Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from <Emphasis Type="Italic">Ficus carica</Emphasis>

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed in E. coli, and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.     

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree)

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches—resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9–194.2 kDa and in isoelectric point values of 4.0–11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.     

STRUCTURE AND ONTOGENY OF LATICIFERS IN CICHORIUM INTYBUS (COMPOSITAE)

The branched anastomosed laticifer system in the primary body of Cichorium intybus L. originates in embryos from files of laticiferous members at the boundary between phloic procambium and ground meristem. Upon seed germination, laticiferous members develop perforations in the end walls which become entirely resorbed. Perforations also develop in the longitudinal walls of contiguous laticiferous members and from lateral connections between developing laticifer branches. Additional laticiferous members originate as procambium differentiation proceeds, and their differentiation follows a continuous acropetal sequence in leaf primordia of the plumule. In roots, laticifers closely associated with sieve tubes in the secondary phloem originate from derivatives of fusiform initials in the vascular cambium. These laticifers develop wall perforations and in a mature condition resemble laticifers in the primary body. As the girth of the root increases, laticifers toward the periphery, unlike associated sieve tubes, resist crushing and obliteration. Laticifers vary in width from about 4 to 22 μm; the widest ones occur in involucral bracts and the narrowest ones in florets. There was no evidence that intrusive growth occurs during development of the laticifer system, although such growth may occur during development of occasional branches which extend through ground tissue independent of phloem and terminate in contact with the epidermis. Presence of amorphous callose deposits is related to aging of laticifers and mechanical injury.     

Laticifers in Camptotheca acuminata Decne: distribution and structure

Summary. In this paper, a system of laticifers in Camptotheca acuminata Decne (Nyssaceae) is described. Laticifers were already present in the leaf primordia of the shoot apex. In the mature leaves, laticifers were found in the midrib and in the larger veins, both in the parenchymatic region delimited by vascular bundles and in the cortex just external to the phloem. In the stem, laticifers were present in both the primary and secondary body, running parallel to the longitudinal axis. They were located in the pith and in the cortex proximal to the phloem. No laticifers were found in the roots. The histochemical analyses indicated that the main compounds accumulated in laticifers were phenols. Neutral lipids and fatty acids were also present. Ultrastructural observations showed osmiophilic globules both in the vacuoles and in the peripheral regions of the cytoplasm of the laticifer cells. Plastids were present, although altered, with some parallel membranes and lacking starch grains. The discovery in C. acuminata of a laticifer system, which had never been described for the order Cornales, could be of taxonomic value, also considering that this order has traditionally represented one of the most problematic groups of flowering plants. Correspondence and reprints: Dipartimento di Biologia Vegetale, Università “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy.     

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis‐1,4‐isoprene) bodies

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex‐containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1–5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1–5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild‐type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3–5 on the dispersity of artificial poly(cis‐1,4‐isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet‐associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.     

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.     

Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum

The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1-5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40-50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.     

FOSSIL LATICIFERS FROM EOCENE BROWN COAL DEPOSITS OF THE GEISELTAL

Thick mats of cellular remains from Eocene brown coal deposits of the Geiseltal near Halle, DDR, were determined to be fossil nonarticulated laticifers. Nuclear magnetic resonance analyses of intact strands showed they consisted of eis-1,4-configuration rubber representing the polymerized isoprenoid contents of individual laticifers. Only remains of laticifers are present; other cells are absent as a result of biodegradation. The long laticifers, often with a surrounding cell wall, retained a tubular shape during their preservation. The isoprenoid content, which filled the entire lumen, possessed a cribriform structural character. The interstices within the rubber represent areas of former protoplasm of the cell. Various configurations in the protoplasm molded by the rubber during the initial phase of fossilization appear as negative images of former nuclei, organelles, and possibly membrane surfaces. The laticifer axes possess branches of several configurations comparable in morphology to those in branched, nonarticulated laticifers in extant plants. Acetone extracts of the rubber contents analyzed by gas-liquid chromatography identified the presence of several hydrocarbons which form a characteristic profile for the laticifer. It is suggested that the distinctive cellular micromorphology, rubber configuration, and hydrocarbon profile of these laticifers can be employed as markers in comparative studies with extant plants to identify the generic or species origin of these laticifers.     

The distribution of plasmodesmata in the phloem ofHevea brasiliensis in relation to laticifer loading

Summary Cell to cell connections, including plasmodesmata and perforations, were examined in the non-conducting secondary phloem ofHevea brasiliensis. Samples were taken from trunks of numerous trees, from several clones, and prepared for thin sectioning and transmission or scanning electron microscopy and as optical sections for fluorescence microscopy. Numerous plasmodesmata were found clustered in primary pit-fields between the ray and axial parenchyma cells. Between the laticifers and adjacent parenchyma sheath cells, structures corresponding to functional plasmodesmata were not observed. But some unusual structural features were occasionally seen in these walls. These observations are discussed in relation to the possible function of the cell types, and to the loss of latex on the tapping ofHevea. It is suggested that the loading of the laticifer might first require a symplastic pathway for the transport of metabolites, at the end of which the assimilates must enter the apoplast. A transmembrane active transport system then transfers the metabolites in the laticifer. The presumable role of parenchyma cells in the loading of laticifers is emphasized.     

Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyses an important step in isoprenoid biosynthesis in plants. In Hevea brasiliensis, HMGR is encoded by a small gene family comprised of three members, hmg1, hmg2 and hmg3. We have previously described hmg1 and hmg2 (Plant Mol Biol 16: 567–577, 1991). Here we report the isolation and characterization of hmg3 genomic and cDNA clones. In comparison to hmg1 which is more highly expressed in laticifers than in leaves, the level of hmg3 mRNA level is equally abundant in laticifers and leaves. In situ hybridization experiments showed that the expression of hmg3 is not cell-type specific while hmg1 is expressed predominantly in the laticifers. Primer-extension experiments using laticifer RNA showed that hmg1 is induced by ethylene while hmg3 expression remains constitutive. The hmg3 promoter, like the promoters of most house-keeping genes, lacks a TATA box. Our results suggest that hmg1 is likely to encode the enzyme involved in rubber biosynthesis while hmg3 is possibly involved in isoprenoid biosynthesis of a housekeeping nature.     

应用酵母双杂交系统筛选橡胶延长因子REF的互作蛋白

利用酵母双杂交系统,以橡胶树(Hevea brasiliensis)橡胶延长因子基因REF的开放阅读框(ORF)构建无自激活性的诱饵表达载体pBD-GAL4-REF,并筛选以pAD-GAL4-2.1载体构建的橡胶树胶乳cDNA文库,对阳性克隆的cDNA插入片段进行测序及生物学功能分析。通过酵母双杂交筛选,共获得5种可能与REF互作的候选蛋白质,它们分别为与诱饵蛋白REF高度同源的REF家族成员、小橡胶粒子蛋白(SRPP)、翻译控制肿瘤蛋白(TCTP)、激发子响应蛋白和泛素耦联酶E2,这表明橡胶延长因子REF除了与自身高度同源蛋白质可能存在相互作用之外,还可能与TCTP和激发子响应蛋白等其它蛋白质发生相互作用。这些结果有助于揭示橡胶粒子的生物学功能。     

Developmental regulation of benzylisoquinoline alkaloid biosynthesis in opium poppy plants and tissue cultures

Summary Opium poppy (Papaver somniferum L.) contains a number of pharmaceutically important alkaloids of the benzylisoquinoline type including morphine, codeine, papaverine, and sanguinarine. Although these alkaloids accumulate to high concentrations in various organs of the intact plant, only the phytoalexin sanguinarine has been found at significant levels in opium poppy cell cultures. Moreover, even sanguinarine biosynthesis is not constitutive in poppy cell suspension cultures, but is typically induced only after treatment with a funga-derived elicitor. The absence of appreciable quantities of alkaloids in dedifferentiated opium poppy cell cultures suggests that benzylisoquinoline alkaloid biosynthesis is developmentally regulated and requires the differentiation of specific tissues. In the 40 yr since opium poppy tissues were first culturedin vitro, a number of reports on the redifferentiation of roots and buds from callus have appeared. A requirement for the presence of specialized laticifer cells has been suggested before certain alkaloids, such as morphine and codeine, can accumulate. Laticifers represent a complex internal secretory system in about 15 plant families and appear to have multiple evolutionary origins. Opium poppy laticifers differentiate from procambial cells and undergo articulation and anastomosis to form a continuous network of elements associated with the phloem throughout much of the intact plant. Latex is the combined cytoplasm of fused laticifer vessels, and contains numerous large alkaloid vesicles in which latex-associated poppy alkaloids are sequestered. The formation of alkaloid vesicles, the subcellular compartmentation of alkaloid biosynthesis, and the tissue-specific localization and control of these processes are important unresolved problems in plant cell biology. Alkaloid biosynthesis in opium poppy is an excellent model system to investigate the developmental regulation and cell biology of complex metabolic pathways, and the relationship between metabolic regulation and cell-type specific differentiation. In this review, we summarize the literature on the roles of cellular differentiation and plant development in alkaloid biosynthesis in opium poppy plants and tissue cultures.     

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.     

夹竹桃科2种引种植物分泌结构的解剖学研究

为了解夹竹桃科(Apocynaceae)植物乳汁管的发生发育,对爱之蔓(Ceropegia woodii)和百万心(Dischidia ruscifolia)营养器官中的分泌结构进行了显微观察。结果表明,爱之蔓和百万心营养器官中均有无节分枝乳汁管的分布,茎皮层中的乳汁管大部分具有明显的分枝,叶中乳汁管具明显分枝,分布与走向多与叶脉维管组织平行。另外,爱之蔓营养器官中的分泌结构除乳汁管外,还有分泌腔。这为夹竹桃科植物的系统分类研究提供了解剖学依据。     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.     

Cytochemical localization of pectinase activity in laticifers ofNerium oleander L.

Summary A method is described for the cytochemical localization of pectinase activity at the ultrastructural level. The procedure involves the use of Benedict's reagent to form an electron-dense copper precipitate when reacted with reducing sugars liberated from exogenously supplied pectin. Using this technique, pectinase activity was examined in the nonarticulated, branched laticifers ofNerium oleander. Electron opaque crystalline deposits indicating the presence of pectolytic enzymes were identified in laticifer central vacuoles. Smaller amounts of reaction product were distributed along the middle lamella between laticifers and adjacent cells. This report represents the first direct evidence for the involvement of pectinase in intrusive growth of nonarticulated laticifers.