Characteristics of protein-storing cells associated with a 67 kDa protein in Hevea brasiliensis

 The protein-storing cells (PSCs) in Hevea brasiliensis were studied by using light- and electron-microscopy and SDS-PAGE. The cells were found in stem and root where secondary phloem was developed. They are a special kind of phloem parenchyma cell which accumulate in their central vacuoles large amounts of protein, fibril-like under an electron microscope, and have few plastids with very small starch grains. Their distribution is strictly restricted to the secondary phloem axial system where they exactly sequestered in functional phloem or slightly over it. A 67 kDa protein was always found in the tissues where the PSCs were observed. During the first seasonal growth flush, the 67 kDa protein in the terminal branchlet exhibits marked quantitative fluctuation which is consistent with the change of the vacuole protein inclusion of the PSCs in the branchlet. These facts suggested that the 67 kDa protein might be the major part of the vacuole protein of the PSCs. Considering the differences between the PSCs in Hevea and the PSCs in the other trees studied, we define two types of PSCs: Hevea-type, which are the cells specialized for protein storage and Populus-type, which are ordinary parenchyma cells accumulating protein and starch. Received: 11 April 1997 / Accepted: 28 July 1997     

Occurrence of Intraxylary Phloem in Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg.

The occurrence of intraxylary phloem in Hevea brasiliensis isreported. The phloem elements were observed as strands associatedwith the protoxylem group in the pericentral region. The natureand importance of such tissue in this species are discussed. Hevea brasiliensis, intraxylary phloem, laticifers, tapping     

Laticifer Differentiation in Hevea brasiliensis: Induction by Exogenous Jasmonic Acid and Linolenic Acid

Laticifer differentiation of Hevea brasiliensis was investigatedby application of lanolin containing jasmonic acid (JA) or otherchemicals to the surface of young stems in epicormic shoots.The young stems had primary laticifers and no secondary laticifers.When applied to extending young stems, JA led to a significantincrease in primary laticifer number but did not induce secondarylaticifer differentiation. Secondary laticifer differentiationand a less significant increase in primary laticifer numberwere caused by JA application to the extended young stems. Theinduction of the secondary laticifers was dependent on the concentrationof JA applied. Cambium cell division leading to the formationof secondary phloem was not accelerated by JA treatment. Treatedbark tissues showed no visible changes except for the additionallaticifers, which were normal in ultrastructure. The secondarylaticifers were also induced by the application of linolenicacid, a precursor of JA biosynthesis. Abscisic acid, ethephonand salicylic acid had no detectable effect on laticifer differentiation.Copyright 2000 Annals of Botany Company Hevea brasiliensis, laticifer differentiation, jasmonic acid, linolenic acid, vascular cambium.     

Another View of the Ultrastructure of Cucurbita Phloem

The primary phloem of young internodes of Cucurbita maxima wasstudied with the electron microscope. Phloem parenchyma cellsare highly vacuolated and contain nuclei, endoplasmic reticulum,ribosomes, mitochondria, chloro-plasts, and occasional dictyosomes.As compared with parenchyma cells, the most distinctive featuresof companion cells are their extremely dense cytoplasm, lowdegree of vacuolation, lack of chloroplasts, and numerous sieve-elementconnexions. Companion cells contain plastids with few internalmembranes. At maturity the enucleate sieve element is linedby a plasmalemma, one or more cistema-like layers of endoplasmicreticulum, and a membrane which apparently delimits the parietallayer of cytoplasm from a large central cavity. In OsO_4–-andglutaraldehyde-fixed elements, the central cavity is traversedby numerous strands, which run from cell to cell through thepores of sieve plates and lateral sieve areas, and which arederived ontogenetically from the slime bodies of immature cells.Numerous normal-appearing mitochondria are present in the parietallayer of cytoplasm. The pores of sieve plates and lateral sieveareas are lined with cytoplasm. The ultrastructural detailsof young sieve elements differ little from those of other youngnucleate cells. During sieve-element development, the sieveelement increases in vacuolation. At the same time, slime bodiesdevelop in the cytoplasm. With glutaraldehyde fixation, thesebodies often exhibit a double-layered limiting membrane. Asthe sieve element continues to differentiate, the slime bodiesincrease in size and the parietal layer of cytoplasm becomesvery narrow. Presently, the slime bodies begin to disperse andtheir contents fuse. This phenomenon occurs in the parietallayer of cytoplasm, while the latter is still delimited fromthe large central vacuole by a distinct tonoplast. The initiationof slime-body dispersal more or less coincides with perforationof the pore sites, and many pores are traversed by slime earlyin their development. Before slime-body dispersal, all dictyosomesand associated vesicles disappear from the cytoplasm. Eventually,the tonoplast diappears and the slime becomes distributed throughoutthe central cavity in the form of strands. Nuclei and ribosomesdisappear before breakdown of the tonoplast. Sieve elementsare connected with companion cells and parenchyma cells by plasmodesmata.     

Hyperplastic Phloem and Its Plastids in Spinach Infected with the Curly Top Virus

The hyperplastic growth induced in the phloem tissue by infectionwith the curly top virus was studied in minor veins of leavesof spinach, Spinacia oleracea L., by the use of the electronmicroscope. Proliferation of cells occurs in the phloem andin the parenchyma bordering the phloem. The arrangement of cellsis less orderly when hyperplasia occurs in older than in youngertissue but in both instances the majority of cells differentiateinto sieve elements. As in normal phloem, sieve element plastidshaving a ring of proteinaceous fibrils are a consistent featurein the hyperplastic phloem. Depending on the kind of cell inwhich hyperplasia is initiated, the plastids may originate fromyoung plastids similar to those in normal sieve elements orfrom more or less completely differentiated chloroplasts. Theprotoplasts of the hyperplastic sieve elements, including theplastids, degenerate during differentiation or after maturation.     

Range of Hevea brasiliensis Pollen Dispersal Estimated by Esterase Isozyme Markers

A study was carried out to estimate the distance Hevea brasiliensispollen could be dispersed under natural conditions. Seeds werecollected at varying distances from the boundary between twoadjacent fields that were each planted with a pure stand ofa genetic clone. Esterase isozyme markers were used to determineif the seeds had been derived from self- or cross-pollination.The incidence of cross-pollination was then examined in relationto the distance from the inter-clonal boundary. A logarithmicmodel gave the best fit (r²=0.864) and suggested that pollencould travel distances in the order of 0.3 to 1.1 km. Copyright1999 Annals of Botany Company Hevea brasiliensis (rubber tree), pollen dispersal, cross-pollination, self-pollination, esterase isozymes.     

Manometric Measurement of Turgor Pressures in Laticiferous Phloem Tissues2

A manometric technique for the determination of turgor pressuresin laticiferous phloem tissues has given reproducible resultsin Hevea brasiliensis and a few other tree species. In Hevea,early morning pressures are in the range 7.9–15.0 atmospheres,falling during the day and recovering at night. These diurnalpressure changes are positively correlated with atmosphericrelative humidity and negatively correlated with changes intemperature, evaporation, leaf water deficit, and stomatal opening.They are not displayed by trees devoid of leaves. Thus the lossin turgor is probably the result of withdrawal of water fromthe phloem tissues under transpirational stress. Pressures at the base of the trunk normally exceed those atthe top, the gradient usually approximating to 1 atmosphere/10metres at night and rising up to six times this figure duringthe day. This increase probably reflects the development oftension gradients in the xylem during transpiration. A generalturgor gradient from base to crown does not preclude mass flowin sieve-tubes in the opposite direction provided that ratesof loading and unloading are such that a sufficient osmoticgradient is maintained in them in the required direction. No marked long-term effects of regular tapping on turgor pressurehave been noted in Hevea trees and there is no evidence forseasonal changes in turgor under our conditions.     

A Crystalline P-Protein in Gmelina arborea

In a light microscope study of the secondary phloem in Gmelinaarborea (Verbenaceae) many sieve elements were found to possessbar-shaped cytoplasmic inclusions of proteinaceous nature Itis suggested that these inclusions represent a type of crystallineP-protein not reported in the family Verbenaceae before Crystalline P-protein, phloem, sieve element inclusion     

Metabolism of 2-Chloroethylphosphonic Acid (Ethephon) in Suspension Cultures of Hevea brasiliensis

Suspension cultures of Hevea brasiliensis cells can metabolizethe growth regulator 2-chloroethylphosphonic acid to producea number of compounds, one of which appears to be an acid-labileconjugate. The general metabolic pattern closely resembles thatfound previously using Hevea leaves. Evidence that the compoundsformed are not chromatographic artefacts of a type describedby other authors is presented.     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.     

Anatomical and Histochemical Aspects of Bark Regeneration in Hevea brasiliensis

Natural rubber is obtained from the bark of Hevea brasiliensis.Both virgin and renewed bark are exploited for this purposeby a process known as tapping which involves controlled woundingand excision of bark tissues. The process of bark renewal andits nature and consequences thus assume importance. Anatomicaland histochemical changes encountered with tapping were thedeposition of lignin and suberin in the peripheral cells, enlargementof tray cells near the cut surface and the formation of a woundperiderm. In the course of development, the wound phellogenmade tangential continuity with the original phellogen in thevirgin bark and functioned as a single phellogen. Vascular cambialactivity was enhanced due to wound stimulus and the newly differentiatedsieve tubes and ray cells were larger in size. The first peridermwas functional for only a short period of time, after whicha new meristematic zone developed in the inner tissues. Virginand renewed bark differed in the proportion of soft and hardbark, amount and distribution of sclereids, tannin cells andcrystals.Copyright 1995, 1999 Academic Press Bark renewal, Hevea brasiliensis, histochemistry, laticifers, para rubber tree, wound periderm     

A Comparison of Abscission of Rubber (Hevea brasiliensis) Leaves Infected with Microcyclus ulei with Leaf Abscission Induced by Ethylene Treatment, Deblading and Senescence

Studies on the histology and on effects of growth substancesand phenols as well as changes in activities of pectinmethylesterase indicated that the mechanism of abscission of Hevealeaflets infected with Microcyclus ulei differed from the mechanismof abscission of debladed, ethylene treated and senescent leaves.An abscission layer which was formed during abscission of debladed,ethylene-treated and senescent leaves was absent during abscissionof heavily diseased leaves. The ratio of pectinmethyl esteraseactivities in tissues distal to the abscission zone to activitiesin tissues proximal to the zone decreased in debladed and ethylenetreated leaves but such decreases were not detected during abscissionof Hevea leaves infected with M. ulei. Hevea brasiliensis Muell. Arg., rubber, leaf abscission, Microcyclus ulei, ethylene, indol-3-ylacetic acid, kinetin     

Distribution and Organization of Non-articulated Laticifers in Mature Tissues of Poinsettia (Euphorbia pulcherrima Willd.)

The distribution and cytological organization of non-articulatedbranched laticifers in the mature root, stem, and leaf tissuesof poinsettia. Euphorbia pulcherrima Willd., were studied bythe use of optical and electron microscopy. The laticifers occurin all parts of the plant body, being well represented in certainparenchymatous tissues and the phloem. The mature region ofthe laticifer has a living protoplast showing a thin parietalcytoplasm, bounded by plasmalemma and a tonoplast, which enclosesa large continuous central vacuole containing the milky latexfluid. The protoplast is multinucleate and possesses large amyloplasts,each enclosing a single elongated starch grain. Sparseness andpoor differentiation of the other components of the protoplast,mostly mitochondria, ribosomes and endoplasmic reticulum, suggestlow metabolism in the mature region of the laticifer. The latexof the central vacuole is dominated by spherical particles,0.3–1 µm in diameter, each with a dense matrix andan eccentric core of lighter staining material. In some laticifersthe latex particles fuse into coagulated masses. Euphorbia pulcherrima Willd, poinsettia, laticifers, ultrastructure, cytology     

Effects of Growth Regulators on Polyamine Content and Peroxidase Activity in Hevea brasiliensis Callus

3, 4-dichlorophenoxyacetic acid (3,4-D) and benzylaminopurine(BAP) at 9 µM (control medium) was compared with 4.5,2.25, and 0.45 µM for ability to induce callogenesis andembryogenesis from seed explants of Hevea brasiliensis. Supplyingthese growth regulators at 4.5 µM for 20 d improved embryogenicpotential compared with the control medium (El Hadrami, Carronand d'Auzac, 1991, Annals of Botany 67, 511–515), sustainedputrescine, spermidine and spermine at a higher level throughoutof much of the culture period (40–70 d), and maintainedlow levels of peroxidase activity. In the control medium, poorcallus embryogenesis is considered a consequence of rapid ageingof tissues characterized by (i) acceleration of an early buttransient production of polyamines, which promoted embryogeniccapacity, and (ii) an early peak in peroxidase activity thatwas positively correlated with callus browning, one of the factorslimiting embryogenesis. Somatic embryogenesis, polyamines, peroxidase, Hevea brasiliensis, rubber-tree     

Ca Fluxes and Membrane Potentials in Nitella translucens

The concentrations of Ca have been measured in the flowing cytoplasmand the vacuole of the single cells of Nitella translucens,the cells being immersed in an artificial pond Water (composition:NaCl, 1.0 mM; KCl, 0.1 mM; CaCl₂, 0. mM). In the flowing cytoplasmthe total concentration is 8 mM and in the vacuole 12 mM. Measurementsof the electrical potential differences across the plasmalemmaand tonoplast membranes show that the cytoplasm is at a potentialof —134 mV with respect to the bathing medium and —24mV with respect to the vacuole. An attempt has been made tomeasure the tracer fluxes of Ca and it is shown that the cellsare not in flux equilibrium. The influx is 0.046 µµmoles cm^–2 sec^–1; the efflux was too small to measurewith any degree of accuracy. The observed potential differences across both membranes arecompared with the Nernst potentials for Ca. This analysis showsthat Ca is not in electrochemical equilibrium across eithermembrane and that the driving forces on Ca are directed fromthe bathing medium and the vacuole into the cytoplasm. It issuggested that there is no necessity for a metabolically drivenCa pump at the plasmalemma because the low cytoplasmic Ca contentcould be due to the low permeability of the plasmalemma; theGoldman flux equation gives a value of P_Ca = 4.3x10^–8cm sec^–1. A Ca pump at the tonoplast appears to be necessaryto explain the steep electrochemical potential gradient fromthe vacuole to the cytoplasm. The efflux of Ca from the isolated cell wall has been measured.From these measurements it was possible to estimate the concentrationof indiffusible anions in the Donnan Free Space; the value obtainedwas 0.74 equiv. 1.^–1.     

Polar Regeneration in Excised Roots of Taraxacum officinale Weber: A Light and Electron Microscopic Study

The day 0 secondary phloem of Taraxacum officinale root segmentscontains wide bands of parenchyma alternating with thin cylindersof conducting tissue composed of discreet conducting strands.At day 1 in the inner distal phloem (and by day 2 proximally)the initially-flattened nuclei of some parenchyma cells becomerounded, more densely stainable and a few have migrated fromthe peripheral cytoplasm to a suspended position in the vacuole.Cell division occurs asynchronously and gradually extends tothe midphloem. By day 4 nodules of primary meristematic cellsoccur proximally and numerous young leaves are visible externallyat days 5–6. Distally, callusing of the phloem is moreextensive and links with that developing from the secondaryxylem. Proximally adventitious buds form and these are bothmore abundant and quicker growing than the distally locatedadventitious roots. Although proliferation is initially mainly confined to the companioncells it increasingly involves activation of the parenchymatissue. These cells undergo a cytological de-differentiationwith the daughter cells showing a progressive decrease in cellsize and vacuome (with cytoplasmic strands, perhaps indicativeof lysosomal activity, often visible in the vacuoles), accompaniedby an increase in nucleolar size and cytoplasmic density.     

A Complex Sub-cellular Component of Widespread Occurrence in Plant Latices

A survey of exudates from a number of latex-bearing plants revealsthe wide-spread occurrence of a complex subcellular componentapparently analogous to the ‘lutoid’ of Hevea brasiliensislatex. Under the conditions of our experiments this componentis usually associated with thread-like material.     

Stomatal Characteristics, Leaf Waxes, and Transpiration Rates of Theobroma cacao and Hevea brasiliensis Seedlings

Stomatal size and frequency as well as structure and distributionof leaf waxes were compared in seedlings of two varieties ofTheobroma cacao and three families of Hevea brasiliensis. Stomataof both species were located on the abaxial leaf surface only.The stomata of Theobroma averaged 45% shorter and 29% narrowerthan those of Hevea, but stomatal frequency was 44% higher inTheobroma. Stomatal size and frequency differed more among Heveafamilies than between Theobroma varieties. The structure ofthe leaf waxes of Theobroma and Hevea differed appreciably.It also varied greatly between the abaxial and adaxial leafsurfaces of Hevea, but only slightly in Theobroma. The structureof leaf waxes varied little between Theobroma varieties or amongHevea families. Leaf conductances and transpiration rates weremuch higher in Hevea than in Theobroma seedlings. The implicationsof water relations in interplanting of Theobroma and Hevea arediscussed. Cacao, Theobroma cacao, rubber, Hevea brasiliensis, stomatal size, stomatal frequency, leaf waxes, transpiration     

Development and Structure of Phloem in the Petiole of Lagenaria siceraria (Mol.) Standl. and Momordica charantia L.

Light microscopic studies of the petioles of Lagenaria sicerariareveal that the external phloem of each bicollateral vascularbundle develops earlier than the internal phloem, and that thesieve elements of the external phloem are arranged in the outerand inner zones. Each sieve element of L. siceraria and Momordicacharantia is vertically associated with a maximum of six andtwo companion cells respectively. Discrete granular bodies seenin the cytoplasm of young sieve elements develop into globular,oval, or elongated slime bodies. Enlargement and fusion of slimebodies, and the subsequent dispersal of slime occur in the parietalcytoplasm. The dispersal of slime coincides with degradationof the nucleus and perforation of the pore sites. Before nucleardisorganization, the sieve-element nucleolus is extruded. Slimeafter its immediate dispersal appears amorphous and uniformlydistributed in the sieve elements. Plugs exhibit varying degreesof condensation of slime near the sieve plates. Certain maturesieve elements in the external phloem of L. siceraria have ovalbodies which we consider reaggregated or undispersed slime.Evidence has been obtained that a central cavity occurs in afew, almost mature, sieve elements wherein the cytoplasm includingthe slime is peripheral.     

Differentiation of Non-articulated Laticifers in Poinsettia (Euphorbia pulcherrima Willd.)

Differentiation of non-articulated laticifers in poinsettia(Euphorbia pulcherrima Willd.) was studied ultra-structurally.Growing laticifers show: (1) a multinucleate apical region containingabundant ribosomes but few other differentiated organelles and(2) a sub-apical zone where the cytoplasm is dominated by vacuolesof diverse morphology with latex particles. These particlesappear first within narrow tubular vacuoles developed especiallyin the peripheral cytoplasm. During vacuolation of the laticifer,portions of cytoplasm, including some of the nuclei, becomeisolated by the enlarging and fusing vacuoles; eventually thesebecome lysed, except the latex particles which remain in thecentral vacuole. During differentiation of a laticifer branch,the cytoplasm contains the usual organelles, including a fewmicrobodies and coated vesicles. The plastids that lie withinthe peripheral cytoplasm differentiate into amyloplasts witha single elongated starch grain. Towards the end of differentiationthe cytoplasm becomes restricted to a thin parietal layer, withthe remaining organelles reduced or degenerate, surroundinga central vacuole filled with latex particles. Euphorbia pulcherrima Willd, poinsettia, ultrastructure, differentiation, laticifers