOhm cm². Illumination caused an increase of the positivePD (after a few oscillations) up to +80 to +100 mV, acidificationof the vacuolar sap, alkalinization of the external medium,and a decrease in the resistance of the protoplast to 7.6 kOhmcm². The kinetics of the changes of PD, pH_v, and pH_o were similarto each other. It is concluded that a light-stimulated activeH⁺ flow occurs from the external medium into the central vacuoleof Valonia ventricosa as a result of the onset of photosyntheticactivity.     

A Possible Mechanism for the Oxidation of Sinapyl Alcohol by Peroxidase-Dependent Reactions in the Apoplast: Enhancement of the Oxidation by Hydroxycinnamic Acids and Components of the Apoplast

Apoplastic peroxidase isoenzymes from stems of Nicotiana tabacumrapidly oxidized sinapic acid and sinapyl alcohol, in additionto 4-coumaric acid, ferulic acid and coniferyl alcohol. By contrast,the peroxidase isoenzymes from stems of Vigna angularis oxidizedsinapic acid and sinapyl alcohol quite slowly but rapidly oxidizedcompounds with a 4-hydroxyphenyl or a guaiacyl group. However,the oxidation of sinapyl alcohol was greatly enhanced by 4-coumaricacid, ferulic acid and an ester of ferulic acid. Intercellularwashing fluid of V. angularis, which contained apoplastic components,also enhanced the oxidation of sinapyl alcohol. Based on theseresults, a possible mechanism for the oxidation of sinapyl alcoholis discussed on the assumption that the biosynthesis of ligninproceeds mainly via peroxidases which cannot oxidize sinapylalcohol in V. angularis. (Received October 23, 1995; Accepted April 3, 1996)     

The Transport and Metabolism of Urea in Chara australis: II. UREASE AND THE DISTRIBUTION OF TRANSPORTED UREA

The ureolytic enzyme in Chara was investigated. This enzymewas shown to be a urease with an unusually high affinity forurea(K_m = 158 mmol m^-3). Little inhibition of urease activitywas found when intact Chara cells were exposed to the ureaseinhibitors hydroxyurea, acetohydroxamic acid and N-ethylmaleimide,although there was some inhibition of urea uptake. The distribution of radioactivity amongst the amino acid, organicacid and sugar/neutral fractions, determined by ion-exchangechromatography, was very similar whether the Chara internodeswere exposed to ¹⁴C-urea or to H¹⁴CO₃. This suggests that thefraction of the urea-carbon liberated by the urease as CO₂ andretained by the cell is used in photosynthetic carbon-fixation.During the initial 15 min of ¹⁴C-urea uptake, label appearsin the vacuole only in the form of unmetabolized urea. Afterthis time a variety of labelled compounds appear in the vacuole,presumably reflecting the gradual movement of carbon-fixationproducts from the chloroplasts to the cytoplasm and thence intothe vacuole. Key words: Urea transport, metabolism, Chara, urease     

Ion Fluxes to the Vacuole of Nitella translucens

The time course of the appearance in the vacuole of Nitellatranslucens and of Tolypella intricata of tracer from the outsidesolution has been studied over short periods of uptake. Thereare two components of chloride transfer to the vacuole, a fastcomponent linear with time and a second component at longertimes whose behaviour is reasonably well described in termsof a single rate constant for exchange; a constant fractionof the total entry is in the fast component and the apparentrate constant for the second component is proportional to theinflux. In Nitella the path of rapid transfer involves chlorideand sodium, and may also involve a small but variable amountof potassium, but in Tolypella potassium has a significant componentof rapid transfer; these correspond to the cations for whichchloride-linked components of cation influx have been shownby another worker. Over both parts of the time course the level of activity inthe cytoplasm specifies, not the rate of transfer to the vacuoleas would be expected, but the rate as a fraction of the influx;the processes of influx to the cell and transfer to the vacuoleare intimately linked. It is difficult to explain the results in terms of static membranesand fixed compartments. An explanation in terms of the sequence,entry of salt by pino-cytotic vesicles at the plasmalemma, fusionof these vesicles with the endoplasmic reticulum after someloss of tracer to the surrounding cytoplasm, and transfer tothe vacuole in minivacuoles formed from the endoplasmic reticulum,is consistent with the time course found. A model of this kind,involving transport by a dynamic membrane system, seems necessaryto explain the results.     

Ultrastructural Aspects of the Glandular Cells from the Secretory Trichomes and from the Cell Suspension Cultures of Achillea millefolium L. ssp. millefolium

The ultrastructure of the glandular cells of the floret secretorytrichomes from Achillea millefolium L. ssp. millefolium (yarrow)was examined before and after anthesis and compared with theultrastructure of the cells from the cell suspension culturesobtained from the same plant. The profuse tubular structuresobserved in the plastids of the glandular cells of the trichomesduring the pre-secretory stage were much reduced in the secretorystage and showed an osmiophilic content. Some endoplasmic reticulumprofiles could be seen adjacent to the plastids. Later in thesecretory stage, the secretion appeared in the periplasmic spacebetween the cells of the upper tiers and in the sub-cuticularspace. Finally the secretion was released by rupture of thecuticle. At the lag phase, the cells from the cell suspensioncultures of yarrow were characterized by the presence or plastidswith tubular structures, similar to those observed in the plastidsof the trichomes in the pre-secretory stage. By the end of thelag phase accumulations of starch were observed inside the plastids.At the beginning of the exponential phase, the tubular structuresof the plastids started to show an osmiophilic content and theaccumulations of starch were still present. At the end of thisphase starch disappeared from the plastids and only osmiophilictubular structures were observed. Rough endoplasmic reticulumas well as smooth endoplasmic reticulum profiles were frequentlyin close association with plastids and mitochondria. At thestationary phase a very large vacuole filled the cells, andin the remaining cytoplasm some endoplasmic reticulum profilesand osmiophilic droplets were observed.Copyright 1994, 1999Academic Press Achillea millefolium L. spp. millefolium, yarrow, ultrastructure, trichomes, glandular cells, plant cell suspension cultures     

Mode of dinitroaniline herbicide action I. Analysis of the colchicine-like effects of dinitroaniline herbicides

Colchicine and a variety of dinitroaniline herbicides (DNHs)produce a similar pattern of inhibition of elongation, inductionof swelling in the elongation zone, depolarization of cell enlargement,and induction of multiple nuclei in corn seedling roots. However,a 1000-fold higher concentration of colchicine is needed toproduce effects quantitatively similar to those of oryzalin.Both colchicine- and DNH-inhibition of elongation start at about3 hr. Since these compounds cause swelling and inhibition ofelongation in -seedling roots, segments from the root elongationzone and intact roots in the presence of cell division inhibitors(all growing without cell division), it appears that the inhibitionof root elongation is caused in part by their effect on cellelongation independent of their effect on cell division. Sincethe growth (increase in fresh weight) of -seedling roots andexcised root segments is not inhibited by these compounds, theireffect on the polarity of cell enlargement must be fairly specific.Unlike colchicine, oryzalin applied to the roots did not causeany significant, visible effect on shoot (mesocotyl and coleoptile)growth. These organs are not resistant to oryzalin, for theIAA-induced elongation of coleoptile segments is inhibited whenthey are floated in oryzalin solutions. As expected, when coleoptilesegments are incubated in ¹⁴C-oryzalin, it is taken up rapidlyand not degraded. The failure of root-applied oryzalin to affectseedling shoot growth is due to lack of transport to the shoots. (Received June 14, 1977; )     

Cycloprodigiosin Hydrochloride Inhibits Acidification of the Plant Vacuole

The effect of cycloprodigiosin hydrochloride (cPrG-HCl), obtainedfrom the marine bacterium Pseudoalteromonas denitnficans, onacidification of the vacuole was studied in Characeae cells.In internodal cells of Nitella, the plasma membrane of whichhad been permeabilized, cPrG-HCl inhibited both ATP- and pyrophosphate-dependentacidification of the vacuole. Application of cPrG-HCl to livinginternodal cells of Chara, induced a significant increase invacuolar pH. The role of cPrG-HCl as a tool for studying thephysiological role of acidic vacuole is discussed. (Received August 26, 1998; Accepted November 17, 1998)     

Ultrastructure of Secretory Cells in the Phloem of Mimosa pudicaL.

The secretory cells arc a prominent component of the phloemtissue of Mimosa pudica L. They form longitudinal files in whichthe common end walls of the cells are perforated. The originof the perforations in relation to protoplast differentiationwas the objectof the study. As the cells emerge from the meristematicstate, portions of cytoplasm including some organelles aretransferredinto the central vacuole and are apparently lysed. This phenomenonis accompanied by an accumulation of flocculent material, possiblythe secretory product referred to in the literature as a carbohydrate.The tonoplast ceases to be definable so that the flocculentmaterialand degraded and non-degraded protoplast componentsare intermingled inthe cell lumen. The perforation of the endwall OCCUR while the tonoplast is still intact. Theplasmalemmasof the contiguous cells withdraw from the part of the wall undergoingdegradation. The space becomes filled with fibrous debris ofthe apparently lysed wall. The degraded wallpart becomes brokenpossibly by a unidirectional pressure flow in the cells, forthe remnants of the wall are pushed into one of the two superimposedcells. At this presumably mature stage, the cells contain someintactprotoplast components among which ribosomes are especially conspicuous.     

Lysosomal nature of plant vacuoles II. Acid hydrolases in the central vacuole of internodal cells of Charophyta

Contents of the central vacuole (cell sap) were separated frominternodal cells of Nitella and Chara. Most of the acid phosphataseand carboxypeptidase, which are marker enzymes for lysosomes,were detected in the separated cell sap. In contrast, most ofthe catalase, cytochrome oxidase, NADPH₂-cytochrome c reductase,and glucose-6-phosphate dehydrogenase were detected in partsof the cell other than the cell sap: which shows there is relativelylittle contamination of the cytoplasm in the separated cellsap. Enzymatic properties of the carboxypeptidase in Nitellaaxilliformis were similar to those of carboxypeptidases in higherplants and those of cathepsin A in animal lysosomes. These resultsindicate that the central vacuole of Charophyta has some propertiesof the lysosome. ¹ Preliminary results of this paper were given in reference(5). (Received February 3, 1975; )     

Vps10p cycles between the TGN and the late endosome via the plasma membrane in clathrin mutants

Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in a chc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport in chc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, in chc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normal VPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.     

Inhibition of Malate Synthesis by Vanadate: Implications for the Cytosolic Alkalinization Induced by Vanadate Concentrations Partially Inhibiting the Plasmalemma H+ Pump

The effects of Na-orthovanadate, at concentrations only partiallyinhibiting net H⁺ extrusion, were determined on vacuolar andcytosolic pH by the weak base and weak acid distribution atequilibrium. Treatment with vanadate induces in Elodea densaleaves and in Arabidopsis thaliana seedlings a moderate acidificationof both cell sap and vacuole. Conversely, it induces an alkalinizationof cytosol, this effect being in apparent contrast with a conditionof reduced activity of the H⁺-transporting plasmalemma ATPase,which should be associated with a cytosolic acidification. InArabidopsis seedlings treated with vanadate, the increase inpH of both cytosol and external medium is associated with adecrease in cell sap buffer capacity, more evident for highervanadate concentrations, and particularly marked in the pH rangebetween 3·5 and 5·5. In these conditions, themalate content is strongly reduced, its decrease almost completelyaccounting for the decrease in cell sap buffer capacity. Anin vitro analysis of the vanadate effect on phosphoenolpyruvatecarboxylase indicates that the decrease in malate content seemssubstantially due to an inhibiting effect of vanadate on thisenzyme. These results stress that the in vivo use of vanadateas an inhibitor of the plasmalemma H⁺-ATPase must be taken withcaution; in particular, for studying the correlations betweenchanges in net H⁺ extrusion and changes in cytosolic pH andrelated processes. Key words: Vanadate, malate, cytosolic pH, Elodea densa, Arabidopsis thaliana     

The Supramolecular Organization of Red Algal Vacuole Membrane Visualized by Freeze-fracture

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost clearly and more frequently in the exoplasmic fracture(EF) face of the vacuole membrane of all red algae investigated.The tetrads lie individually within the vacuole membrane orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

The Reproductive Biology of Totara (Podocarpus totara) (Podocarpaceae)

A reproductive cycle of totara (Podocarpus totara) in New Zealandis complete within 2 years. After strobilus initiation in September,there is a 9 month period of winter dormancy until emergenceduring the growth flush in July–August of the followingyear. Female strobili bear only one or two ovules which arepollinated mid-October to mid-November at the megaspore tetradstage. Pollen germination and fertilization occur rapidly duringDecember. The pollen tube carries the body cell, sterile andtube nuclei with at least three prothallial nuclei, and branchesout after reaching the archegonia. The four–six archegoniacontain egg nuclei with no distinct perinuclear zone and a largechalazal vacuole. Fertilization by the larger of the two unequalmale gametes is accompanied by a degradation of egg cell cytoplasm.The three-tier proembryo contains a binucleate embryonal tierof only one cell. Soon after embryo penetration into the megagametophyte,the binucleate embryonal-tier cell undergoes mitosis and cellwall formation resulting in four uninucleate cells. Some ovulescontained secondary embryos thought to be the product of suspensorcleavage. Embryo maturation occurs by February.Copyright 1999Annals of Botany Company Podocarpus totara, totara, conifer, podocarp, reproductive biology, embryology.