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     

Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using spectral confocal laser scanning microscopy

In Hevea brasiliensis, laticifers produce and accumulate rubber particles. Despite observation using histochemical methods, development stage structure and structures with ceasing functions have rarely been described. Spectral confocal laser scanning microscopy with Nile red staining simplifies laticifer structure observation in tangential sections while enhancing the resolution. Laticifer and ray images were extracted from unmixed images and used to monitor changes during growth. A laticifer network structure developed from increased anastomoses between adjoining laticifers outside of the conducting phloem, but because of increased radial division and growth of rays, the network structure ruptured and disintegrated. We also investigated immunohistochemical localization of two rubber particle-associated proteins in the laticifers: small rubber particle protein (SRPP) and rubber elongation factor (REF). Mature bark test results show that SRPP is localized only in the laticifer layers in the conducting phloem; REF is localized in all laticifer layers. Because SRPP plays a positive role in rubber biosynthesis, results show that the rubber biosynthesis capability of laticifers is concentrated where rays and the sieve tube actively transport metabolites.     

Ultrastructure of Non-Articulated Laticifers in Allamanda violacea

Ultrastructure studies on the differentiation of non-articulatedbranched laticifers in Allamanda violacea Gardn. were carriedout. Growing laticifers show sequential changes. In the earlystage, the laticifers possess electron dense cytoplasm, abundantmitochondria, ER, ribosomes, small vacuoles, nucleus and plastidwith starch-grains. The ER dilates to form small vacuoles whichcoalesce at the later stages. A large central vacuole is formedin the mature laticifers due to the cellular autophagy of cytoplasmincluding the cell organelles. At this stage, the mitochondriapossess a few cristae and plastids with plastoglobuli and smallstarch grains. Towards the end of differentiation the cytoplasmis restricted to a thin parietal layer along the cell wall,the remaining organelles being either reduced in number or degenerate.Plasmodesmata and primary pit fields are occasionally observedbetween the laticifer and the adjacent parenchyma cell. Allamanda violacea, laticifers, ultrastructure     

Investigations of Laticifer Differentiation in Tissue Cultures Derived from Asclepias syriaca L.

Callus cultures of Asclepias syriaca were established from stemexplants and grown in tissue culture. The culture medium onwhich the callus was grown was modified to produce either planfletsof superficial origin on the callus or embryoids which wereanalyzed to determine whether laticifers differentiated in thesestructures. Mature zygotic embryos and adult plants of A. syriacanormally possess a well-developed network of intrusively-growingnon-articulated branched laticifers that arise only once duringplant develop ment from initials differentiated in the youngheart stage embryo. Embryoids were derived from two differentculture media. These embryoids were observed to lack laticifers,although they were similar in their morphology in other respectsto zygotic embryos. Plantlets of superficial origin were formedon each of the media employed in this study. These plantletswere observed to possess laticifers that resemble those in normalshoots. Embryoids and induced shoots represent experimentalsystems in which it may be possible to control for the firsttime the differentiation of the laticifer as a cell type instructures similar to those present in the normal plant.     

Laticifers in Nelumbo nucifera Gaertn.: Distribution and Structure

The laticifers of Nelumbo nucifera Gaertn. (Nelumbonaceae) occurin vascular bundles and separately from these in the groundparenchyma. In common with laticifers in some other plants,they pass through a stage when they contain numerous small vacuoles,or vesicles, which later merge with the central vacuole. Osmiophilicglobules are formed in the small vacuoles and later releasedinto the central one. Partial disintegration of protoplaststakes place in the central vacuole, apparently by autophagy.The globules are retained. The laticifers are long thin-walledcells. Many show single perforations in the end walls.     

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     

Natural and Experimentally Induced Dispersion of Aggregate Rays in Shoots of Quercus ithaburensis Decne. and Q. calliprinos Webb

There are two types of sites where aggregate rays in Quercusithaburensis Decne. and Q. calliprinos Webb do not occur naturallyin the tree: branch junctions and small ridges of wood. Partialgirdling of Quercus trunks resulted in the dispersion of aggregaterays which diffused into numerous simple rays. The breakup ofaggregate rays following partial girdling is not a typical woundresponse since wounding is known to stimulate ray enlargement.The disaggregation of the huge rays reflects changes in therelationship between axial and radial signal flows. Aggregate rays, Quercus ithaburensis Decne., Quercus calliprinos Webb, ray differentiation, wood formation, xylem anatomy     

Ultrastructure and Development of the Laticifers of Ficus carica L

Laticifers of Ficus caricaL. are of the branched, non-articulatedtype. They occur in the cortex and pith of the plant axis andpenetrate leaves and inflorescences. Observations were madeon laticifers located in shoot apices. Growing regions of laticifers undergo a sequence of ultrastructuralchanges. These are: (a) a pronounced increase in the vacuolarspace which divides the cytoplasm into separated masses; (b)a development of numerous vesicular structures in the cytoplasm.The vesicular structures are released into the vacuolar space.The whole process is accompanied by disintegration of cytoplasm.Apparently isolated masses of cytoplasm occur in the luminaof laticifer tips in sections taken from dormant apices. Itis assumed that these masses have a role in the initiation ofnew laticifer regions in the next growing season. Ficus caricaL., laticifers, ultrastructure, development differentiation     

Anatomy of the Latex System in the Dormant Embryo of Euphorbia marginata Purch : Correlative Analysis of the Histological Organization and Topographic Distribution of Laticifers

The anatomy, morphology and topography of laticifers in themature embryo of Euphorbia marginata have been studied by conventionaland fluorescence microscopy. The mature embryo consists of awell-developed embryonic axis and two cotyledons and it containsdifferentiating vascular tissue. In the hypocotyl, six procambialstrands are present which branch and extend into the cotyledons,whereas in the root they are fused in a triarch structure. Inthe embryonic axis, the latex system consists of stelar andcortical components that are first anastomized in a cotyledonaryplexus and then are joined above in a cotyledonary system. Thestelar and cortical systems are linked by horizontal branchesto a nodal latex plexus placed under the epicotylary meristem.All the vascular laticifers, 24 in number, end unbranched inthe apex of the radicle. The cortical laticifers are profuselybranched toward the epidermis in tiny veins as far as the sub-epidermallevel. The cotyledonary laticifers follow the vascular tissueand form a network surrounding the mesophyllary areoles. Thetopography of the latex system leads one to hypothesize thatthe criteria which determine the constitution of the latex systemlareonly the essential peculiarities of idioblasts, but also correlativephenomena between growing laticifers and surrounding tissues.The stelar system appears to be under the control of vascularpolar growth, while the cortical and cotyledonary latex systemsseem to be subjected both to endogenous polar stimuli and toextra-embryonal influences. Euphorbia marginata, anatomy, embryo, laticifers     

Molecular and biochemical characterization of a cyanogenic β-glucosidase in the inner bark tissues of rubber tree (Hevea brasiliensis Muell. Arg.)

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of β-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both β-glucosidase and linamarase and was thus characterized as a cyanogenic β-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic β-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.     

Laticifer Differentiation of Calotropis gigantea R. Br. ex Ait. in Cultures

The initiation and subsequent development of laticifers werestudied in callus tissues of Calotropis gigantea grown on MS(Murashige and Skoog) medium supplemented with 1 mg l^–1IAA. Laticifer development was related to the age of the cultureand could be preserved by repeated subculturing on similar mediumwith IAA. Our previous work had established that cardenolidebiosynthesis was related to rhizogenesis and here we reportthe culture system of laticifer, long term preservation anddifferentiation of this hydrocarbon producing energy plant. Calotropis gigantea, Asclepiadaceae, laticifer, differentiation, cardenolides, tissue culture     

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.     

Studies on Differentiation of Laticifers through Light and Electron Microscopy in Calotropis gigantea (Linn.) R.Br.

The distribution, cytological organization and differentiationof non-articulated laticifers in the primary and mature tissuesof Calotropis gigantea (Linn.) R.Br., were studied by the useof optical and electron microscopy. Laticifers occur in thecortex, vascular bundle and pith of the plant axis. At the earliestdetectable stage a laticifer is a cell which undergoes rapidelongation and nuclear division. This results in a multinucleateelongated cell which undergoes further increase in length withgradual degeneration of the cytoplasm. At the electron microscopiclevel the presumptive laticifer cell shows increasing vacuolationwhich forms a large central vacuole. Simultaneously the cytoplasmicorganelles undergo degeneration by autophagic processes. Laternumerous vesicles can be observed in the large central vacuole,the remaining cytoplasm being pushed to a thin layer. Maturelaticifers show three types of spherical structures of whichthe highly electron dense globules are the latex particles. Calotropis gigantea (Linn.), R.Br., laticifers, ultrastructure, differentiation     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.     

Histochemical and immunohistochemical identification of laticifer cells in callus cultures derived from anthers of Hevea brasiliensis

Laticifers are highly specialized cells present in over 20 plant families. They are well defined in planta. In vitro development of laticifers was also observed in some plants, but uncertain in the callus cultures of rubber tree, one of the most economically important latex producing plants. In the present study, we provide evidence that laticifer cells present in the callus cultures of rubber tree by histochemical and immunohistochemical studies. They present in the callus mainly as separate non-elongated form, a novel morphology different from the morphology of laticifer cells in planta, excluding their origin from explants. The occurring frequency of laticifer cells in the callus was genotype-dependent and negatively correlated with the somatic embryogenetic ability, suggesting that the presence of laticifer cells in the callus inhibit somatic embryogenesis in tissue culture of rubber tree. The genotypes PR107, RRIM600, Reyan8-79, and Reyan7-33-97 with lower embryogenetic ability compared to Haiken 2 had more laticifer cells, and laticifer clusters were only observed in these genotypes.     

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