A comparative study of latex-producing tissues in genera of Liabeae (Asteraceae)

Laticifers are highly specialized living plant cells which produce and contain latex. Occurrence of latex was used to establish morphological affinities (i) between Liabeae and other Asteracean tribes, (ii) among the Liabean genera, and (iii) in order to obtain phylogenies within Liabeae. However, structures and types of latex-producing tissues in this tribe have not yet been studied anatomically. In the present paper latex-producing structures of aerial parts in species of Microliabum, Munnozia, and Paranephelius (Liabeae), from open areas in mid-elevation Andean forests and in Andean high-elevation habitats, were studied. In all the analyzed species, latex secretion was easily observed in stem and leaf blade hand sections. Laticifers accompanied vascular tissues in all the cases, throughout stems and leaves, and they were of the articulated anastomosed type, at least in fully developed stages. Laticifers were found facing both, the xylem and the phloem, except for Paranephelius stems, in which they occur merely next to the phloem. Leaf laticifers form a reticulum accompanying the vein system. The type of latex-producing tissue shared by Microliabum and Munnozia could be a character shared by common ancestry whereas the laticifer system of Paranephelius stems could represent an evolutionary novelty for this genus. The laticifer type described in this study in aerial parts of Liabeae may allow establishing morphological affinities with tribes Cichorieae and Arctoteae.     

Age-dependent and jasmonic acid-induced laticifer-cell differentiation in anther callus cultures of rubber tree

Main conclusion

Callus cultures of rubber tree may serve as an efficient model to screen and study environmental factors and phytohormones that stimulate laticifer cell differentiation and improve latex yield. The number of laticifer cells in bark is one of the most important factors determining the biosynthesis and economic value of rubber trees (Hevea brasiliensis). The differentiation of laticifer cells in planta has been characterized, whereas laticifer-cell differentiation in callus cultures in vitro is largely unknown. In this study, we present molecular and physiological evidences for laticifer-cell differentiation in calli derived from rubber tree anthers. RT-PCR analysis showed that three key genes rubber elongation factor (REF), small rubber particle protein (SRPP), and cis-prenyl transferase (CPT) that are essential in latex biosynthesis in rubber tree bark also were transcribed in anther calli. Laticifer cell development in callus cultures was age-dependent; the cells began to appear at 58 days after initiation of culture, and the percentage of laticifer cells increased steadily with increasing callus age. Addition of 0–2 mg/L jasmonic acid (JA) to the media significantly promoted the differentiation of laticifer cells in callus cultures. However, JA concentrations higher than 3 mg/L were not optimum for laticifer cells differentiation; this result was not observed in previous in planta studies. Laticifer cells differentiated on media with pH 5.8–7.0, with an optimum of pH 6.2, whereas a higher pH inhibited differentiation. These results indicate that the anther-derived rubber tree callus may serve as a new and more efficient model to study environmental factors that influence laticifer cell differentiation, and may be useful for research on new technologies to improve latex yield, and to screen for commercially useful phytohormones.     

橡胶树形成层组织的酵母双杂交cDNA文库构建及HbHDA6互作蛋白筛选

次生乳管是天然橡胶合成和贮存的场所,是由橡胶树树干树皮中维管形成层细胞分裂分化而来。次生乳管的数量与天然橡胶产量直接相关,而这些乳管的数量取决于形成层分化次生乳管的频率(乳管分化能力),是橡胶树产量育种的主要指标。前期研究中,我们发现组蛋白去乙酰化酶(HDA)抑制剂曲古抑菌素A(TSA)能诱导橡胶树乳管分化且组蛋白去乙酰化酶基因(HbHDA6)能够参与橡胶树乳管分化调控。由于组蛋白乙酰化修饰调控橡胶树次生乳管分化的分子机制尚未阐明,因此该文使用冠菌素(COR)诱导橡胶树形成层分化产生次生乳管的实验系统,以分离形成层组织为材料,构建酵母双杂交cDNA文库,以HbHDA6基因为诱饵来筛选酵母双杂交文库,确定与HbHDA6相互作用的蛋白。结果表明：(1)利用Gateway技术构建的均一化COR诱导橡胶树形成层组织的酵母双杂交cDNA文库,初级文库的容量为6.34×10⁶ CFU·mL^-1,总单克隆数为1.27×10⁷,文库重组率为100%;次级文库的容量为7.72×10⁶ CFU·mL^-1     

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different <Emphasis Type="Italic">Hevea</Emphasis> clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.     

Ontogeny of anastomosed laticifers in the stem apex of <Emphasis Type="Italic">Hancornia speciosa</Emphasis> (Apocynaceae): a topographic approach

Latex is a complex plant secretion with both ecological and economic importance. There is little information currently available on the cytological aspects of the ontogenesis of anastomosed laticifers, the ducts originating through the lysis of adjacent cell walls. Hancornia speciosa is a tree typical of the Cerrado (neotropical savanna) biome. Its latex has medicinal value and is also used to produce rubber. The ontogenesis of its laticifers and the process of latex synthesis are described here. Structural, cytochemical, and ultrastructural analyses of the stem apex and phytochemical analyses of the latex were performed. Laticifer ontogenesis begins early in promeristem cells and subsequently extends through the procambial region. The laticifer precursor cells demonstrate intense metabolic activity, evidenced by starch accumulation and the proliferation of mitochondria, dictyosomes, endoplasmic reticulum, and ribosomes—resulting in the thickening of the cell walls and accumulations of oil droplets in the cytoplasm and fibrous materials in the vacuoles. The ontogenetic process culminates with the partial dissolution of adjacent cell walls and the collapse of the cytoplasm, giving rise to anastomosed laticifers distributed throughout the phloem and adjacent regions of the cortex and medulla. The latex itself is composed of terpenes, mucilage, proteins, alkaloids, and organelle residues that form an emulsion. Laticifer development takes place in three phases: (1) the formation of the emulsion in the promeristem, (2) anastomosis and the collapse of the cytoplasm in the distal region of the procambium, and (3) the maturation of laticifers and latex storage in a central vacuole in the proximal region of the procambium.     

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.     

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.     

Bark anatomy in Croton draco var. draco (Euphorbiaceae)

Many arboreal forms of the genus Croton (ca. 800 spp.), amply distributed in the Americas, have latex-producing cells in their bark, which is widely used in traditional medicine to treat skin infections and some forms of cancer. Studies validate its ethnomedicinal use-more than 20 pharmaceutically important secondary metabolites have been reported for its latex and bark-but anatomical and ecological studies are scarce. Given this species' ample distribution, laticifer abundance could be affected by the environment. We tested this for genetically similar trees growing in two types of vegetation in Veracruz, Mexico at sites commonly visited by traditional doctors. We describe the bark anatomy of C. draco, focusing on the laticifers, histochemically characterize the bark and the latex extracted from it, and document differences in laticifer abundance in the two environments. We have also identified another cell type (what we call type B) in the secretory latex system and describe it histochemically and microscopically. The location of bark cells that contain essential oils is reported here for the first time. Given the genetic similarity of the trees at both sites, the between-site variation in the number of laticifers in stem and branch bark appears to be an effect of the environment.     

Ethephon Increases Rubber Tree Latex Yield by Regulating Aquaporins and Alleviating the Tapping-Induced Local Increase in Latex Total Solid Content

The concentration of phloem solute generally falls from leaves to roots. However, a local increase in latex total solid content (LILTSC) was identified near the tapping cut of rubber trees. To understand the mechanism of ethephon-stimulated latex yield, the formation and ethephon (an ethylene releaser) alleviation of the LILTSC near the tapping cut were examined. It was found that the LILTSC near the tapping cut of a tapped rubber tree was caused by the tapping-accelerated rubber biosynthesis which began following the first tapping and became significant after the fourth tapping. Ethephon stimulation markedly reduced the LILTSC. The latex yield change pattern upon ethephon stimulation was associated with the kinetic change of LILTSC and the decomposition dynamic of ethephon into ethylene. Once the LILTSC was reduced by ethylene release upon ethephon stimulation, the latex yield increased; however, when the ethylene release upon ethephon stimulation receded, the LILTSC was restored and the effect of ethephon stimulation dissipated. The reduction of LILTSC by ethephon stimulation could be ascribed to the translocation property of ethylene in plants and its regulation of aquaporins. Because maximum ethylene release upon tapping-cut-ethephon-application occured close to the tapping cut, the aquaporins were more up-regulated in this region, leading to a reduction of the LILTSC and an increase in latex yield. All these results suggest that the LILTSC near the tapping cut was caused by tapping; the ethephon-induced aquaporin up-regulation and LILTSC reduction are involved in the mechanism of ethephon-promoted latex yield.     

Laser diffraction: A new tool for identification and studies of physiological effectors involved in aggregation-coagulation of the rubber particles from Hevea latex

Latex coagulation is the main limiting factor of rubber yield in Hevea brasiliensis (rubber tree). Using laser diffraction, we set up and optimized a new method for monitoring the kinetics of rubber particle (RP) aggregation, a prerequisite for latex coagulation. In contrast to any previous method used, laser diffraction allows continuous monitoring changes in size of RP aggregates, thereby allowing characterization and quantification of the processes involved in latex coagulation. Using this technique, we confirm that RP aggregating factors are proteins compartmentalized within latex cell vacuoles (lutoids), which, especially at relatively acidic physiological pH, can induce formation of RP aggregates large enough to induce plugging of severed latex vessel extremities. Conversely, latex cytosol was found to harbor anti-aggregating proteins. Further, we were able to titrate the RP-aggregating efficiency of the intralutoidic serum and the anti-aggregating efficiency of the cytosol. Preliminary assays showed that these two parameters were correlated with the yield potential of the tested rubber clones. This method will allow identification and characterization of proteins involved in latex coagulation, hence in rubber yield. We suggest that laser diffraction could be used to monitor the kinetics and characterize the physiological processes involved in aggregation of any particles, organelles or cells.     

A method for protein extraction from different subcellular fractions of laticifer latex in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> compatible with 2-DE and MS

Background  

Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS.     

橡胶树茉莉酸信号途径相关基因表达与橡胶产量的相关性

割胶促进橡胶树合成天然橡胶与激活乳管细胞的茉莉酸信号途径密切相关,但茉莉酸信号途径关键环节的基因表达水平与干胶产量的相关性尚不清楚。为了找到与产量相关的分子标记,该研究采用qPCR技术,分析了割胶条件下茉莉酸信号途径关键环节的9个相关基因在5个橡胶树魏克汉种质和5个1981’IRRDB种质乳管细胞中的表达。结果表明:大多魏克汉种质的株次干胶产量显著高于1981’IRRDB种质。在9个基因中,除了HbMYC4和HbMYC5,其余7个基因在大多橡胶树魏克汉种质中的表达量均显著高于1981’IRRDB种质,尤其是HbMYC3基因表达差异性好,与干胶产量相关性高,有望作为橡胶树产量育种的一个分子标记。这对育种周期长的橡胶树产量育种具有实际应用价值。     

三种植物生长物质对大豆叶茎解剖结构的影响

在大田栽培条件下,大豆‘垦农4号’于开花始期叶面喷施植物生长物质2-N,N-二乙氨基乙基己酸酯（DTA）、氯化胆碱（CC）和SOD模拟物（SODM）,并比较不同植物生长物质影响大豆叶片、叶柄和茎的解剖结构。结果表明,喷施植物生长物质后30d,叶中栅栏组织厚度及栅海比均增加;喷施SODM、DTA的叶中主脉维管束横截面积和木质部导管数目增加,CC对主脉维管柬横截面积和木质部导管数目的影响不明显;喷施3种植物生长物质的叶柄表皮细胞厚度、叶柄维管束横截面积和导管数量增加,茎部薄壁组织、韧皮部和木质部厚度增加,茎的直径也增加。     

Chromosome-level wild Hevea brasiliensis genome provides new tools for genomic-assisted breeding and valuable loci to elevate rubber yield

The rubber tree (Hevea brasiliensis) is grown in tropical regions and is the major source of natural rubber. Using traditional breeding approaches, the latex yield has increased by sixfold in the last century. However, the underlying genetic basis of rubber yield improvement is largely unknown. Here, we present a high-quality, chromosome-level genome sequence of the wild rubber tree, the first report on selection signatures and a genome-wide association study (GWAS) of its yield traits. Population genomic analysis revealed a moderate population divergence between the Wickham clones and wild accessions. Interestingly, it is suggestive that H. brasiliensis and six relatives of the Hevea genus might belong to the same species. The selective sweep analysis found 361 obvious signatures in the domesticated clones associated with 245 genes. In a 15-year field trial, GWAS identified 155 marker–trait associations with latex yield, in which 326 candidate genes were found. Notably, six genes related to sugar transport and metabolism, and four genes related to ethylene biosynthesis and signalling are associated with latex yield. The homozygote frequencies of the causal nonsynonymous SNPs have been greatly increased under selection, which may have contributed to the fast latex yield improvement during the short domestication history. Our study provides insights into the genetic basis of the latex yield trait and has implications for genomic-assisted breeding by offering valuable resources in this new domesticated crop.     

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.