Laticifer Differentiation Research Articles

Novel Insights into the Organization of Laticifer Cells: A Cell Comprising a Unified Whole System.

Laticifer cells are specialized plant cells that synthesize and accumulate latex. Studies on laticifers have lagged behind in recent years, and data regarding the functional role of laticifers and their fitness benefit still remain elusive. Laticifer differentiation and its impact on plant growth and development also remain to be investigated. Here, cellular, molecular, and genetic tools were developed to examine the distribution, differentiation, ontogeny, and other characteristic features, as well as the potential developmental role of laticifer cells in the latex-bearing plant Euphorbia lathyris. The organization of the laticiferous system within the E. lathyris plant body is reported, emerging as a single elongated and branched coenocytic cell, constituting the largest cell type existing in plants. We also report the ontogeny and organization of laticifer cells in the embryo and the identification of a laticifer-associated gene expression pattern. Moreover, the identification of laticifer- and latex-deficient mutants (pil mutants) allowed for the identification of distinct loci regulating laticifer differentiation, growth, and metabolic activity. Additionally, pil mutants revealed that laticifer cells appear nonessential for plant growth and development, thus pointing toward their importance, instead, for specific ecophysiological adaptations of latex-bearing plants in natural environments.

Identification of differentially expressed genes and signalling pathways in bark of Hevea brasiliensis seedlings associated with secondary laticifer differentiation using gene expression microarray

The natural rubber of Para rubber tree, Hevea brasiliensis, is the main crop involved in industrial rubber production due to its superior quality. The Hevea bark is commercially exploited to obtain latex, which is produced from the articulated secondary laticifer. The laticifer is well defined in the aspect of morphology; however, only some genes associated with its development have been reported. We successfully induced secondary laticifer in the jasmonic acid (JA)-treated and linolenic acid (LA)-treated Hevea bark but secondary laticifer is not observed in the ethephon (ET)-treated and untreated Hevea bark. In this study, we analysed 27,195 gene models using NimbleGen microarrays based on the Hevea draft genome. 491 filtered differentially expressed (FDE) transcripts that are common to both JA- and LA-treated bark samples but not ET-treated bark samples were identified. In the Eukaryotic Orthologous Group (KOG) analysis, 491 FDE transcripts belong to different functional categories that reflect the diverse processes and pathways involved in laticifer differentiation. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and KOG analysis, the profile of the FDE transcripts suggest that JA- and LA-treated bark samples have a sufficient molecular basis for secondary laticifer differentiation, especially regarding secondary metabolites metabolism. FDE genes in this category are from the cytochrome (CYP) P450 family, ATP-binding cassette (ABC) transporter family, short-chain dehydrogenase/reductase (SDR) family, or cinnamyl alcohol dehydrogenase (CAD) family. The data includes many genes involved in cell division, cell wall synthesis, and cell differentiation. The most abundant transcript in FDE list was SDR65C, reflecting its importance in laticifer differentiation. Using the Basic Local Alignment Search Tool (BLAST) as part of annotation and functional prediction, several characterised as well as uncharacterized transcription factors and genes were found in the dataset. Hence, the further characterization of these genes is necessary to unveil their role in laticifer differentiation. This study provides a platform for the further characterization and identification of the key genes involved in secondary laticifer differentiation.

The secondary laticifer differentiation in rubber tree is induced by trichostatin A, an inhibitor of histone acetylation

The secondary laticifer differentiation in rubber tree is induced by trichostatin A, an inhibitor of histone acetylation

Comparative proteomic analysis of latex from Hevea brasiliensis treated with Ethrel and methyl jasmonate using iTRAQ-coupled two-dimensional LC–MS/MS

Ethrel (ET) is an effective and widely used latex yield stimulant of Hevea brasiliensis (Pará rubber tree), and jasmonate (JA) is a key inducer of laticifer differentiation in this plant. To examine variations in the latex proteome caused by these phytohormones, ET and methyl jasmonate (MeJA) were applied to Reyan 7-33-97 rubber tree clones, and comparative proteomic analyses were conducted. On the basis of a transcriptome shotgun assembly (TSA) sequence database and an iTRAQ-coupled two-dimensional LC–MS/MS approach, 1499 latex proteins belonging to 1078 clusters were identified. With a 1.5-fold cut-off value to determine up- and down-regulated proteins, a total of 101 latex proteins were determined to be regulated by ET and/or MeJA via pairwise comparisons among the three exposure durations (0h, 6h, and 48h). Proteins associated with latex regeneration, including phosphoenolpyruvate carboxylase and acetyl-CoA C-acetyltransferase, and those associated with latex flow, such as chitinase and a sieve element occlusion protein, were affected by the application of ET. Chitinase and polyphenol oxidase were also found to be regulated by MeJA. The findings of this study may provide new insight into the roles of phytohormones in latex yield and the causative mechanisms of laticifer differentiation in rubber trees. SignificanceOn the basis of a transcriptome shotgun assembly (TSA) sequence database and an iTRAQ-coupled two-dimensional LC–MS/MS approach, the most comprehensive proteome of the latex was profiled, and the ethylene-/jasmonate-responsive proteins were identified in the latex of H. brasiliensis. The findings of this study may provide new insight into the role of phytohormones in latex yield and the causative mechanisms of laticifer differentiation in rubber trees.

Analysis of Differentially Expressed Genes Associated with Coronatine-Induced Laticifer Differentiation in the Rubber Tree by Subtractive Hybridization Suppression.

The secondary laticifer in the secondary phloem is differentiated from the vascular cambia of the rubber tree (Hevea brasiliensis Muell. Arg.). The number of secondary laticifers is closely related to the rubber yield potential of Hevea. Pharmacological data show that jasmonic acid and its precursor linolenic acid are effective in inducing secondary laticifer differentiation in epicormic shoots of the rubber tree. In the present study, an experimental system of coronatine-induced laticifer differentiation was developed to perform SSH identification of genes with differential expression. A total of 528 positive clones were obtained by blue-white screening, of which 248 clones came from the forward SSH library while 280 clones came from the reverse SSH library. Approximately 215 of the 248 clones and 171 of the 280 clones contained cDNA inserts by colony PCR screening. A total of 286 of the 386 ESTs were detected to be differentially expressed by reverse northern blot and sequenced. Approximately 147 unigenes with an average length of 497 bp from the forward and 109 unigenes with an average length of 514 bp from the reverse SSH libraries were assembled and annotated. The unigenes were associated with the stress/defense response, plant hormone signal transduction and structure development. It is suggested that Ca2+ signal transduction and redox seem to be involved in differentiation, while PGA and EIF are associated with the division of cambium initials for COR-induced secondary laticifer differentiation in the rubber tree.

Mechanical wounding-induced laticifer differentiation in rubber tree: An indicative role of dehydration, hydrogen peroxide, and jasmonates

The secondary laticifer in the secondary phloem of rubber tree are a specific tissue differentiating from vascular cambia. The number of the secondary laticifers is closely related to the rubber productivity of Hevea. Factors involved in the mechanical wounding-induced laticifer differentiation were analyzed by using paraffin section, gas chromatography–mass spectrometry (GC–MS), and Northern-blot techniques. Dehydration of the wounded bark tissues triggered a burst of hydrogen peroxide, abscisic acid, and jasmonates and up-regulated the expression of HbAOSa, which was associated with the secondary laticifer differentiation strictly limited to the wounded area. Application of exogenous hydrogen peroxide, methyl jasmonate, and polyethylene glycol 6000 (PEG6000) could induce the secondary laticifer differentiation, respectively. Moreover, 6-Benzylaminopurine, a synthetic cytokinin, enhanced the methyl jasmonate-induced secondary laticifer differentiation. However, the dehydration-induced secondary laticifer differentiation was inhibited by exogenous abscisic acid. Diphenyleneiodonium chloride (DPI), a specific inhibitor of NADPH oxidase, was effective in inhibiting the accumulation of hydrogen peroxide as well as of jasmonates upon dehydration. It blocked the dehydration-induced but not the methyl jasmonate-induced secondary laticifer differentiation. The results suggested a stress signal pathway mediating the wound-induced secondary laticifer differentiation in rubber tree.

Transcriptional and post-transcriptional regulation of the jasmonate signalling pathway in response to abiotic and harvesting stress in Hevea brasiliensis.

BackgroundLatex harvesting in Hevea brasiliensis amounts to strong abiotic stress that can cause a halt in production in the most susceptible clones. Although the role of jasmonic acid has been suggested in laticifer differentiation, its role in latex production and in the response to harvesting stress has received very little attention. Only a few key genes acting in the COI-JAZ-MYC module have been isolated and studied at transcriptional level.ResultsUse of a reference transcriptome obtained on rubber clone PB 260 covering a large number of tissues under different environmental conditions enabled us to identify 24 contigs implicated in the jasmonate signalling pathway in the rubber tree. An analysis of their expression profile by qPCR, combined with hierarchical clustering, suggested that the jasmonate signalling pathway is highly activated in laticifer cells and, more particularly, in the response to harvesting stress. By comparison with their genomic sequences, the existence of regulation by alternative splicing was discovered for JAZ transcripts in response to harvesting stress. Lastly, positive transcriptional regulation of the HbJAZ_1405 gene by MYC was demonstrated.ConclusionThis study led to the identification of all actors of jasmonate signalling pathway and revealed a specific gene expression pattern in latex cells. In-depth analysis of this regulation showed alternative splicing that has been previously shown in Arabidopsis. Interestingly, genotypic variation was observed in Hevea clones with contrasting latex metabolism. This result suggests an involvement of jasmonate signalling pathway in latex production. The data suggest that specific variability of the JA pathway may have some major consequences for resistance to stress. The data support the hypothesis that a better understanding of transcriptional regulations of jasmonate pathway during harvesting stress, along with the use of genotypic diversity in response to such stress, can be used to improve resistance to stress and rubber production in Hevea.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0341-0) contains supplementary material, which is available to authorized users.

Characterisation of recombinant Hevea brasiliensis allene oxide synthase: Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase ( AOS) cDNA and biochemical characterisation of the recombinant AOS (His 6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K m value of 4.02 ± 0.64 μM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His 6-HbAOS enzyme activity. Although JA had no effect on His 6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His 6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.

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

Abstract Laticifer differentiation of Hevea brasiliensis was investigated by application of lanolin containing jasmonic acid (JA) or other chemicals 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 significant increase in primary laticifer number but did not induce secondary laticifer differentiation. Secondary laticifer differentiation and a less significant increase in primary laticifer number were caused by JA application to the extended young stems. The induction of the secondary laticifers was dependent on the concentration of JA applied. Cambium cell division leading to the formation of secondary phloem was not accelerated by JA treatment. Treated bark tissues showed no visible changes except for the additional laticifers, which were normal in ultrastructure. The secondary laticifers were also induced by the application of linolenic acid, a precursor of JA biosynthesis. Abscisic acid, ethephon and salicylic acid had no detectable effect on laticifer differentiation.

In vitro Hormonal Regulation of Laticifer Differentiation in Calotropis procera

Cultures of Calotropis procera were maintained on MS medium supplemented with 4·6 μM FAP and 3 μM NAA. Laticifer initials were observed in 2-week-old cultures which were converted into matured, branched, non-articulated laticifers in 4 weeks. It was observed that laticifer differentiation increased with the age of cultures, from 2·78% (in the second passage) to 15·11% (in the 12th passage). It has been established that laticifer differentiation in vitro is a cytokinin-dependent process and among the cytokinins, FAP was more effective than BA and 2-iP. But the type of auxin and its concentration also play an important role in modifying the effect of cytokinin. Among the different auxins used IAA was more effective for laticifer differentiation than IBA and NAA, while 2,4D was inhibitory. Maximum laticifer differentiation (17·01% was observed on MS medium supplemented with 4·6 μM FAP and 1 μM IAA.

A β- d-fucosidase from Asclepias curussavica latex

A β- d-fucosidase was isolated from Asclepias curassavica latex by anion exchange and gel filtration chromatography. The enzyme was purified 136-fold. The enzyme is a monomer and its M r is close to 50 000. It shows optimal activity at pH 5.5 and at 50°. The enzyme hydrolyses p-nitrophenyl-β- d-fucopyranoside with apparent K m and V max values of 6.58 mM and 304 mM min − 1 mg − 1 of protein, respectively, at the optimum pH. p-Nitrophenyl-β- d-fucopyranoside is the best substrate. d(+)-fucose acts as competitive inhibitor with a K i value of 7.36 mM at pH 5.5. The hypothesis that β- d-fucosidase represents an enzymatic marker of laticifer differentiation and of the cell wall degradation process is discussed.

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

The distribution, cytological organization and differentiation of non-articulated laticifers in the primary and mature tissues of Calotropis gigantea (Linn.) R.Br., were studied by the use of optical and electron microscopy. Laticifers occur in the cortex, vascular bundle and pith of the plant axis. At the earliest detectable stage a laticifer is a cell which undergoes rapid elongation and nuclear division. This results in a multinucleate elongated cell which undergoes further increase in length with gradual degeneration of the cytoplasm

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

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

PLASTIDS IN LATICIFERS OF PAPAVER. I. DEVELOPMENT AND CYTOCHEMISTRY OF LATICIFER PLASTIDS IN P. SOMNIFERUM L. (PAPAVERACEAE)

Plastids were observed in all stages of laticifer differentiation in Papaver somniferum L. Plastids in laticifer initials were present as proplastids that later developed electron‐dense inclusions, but never possessed the thylakoids or starch grains that characterize chloroplasts in other cells. Electron‐dense inclusions in laticifer plastids were membrane‐bound and appeared to arise from the accumulation of material within an invagination of the inner plastid membrane. Cytochemical studies of these plastid inclusions indicated that their matrix was not composed of crystalline protein, α‐amylose, amylopectin or polysaccharide. The results suggest that the electron‐dense, membrane‐bound inclusions in laticifer plastids may be composed of lipoprotein.

Fine structure and development of laticifers inGnetum gnemon L.

Articulated laticifers are a regular component of stem cortex and pith ofGnetum gnemon L. Differentiation of laticifers starts very early and is completed within the very first centimeters adjacent to the apical meristem.

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

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

ONTOGENY AND CYTOCHEMISTRY OF ALKALOIDAL VESICLES IN LATICIFERS OF PAPAVER SOMNIFERUM L. (PAPAVERACEAE)

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide‐osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron‐dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle.

Ontogeny and Cytochemistry of Alkaloidal Vesicles in Laticifers of Papaver somniferum L. (Papaveraceae)

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide-osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron-dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle.