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.

Detection of autophagy processes during the development of nonarticulated laticifers in Euphorbia kansui Liou.

MAIN CONCLUSION: Autophagy is involved in cytoplasmic degradation through directly engulfing cytosol and organelles by autophagosomes and then fusing with lysosome-like vesicles during the development of nonarticulated laticifers in Euphorbia kansui Liou. Autophagy has been reported to play an important role in a wide range of eukaryotic organisms during responses to various abiotic and biotic stresses. However, until recently, the functions of autophagy in normal plant differentiation and development were still in their infancy. Nonarticulated laticifers, a type of secretory tissue in plants, undergo the degradation of cytosol and organelles during their development. However, little evidence of autophagy in laticifer differentiation has been provided. In the present study, using anti-ATG8 antibody-Alexa Fluor 488, Lyso-Tracker Red (LTR) and monodansylcadaverine (MDC) as markers for detecting autophagosomes, as well as autophagy-related structures, we observed that the green fluorescence of ATG8a largely colocalized with the red fluorescence of LTR and purple fluorescence of MDC and the quantity of autophagosomes experienced a trend from less to more to less during laticifer development. Additionally, we described the autophagy process during the development of nonarticulated laticifers in Euphorbia kansui Liou at the ultrastructural level in detail. In addition, further immunogold TEM studies also verified the presence of autophagosomes, autolysosomes and lysosome-like structures in laticifers. Taken together, these results suggest that autophagy contributes to the development of the nonarticulated laticifers in E. kansui Liou and that autophagosomes fuse with lysosome-like structures for degradation. These results will lay an important foundation for further studies on laticifer regulation.

The occurrence of C(2) photosynthesis in Euphorbia subgenus Chamaesyce (Euphorbiaceae).

This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C(3)-C(4) intermediate species utilizing C(2) photosynthesis, the process where photorespired CO(2) is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C(4), but three species in subsection Acutae (E. acuta, E. angusta, and E. johnstonii) have C(3) isotopic ratios. Phylogenetically, subsection Acutae branches between basal C(3) clades within Euphorbia and the C(4) clade in subgenus Chamaesyce. Euphorbia angusta is C(3), as indicated by a photosynthetic CO(2) compensation point (Г) of 69 µmol mol(-1) at 30 °C, a lack of Kranz anatomy, and the occurrence of glycine decarboxylase in mesophyll tissues. Euphorbia acuta utilizes C(2) photosynthesis, as indicated by a Г of 33 µmol mol(-1) at 30 °C, Kranz-like anatomy with mitochondria restricted to the centripetal (inner) wall of the bundle sheath cells, and localization of glycine decarboxlyase to bundle sheath mitochondria. Low activities of PEP carboxylase, NADP malic enzyme, and NAD malic enzyme demonstrated no C(4) cycle activity occurs in E. acuta thereby classifying it as a Type I C(3)-C(4) intermediate. Kranz-like anatomy in E. johnstonii indicates it also utilizes C(2) photosynthesis. Given the phylogenetically intermediate position of E. acuta and E. johnstonii, these results support the hypothesis that C(2) photosynthesis is an evolutionary intermediate condition between C(3) and C(4) photosynthesis.

Authentication of the 31 species of Toxic and Potent Chinese Materia Medica (T/PCMM) by microscopic technique, part 2: Three species of seed T/PCMM.

Toxic and Potent Chinese Materia Medica (T/PCMM) are being used more and more in the treatment of various diseases. In view of their toxic side effects and to ensure their safe use, accurate and reliable authentication is indispensable. However, identifying characteristics of T/PCMM are seldom reported, even though modern microscopy can provide ample, unique identifying characteristics from cells found in transverse sections and powders. In particular, no systematic authentication studies on seed T/PCMM have been conducted. In the course of our study on 31 T/PCMM originating from plants, animals, minerals, and secreta, an accurate and convenient method, based on microscopic techniques, has been developed and reported for the authentication of animal T/PCMM. The present study deals with detailed investigations on three species of seed T/PCMM, namely Semen Hyoscyami (Hyoscyamus niger L.), Semen Euphorbiae (Euphorbia lathyris L.), and Semen Strychni (Strychnos nux-vomica L.). The macroscopic characters are here described in detail, and the microscopic characters were conclusively determined by common and polarized light microscopy. Results showed that these three T/PCMM can be easily identified by the present method even when powdered and combined. Thus, the microscopic method is applicable for authentication of the earlier three T/PCMM, and the morphological and microscopic characteristics described here are proposed as parameters to establish the authenticity of these three T/PCMM.

Characterisation of surface wettability based on nanoparticles.

Nanoparticles are becoming frequently used in the research area of creating functional surfaces because they can be more versatile than just making dimensions smaller. Particularly, a variety of nanoparticles have been applied for the construction of superhydrophobic and superhydrophilic surfaces with micro- and nano-scaled structures. As nanoparticles can also be fashioned and modified, their effects will be of great importance to the formed surface structures. In the present paper, we review the recent research progress in the utilization of nanoparticles to form extremely wettable/non-wettable surface structures and their influence on surface wettability. This report manifests an apparent inclination of nanoparticle structured surfaces using the multidisciplinary approaches, from the viewpoint of engineer/scientist. Therefore, the typical methodologies with regard to the use of nanoparticles, including the preparation and functionalisation processes, for the realization of surface wettabilities are discussed in this work. The discussions also represent some of the size-determined phenomena that are related to wettable/non-wettable surfaces. This Review thus provides an insight into the connection between nanoparticles and surface wettability.

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou.

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.

Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM).

The cuticle of terrestrial vascular plants and some bryophytes is covered with a complex mixture of lipids, usually called epicuticular waxes. Self-assembly processes of wax molecules lead to crystalline three-dimensional micro- and nanostructures that emerge from an underlying wax film. This paper presents the first AFM study on wax regeneration on the surfaces of living plants and the very early stages of wax crystal formation at the molecular level. Wax formation was analysed on the leaves of Euphorbia lathyris, Galanthus nivalis, and Ipheion uniflorum. Immediately after wax removal, regeneration of a wax film began, consisting of individual layers of, typically, 3-5 nm thickness. Subsequently, several different stages of crystal growth could be distinguished, and different patterns of wax regeneration as well as considerable variation in regeneration speed were found.