iTRAQ-Based Proteomics Analysis of Autophagy-Mediated Responses against MeJA in Laticifers of Euphorbia kansui L.

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. Methyl jasmonate (MeJA), a plant hormone, mediates diverse developmental process and defense responses which induce a variety of metabolites. In plants, little is known about autophagy-mediated responses against MeJA. In this study, we used high-throughput comparative proteomics to identify proteins of latex in the laticifers. The isobaric tags for relative and absolute quantification (iTRAQ) MS/MS proteomics were performed, and 298 proteins among MeJA treated groups and the control group of Euphorbia kansui were identified. It is interesting to note that 29 significant differentially expressed proteins were identified and their associations with autophagy and ROS pathway were verified for several selected proteins as follows: α-L-fucosidase, ß-galactosidase, cysteine proteinase, and Cu/Zn superoxide dismutase. Quantitative real-time PCR analysis of the selected genes confirmed the fact that MeJA might enhance the expression of some genes related to autophagy. The western blotting and immunofluorescence results of ATG8 and ATG18a which are two important proteins for the formation of autophagosomes also demonstrated that MeJA could promote autophagy at the protein level. Using the electron microscope, we observed an increase in autophagosomes after MeJA treatment. These results indicated that MeJA might promote autophagy in E. kansui laticifers; and it was speculated that MeJA mediated autophagy through two possible ways: the increase of ROS induces ATG8 accumulation and then aotophagosome formation, and MeJA promotes ATG18 accumulation and then autophagosome formation. Taken together, our results provide several novel insights for understanding the mechanism between autophagy and MeJA treatment. However, the specific mechanism remains to be further studied in the future.

Report: Pharmacognostic and physicochemical screening of Euphorbia nivulia Buch.-Ham.

Euphorbia nivulia Buch.-Ham. (Euphorbiaceae) is commonly known as Indian Spurge Tree in English, and "Saj Thor" or "Jhanami booti" in local language. The plant is used traditionally in the treatment of various diseases like inflammation, fever, worm infection, asthma, cough, wounds and diabetes. In current study fresh as well as dried aerial parts of the plant and cut sections were examined, both macroscopically and microscopically. The study also deals with fluorescence analysis and phytochemical characteristics and other WHO recommended methods for standardization. WHO guidelines on quality control for medicinal plants materials were used for pharmacognostical evaluation of E. nivulia, phytochemical screening helps in determining the predominant classes of active constituents responsible for the activity. The present work will be helpful in identification of the fresh and dried samples of aerial parts pharmacognostically and anatomically. These studies will serve as a reference for correct identification and may be helpful in checking any type of adulteration. These observations will also help in differentiating this species from closely related species of the same genus and family.

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.

Foldable structures and the natural design of pollen grains.

Upon release from the anther, pollen grains of angiosperm flowers are exposed to a dry environment and dehydrate. To survive this process, pollen grains possess a variety of physiological and structural adaptations. Perhaps the most striking of these adaptations is the ability of the pollen wall to fold onto itself to prevent further desiccation. Roger P. Wodehouse coined the term harmomegathy for this folding process in recognition of the critical role it plays in the survival of the pollen grain. There is still, however, no quantitative theory that explains how the structure of the pollen wall contributes to harmomegathy. Here we demonstrate that simple geometrical and mechanical principles explain how wall structure guides pollen grains toward distinct folding pathways. We found that the presence of axially elongated apertures of high compliance is critical for achieving a predictable and reversible folding pattern. Moreover, the intricate sculpturing of the wall assists pollen closure by preventing mirror buckling of the surface. These results constitute quantitative structure-function relationships for pollen harmomegathy and provide a framework to elucidate the functional significance of the very diverse pollen morphologies observed in angiosperms.

The Caribbean slipper spurge Euphorbia tithymaloides: the first example of a ring species in plants.

A ring species arises when a parental population expands around an area of unsuitable habitat in such a way that when the two fronts meet they behave as distinct species while still being connected through a series of intergrading populations. Ring species offer great possibilities for studying the forces causing species divergence (e.g. the nature of pre-zygotic or post-zygotic reproductive isolation) or helping to maintain species integrity (e.g. reinforcement). Yet, ring species are extremely rare, and have only been documented convincingly in animals. Here, we present phylogenetic analyses of two nuclear gene regions from the Caribbean slipper spurge (Euphorbia tithymaloides) species complex that provide evidence that this group forms a ring species. These data show that the species complex originated in the area where Mexico and Guatemala meet, and expanded around the Caribbean basin along two distinct fronts: one eastward through the Yucatan Peninsula and into the Greater Antilles (GA); one southeastward through northern South America and then northward to the Lesser Antilles and eastern GA. The two terminal forms co-occur in the Virgin Islands and appear to be morphologically and ecologically distinct. Thus, our results suggest that Euphorbia tithymaloides is the first compelling example of a ring species in plants.

Sequential cell wall transformations in response to the induction of a pedicel abscission event in Euphorbia pulcherrima (poinsettia).

Alterations in the detection of cell wall polysaccharides during an induced abscission event in the pedicel of Euphorbia pulcherrima (poinsettia) have been determined using monoclonal antibodies and Fourier transform infrared (FT-IR) microspectroscopy. Concurrent with the appearance of a morphologically distinct abscission zone (AZ) on day 5 after induction, a reduction in the detection of the LM5 (1-->4)-beta-D-galactan and LM6 (1-->5)-alpha-L-arabinan epitopes in AZ cell walls was observed. Prior to AZ activation, a loss of the (1-->4)-beta-D-galactan and (1-->5)-alpha-L-arabinan epitopes was detected in cell walls distal to the AZ, i.e. in the to-be-shed organ. The earliest detected change, on day 2 after induction, was a specific loss of the LM5 (1-->4)-beta-D-galactan epitope from epidermal cells distal to the region where the AZ would form. Such alteration in the cell walls was an early, pre-AZ activation event. An AZ-associated de-esterification of homogalacturonan (HG) was detected in the AZ and distal area on day 7 after induction. The FT-IR analysis indicated that lignin and xylan were abundant in the AZ and that lower levels of cellulose, arabinose and pectin were present. Xylan and xyloglucan epitopes were detected in the cell walls of both the AZ and also the primary cell walls of the distal region at a late stage of the abscission process, on day 7 after induction. These observations indicate that the induction of an abscission event results in a temporal sequence of cell wall modifications involving the spatially regulated loss, appearance and/or remodelling of distinct sets of cell wall polymers.

Multiple facets of laticifer cells.

In the latex-bearing plants, the laticiferous system is the tubing structure that contains the latex and is constituted of living cells (laticifers). While laticifers are present only in a small percentage of the flowering plant species, they represent a type of specialized tissue within the plant where a myriad of metabolites are synthesized, some of them of considerable commercial importance. In this mini-review we synopsize the present knowledge about laticifer cells and discuss about their particular features as well as some evolutionary and ecophysiological cues and the potential exploitation of the knowledge generated around this peculiar type of plant cell. We illustrate some of these questions with the experience in Euphorbia lathyris laticifers and latex.

Lathyrol diterpenes as modulators of P-glycoprotein dependent multidrug resistance: structure-activity relationship studies on Euphorbia factor L3 derivatives.

Five series of 37 new acylate and epoxide derivatives (3-39) of Euphorbia factor L3, a lathyrol diterpene isolated from Euphorbia lathyris, were designed by modifying the hydroxyl moiety of C-3, C-5, or C-15. Chemoreversal effects of the acylates on multidrug resistance (MDR) were evaluated in breast cancer multidrug-resistant MCF-7/ADR cells that overexpress P-glycoprotein (P-gp). Eight derivatives exhibited greater chemoreversal ability than verapamil (VRP) against adriamycin (ADR) resistance. Compounds 19 and 25 exhibited 4.8 and 4.0 times, respectively, more effective reversal ability than VRP against ADR resistance. To determine the key characteristics of Euphorbia factor L3 derivatives that contribute to MDR reversal, we conducted a structure-activity relationship study of these compounds. The simulation studies indicated different possible mechanisms and revealed the important influence of hydrophobic interactions and hydrogen bonds in the flexible cavity of P-gp.

[Pharmacognostical studies on the roots of Euphorbia stracheyi].

Morphological and histological characters of the roots of Euphorbia stracheyi Boiss., a traditional Chinese medicine, were described and illustrated with line drawings. TLC analysis of the above drug was also undertaken. These studies provide referencial information for clinics, quality control, development and identification of this crude drug.

[Pharmacognostical studies on the roots of Euphorbia fischeriana].

Morphological and histological characters of the roots of Euphorbia fischeriana Steud., used as one of the origins of traditional Chinese medicine Langdu, were described and illustrated with line drawings. TLC analysis of the above drug was also undertaken. These studies provide referencial informations for clinics, quality control, development and identification of this crude drug.

Expression of the gene for sterol-biosynthesis enzyme squalene epoxidase in parenchyma cells of the oil plant, Euphorbia tirucalli.

In plants, phytosterols and triterpenes are major secondary metabolites. In an attempt to reveal the mechanism for synthesis and storage of these compounds, we isolated and characterized cDNA clones for squalene epoxidase (SE), from a succulent shrub, Euphorbia tirucalli. Southern-blot analysis of total DNA using cDNA fragment as a probe showed that the E. tirucalli squalene epoxidase gene (EtSE) is single-copy type in terms of restriction fragment length polymorphism (RFLP). Deduced amino-acid sequence of the cDNA showed 83 and 75% identity to those of rice and ginseng, respectively, in an area excluding a less homologous putative transmembrane region in the N-terminal end. Functional characterization with heterologous expression using an erg1-disrupted yeast mutant KLN1 indicated that the EtSE recovered ergosterol auxotrophy of the mutant, and gave rise to an ergosterol accumulation in the EtSE transformant. RT-PCR analysis showed the EtSE transcripts in leaves and stem internodes accumulated in almost equal amounts, which were more abundant than those in roots. In situ hybridization using EtSE antisense probe revealed prominent EtSE expression on a parenchyma cell adjacent to primary laticifers that were located in a rosary orientation in the inner region of cortex. This is the first report of expression of a gene for a rate-limiting enzyme in mevalonate pathway in organs and tissues of a plant.

Localization of nonspecific lipid transfer proteins correlate with programmed cell death responses during endosperm degradation in Euphorbia lagascae seedlings.

When the storage materials have been depleted, the endosperm cells undergo programmed cell death. Very little is known about how the components of the dying cells are recycled and used by the growing seedling. To learn more about endosperm degradation and nutrient recycling, we isolated soluble proteins from the endosperm of Euphorbia lagascae seedlings collected 2, 4, and 6 d after sowing. The protein extracts were subjected to two-dimensional gel electrophoresis. Proteins that increased in amount in the endosperm with time were selected for further analysis with mass spectrometry. We successfully identified 17 proteins, which became more abundant by time during germination. Among these proteins were three E. lagascae lipid transfer proteins (ElLTPs), ElLTP1, ElLTP2, and ElLTP3. Detailed expressional studies were performed on ElLTP1 and ElLTP2. ElLTP1 transcripts were detected in endosperm and cotyledons, whereas ElLTP2 transcripts were only detected in endosperm. Western blots confirmed that ElLTP1 and ElLTP2 accumulate during germination. Immunolocalization experiments showed that ElLTP1 was present in the vessels of the developing cotyledons, and also in the alloplastic space in the endosperm. ElLTP2 formed a concentration gradient in the endosperm, with higher amounts in the inner regions close to the cotyledons, and lesser amounts in the outer regions of the endosperm. On the basis of these data, we propose that ElLTP1 and ElLTP2 are involved in recycling of endosperm lipids, or that they act as protease inhibitors protecting the growing cotyledons from proteases released during programmed cell death.