Timothy C. Baldwin

Traditional uses and potential health benefits of Amorphophallus konjac K. Koch ex N.E.Br.

Amorphophallus konjac (konjac) has long been used in China, Japan and South East Asia as a food source and as a traditional medicine. Flour extracted from the corm of this species is used in Far Eastern cuisine to make noodles, tofu and snacks. In traditional Chinese medicine (TCM), a gel prepared from the flour has been used for detoxification, tumour-suppression, blood stasis alleviation and phlegm liquefaction; and for more than 2000 years has been consumed by the indigenous people of China for the treatment of asthma, cough, hernia, breast pain, burns as well as haematological and skin disorders. Over the past two decades, purified konjac flour, commonly known as konjac glucomannan (KGM) has been introduced on a relatively small scale into the United States and Europe, both as a food additive and a dietary supplement. The latter is available in capsule form or as a drink mix and in food products. Clinical studies have demonstrated that supplementing the diet with KGM significantly lowers plasma cholesterol, improves carbohydrate metabolism, bowel movement and colonic ecology. Standards for the classification of both konjac flour and KGM have been established by the Chinese Ministry of Agriculture, the European Commission and the U.S. Food Chemicals Codex. However, to date, there is no worldwide agreed regulatory standard for konjac flour or KGM. This highlights the need for harmonization of konjac commercial standards to assess and ensure the quality of existing and future KGM products. Despite the widespread consumption of konjac derived products in East and South East Asia, there has been limited research on the biology, processing and cultivation of this species in the West. Most studies performed outside Asia have focussed on the structural characterisation and physicochemical properties of KGM. Therefore, the objective of this monograph is to review the literature covering the ethnic uses, botany and cultivation of konjac corms, together with the health benefits of KGM with the associated requirements for quality control. Possible directions for future research and development and standardisation of production and classification of this versatile natural product will be discussed.

Localisation and characterisation of cell wall mannan polysaccharides in Arabidopsis thaliana

Polysaccharides containing β-1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.

Abnormal Glycosphingolipid Mannosylation Triggers Salicylic Acid–Mediated Responses in Arabidopsis

We showed that a Golgi sugar nucleotide transporter (GONST1) is not required for polysaccharide biosynthesis as previously hypothesized. Instead, we found that GONST1 provides substrate for the glycosylation of an abundant class of sphingolipid. gonst1 plants are stunted and display a constitutive defense response, including elevated salicylic acid and hydrogen peroxide levels. The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi.

Distribution of lipid transfer protein 1 (LTP1) epitopes associated with morphogenic events during somatic embryogenesis of Arabidopsis thaliana

AbstractUsing immunocytochemical methods, at both the light and electron microscopic level, we have investigated the spatial and temporal distribution of lipid transfer protein 1 (LTP1) epitopes during the induction of somatic embryogenesis in explants of Arabidopsis thaliana. Immunofluorescence labelling demonstrated the presence of high levels of LTP1 epitopes within the proximal regions of the cotyledons (embryogenic regions) associated with particular morphogenetic events, including intense cell division activity, cotyledon swelling, cell loosening and callus formation. Precise analysis of the signal localization in protodermal and subprotodermal cells indicated that cells exhibiting features typical of embryogenic cells were strongly labelled, both in walls and the cytoplasm, while in the majority of meristematic-like cells no signal was observed. Staining with lipophilic dyes revealed a correlation between the distribution of LTP1 epitopes and lipid substances within the cell wall. Differences in label abundance and distribution between embryogenic and non-embryogenic regions of explants were studied in detail with the use of immunogold electron microscopy. The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon. The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed. Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

Differences in protodermal cell wall structure in zygotic and somatic embryos of Daucus carota (L.) cultured on solid and in liquid media

The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.

An apoplastic chitinase CpCHT1 isolated from the corolla of wintersweet exhibits both antifreeze and antifungal activities

The shrub Chimonanthus praecox L. (wintersweet) which is native to Chinese montane forests produces its flowers in the midst of winter. This indicates that the floral organs of this species are adapted to growth and development under freezing temperatures. Here, we report the isolation and preliminary characterisation of a 33 kDa apoplastic antifreeze chitinase (CpCHT1) from the petals and its corresponding cDNA. The chitinase activity of CpCHT1 was confirmed by activity staining. Antifreeze activity was validated in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. CpCHT1 was also found to affect the germination of fungal spores of four major plant pathogens. In addition, the gene and protein are expressed constitutively not only in flowers, but also in leaves, bark and root tissues. From these data we hypothesize that this protein is multifunctional and may protect wintersweet from freezing injury and provide nonspecific disease resistance.

Axenic Seed Germination and in Vitro Seedling Development of Restrepia brachypus (Orchidaceae)

Abstract Montane rain forests in Central and South America are threatened by human activities and climate change. Consequently, epiphytic plant genera such as Restrepia are also endangered, making their ex-situ conservation vital. For success, this conservation strategy requires affordable, efficient, and reliable protocols for axenic seed germination as well as protocorm and seedling development prior to establishment ex vitro. In our study, effects of four asymbiotic media (Murashige and Skooge, Phytotech P668, Vacin and Went, and Western) on seed germination and early protocorm development of Restrepia brachypus were compared. In addition, their effects with and without banana pulp were examined on in vitro seedling development. Western medium produced the highest mean germination rate (53%), the second highest mean protocorm diameter (325 µm) and, with banana, the largest mean seedling length (3.6 mm). These data provide a simple protocol using commercially available media that is suitable for ex-situ conservation of Restrepia. These media may also be of use for the micropropagation and conservation of other related orchid genera.

An Improved Method for the Fixation, Embedding and Immunofluorescence Labeling of Resin-Embedded Plant Tissue

The use of antibodies as direct probes for specific macromolecules in plant cells and tissue is a well-established and extremely powerful technique and is of particular use in the post-genomics era. In this paper, we present an improved fixation, embedding, and immunofluorescence technique suitable for fixing “difficult” plant tissues such as pistils and inflorescence stems, which possess many trichomes and a thick hydrophobic cuticle. The key modification of the fixative used in the current study was the addition of a small amount of sucrose, CaCl2, and detergent into a 4% (v/v) formaldehyde and 1% (v/v) glutaraldehyde mixture without the requirement to vacuum infiltrate. The modified immunofluorescence labeling method featured an amended blocking buffer, increased number of washing steps, and the use of an aqueous mounting medium which produced intense immunolabeling signals with extremely low background. Moreover, the immunocytochemistry methodology described in this study has proven to be suitable for use on two widely studied plant species, namely, Vicia faba and Arabidopsis thaliana, and may, therefore, be applicable for use in studies of a wide range of angiosperms.

Temporal and spatial regulation of glucomannan deposition and mobilization in corms of Amorphophallus konjac (Araceae)

PREMISE OF THE STUDY Konjac glucomannan (KGM), the main biologically active constituent of konjac flour extracted from corms of Amorphophallus konjac (konjac), has potential to be used as a nutraceutical (satiety agent) to combat obesity. Here we present the results of an immunocytochemical investigation of the developmental regulation of the deposition and mobilization of glucomannan in corm tissues of konjac, using an antiheteromannan (mannan/glucomannan) antiserum. METHODS The intensity of antibody binding to glucomannan idioblasts at six developmental stages (i.e., dormancy, leaf bud emergence, leaf bud elongation, leaflet emergence, leaf expansion, and shoot senescence) was compared. KEY RESULTS A temporally regulated pattern of glucomannan deposition and mobilization within the glucomannan idioblasts was observed. A source-sink transition in the corm was shown to occur after leaflet emergence, prior to complete expansion of the leaves. Our data also suggest that the mobilization of KGM initiates at the periphery of the corm and proceeds inward toward the center of the corm. CONCLUSIONS This study represents a significant milestone in our understanding of the mechanisms involved in the physiological and biochemical control of KGM biosynthesis, partitioning, storage, and remobilization. Moreover, this information and the methodology presented provide valuable data for future improvement of the yield and productivity of this important crop.

Investigating the Postmortem Molecular Biology of Cartilage and its Potential Forensic Applications

This study investigated the postmortem molecular changes that articular cartilage undergoes following burial. Fresh pig trotters were interred in 30‐cm‐deep graves at two distinct locations exhibiting dissimilar soil environments for up to 42 days. Extracts of the metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joint cartilage from trotters disinterred weekly over 6 weeks were analyzed by Western blot against the monoclonal antibody 2‐B‐6 to assess aggrecan degradation. In both soil conditions, aggrecan degradation by‐products of decreasing molecular size and complexity were observed up to 21 days postmortem. Degradation products were undetected after this time and coincided with MCP/MTP joint exposure to the soil environment. These results show that cartilage proteoglycans undergo an ordered molecular breakdown, the analysis of which may have forensic applications. This model may prove useful for use as a human model and for forensic investigations concerning crimes against animals and the mortality of endangered species.