Differential responses of Hollyhock (Alcea rosea L.) varieties to salt stress in relation to physiological and biochemical parameters.

The response of 14 Hollyhock (Alcea rosea L.) varieties to salinity were evaluated in a field experiment over two growing seasons. Carotenoid, Chl a, Chl b, total Chl, proline and MDA content, CAT, APX and GPX activity and petal and seeds yields were determined in order to investigate the mechanism of salt tolerance exhibited by Hollyhock, and too identify salt tolerant varieties. Overall, the photosynthetic pigment content,petal and seed yields were reduced by salt stress. Whereas the proline and MDA content, and the CAT, APX and GPX activities increased as salt levels increased. However, the values of the measured traits were dependent upon the on the level of salt stress, the Varietie and the interaction between the two variables. Based upon the smallest reduction in petal yield, the Masouleh variety was shown to be the most salt tolerant, when grown under severe salt stress. However, based upon the smallest reduction in seed yield, Khorrmabad was the most tolerant variety to severe salt stress. These data suggest that the selection of more salt tolerant Hollyhock genotypes may be possible based upon the wide variation in tolerance to salinity exhibited by the varieties tested.

Evaluation of the Effect of Plectranthus amboinicus L. Leaf Extracts on the Bacterial Antioxidant System and Cell Membrane Integrity of Pseudomonas aeruginosa PA01 and Staphylococcus aureus NCTC8325.

Plectranthus amboinicus (Indian borage) has been extensively studied for its medicinal properties, which can be exploited to develop new antimicrobial therapeutics. The current study investigated the effect of Plectranthus amboinicus leaf extracts on the catalase activity, reactive oxygen species, lipid peroxidation, cytoplasmic membrane permeability, and efflux pump activity in S. aureus NCTC8325 and P. aeruginosa PA01. As the enzyme catalase protects bacteria against oxidative stress, disruption of its activity creates an imbalance in reactive oxygen species (ROS) levels, which subsequently oxidizes lipid chains, leading to lipid peroxidation. In addition, bacterial cell membranes are a potential target for new antibacterial agents, as efflux pump systems play a crucial role in antimicrobial resistance. Upon exposure of the microorganisms to Indian borage leaf extracts, the observed catalase activity decreased by 60% and 20% in P. aeruginosa and S. aureus, respectively. The generation of ROS can cause oxidation reactions to occur within the polyunsaturated fatty acids of the lipid membranes and induce lipid peroxidation. To investigate these phenomena, the increase in ROS activity in P. aeruginosa and S. aureus was studied using H2DCFDA, which is oxidized to 2',7'-dichlorofluorescein (DCF) by ROS. Furthermore, the concentration of lipid peroxidation product (malondialdehyde) was assessed using the Thiobarbituric acid assay and was shown to increase by 42.4% and 42.5% in P. aeruginosa and S. aureus, respectively. The effect of the extracts on the cell membrane permeability was monitored using diSC3-5 dye and it was observed that the cell membrane permeability of P. aeruginosa increased by 58% and of S. aureus by 83%. The effect on efflux pump activity was investigated using Rhodamine-6-uptake assay, which displayed a decrease in efflux activity of 25.5% in P. aeruginosa and 24.2% in S. aureus after treatment with the extracts. This combination of different methods to study various bacterial virulence factors provides a more robust, mechanistic understanding of the effect of P. amboinicus extracts on P. aeruginosa and S. aureus. This study thus represents the first report of the assessment of the effect of Indian borage leaf extracts on bacterial antioxidant systems and bacterial cell membranes, and can facilitate the future development of bacterial resistance modifying agents derived from P. amboinicus.

Evaluation of the Effect of Leaf Development in Plectranthus amboinicus L. on Antimicrobial Activity and Virulence Factors of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus NCTC8325.

Plectranthus amboinicus is widely recognized as a potential source of antimicrobial compounds due to the presence of bioactive components (essential oils) secreted by the glandular trichomes borne on the leaves. As such, an understanding of the effect of leaf development on the production of these essential oils (EOs) is of crucial importance to its medicinal applications. The current study represents the first comparative investigation of the effect of different stages of leaf development (lag, log, and stationary phase) upon the yield and bioactivity of phytochemicals produced. The effects of leaf extracts on the antimicrobial activity, cell surface hydrophobicity, biofilm formation, and motility of P. aeruginosa and Staphylococcus aureus were evaluated. Cryo-scanning electron microscopy was used to record the abundance and distribution of both glandular and non-glandular trichomes during leaf development. Gas chromatography-mass spectrometry analysis revealed that the potent phytochemical thymol is present primarily in log (30.28%) and stationary phase (20.89%) extracts. Log phase extracts showed the lowest minimum inhibitory concentration (25 mg/ml) when compared to other phases of development. Stationary phase extracts were shown to exhibit the highest biofilm dispersal activity against P. aeruginosa (80%), and log phase extracts against biofilms of S. aureus (59%). Log phase extracts showed the highest biofilm inhibitory activity against P. aeruginosa (66%) and S. aureus (63%). In conclusion, log phase leaf extracts of P. amboinicus exhibited a multimodal mechanism of action by displaying antimicrobial, antibiofilm activities and reducing the motility and hydrophobicity, which are important virulence factors in P. aeruginosa and S. aureus pathogenesis.

Effective Control against Broadleaf Weed Species Provided by Biodegradable PBAT/PLA Mulch Film Embedded with the Herbicide 2-Methyl-4-Chlorophenoxyacetic Acid (MCPA).

Biodegradable mulches are considered a promising alternative to polyethylene-based, nonbiodegradable mulch for sustainable agriculture. In the present study, a bioactive 2-methyl-4- cholorophenoxyacetic acid/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (MCPA-PHBV) conjugate blended with biodegradable poly(butylene adipate-co-terephthalate/polylactide (PBAT/PLA) was developed and used as mulch under controlled condition greenhouse pot experiment with fava bean (Vicia faba) as the nontarget crop species. The objectives were to examine the effectiveness of sustained-release of MCPA herbicide from biodegradable mulch for broadleaf weed suppression and to assess any adverse effects of the herbicide on the nontarget species (fava bean). The energy-dispersive X-ray spectroscopy analysis (EDS) suggests that a substantial quantity of the herbicide was released from the biodegradable mulch which effectively killed the broadleaf weed species even at 1% MCPA concentration. However, the higher concentrations of the herbicide adversely affected several physiological parameters of fava bean growth and development. Stomatal conductance decreased, while leaf temperature subsequently rose (at MCPA concentrations 5, 7.5, and 10%). The quantum yield of the Photosystem II (PSII) indicates that the photosynthetic efficiency was also restricted at concentrations 7.5% and 10%. Evidently, this slow-release herbicide system worked efficiently for broadleaf weed control but at higher concentrations, resulted in adverse physiological effects on the nontarget crop species. This study has demonstrated that biodegradable mulches containing MCPA herbicide are able to effectively inhibit the growth of broad leaf weed species and may be of potential importance in a wide variety of horticultural and agricultural applications.

Biodegradable PBAT/PLA Blend with Bioactive MCPA-PHBV Conjugate Suppresses Weed Growth.

The herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) conjugated with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was prepared via a melt transesterification route. The resultant bioactive oligomer was then mixed with a blend of polylactide (PLA) and poly(butylene adipate-co-terephthalate) (PBAT) with different loadings to manufacture films to be used as a bioactive, biodegradable mulch to deliver the herbicide to target broadleaf weed species. The biological targeting of the MCPA-PHBV conjugate in the mulch film was investigated under glasshouse conditions using faba bean (Vicia faba) as a selective (nontarget) model crop species having broadleaf morphology. The presence of the MCPA-PHBV conjugate in the biodegradable PBTA/PLA blend was shown to completely suppress the growth of broadleaf weed species while displaying only a mild effect on the growth of the model crop. The degradation of the mulch film under glasshouse conditions was quite slow. The release of the MCPA-PHBV during this process was detected using NMR, GPC, EDS, and DSC analyses, indicating that the majority of the MCPA diffused out after MCPA-PHBV conjugate bond scission. These data provide a strong "proof of concept" and show that this biodegradable, bioactive film is a good candidate for future field applications and may be of wide agricultural applicability.

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.

An investigation of self-incompatibility within the genus Restrepia.

UNLABELLED: • PREMISE OF THE STUDY: The genus Restrepia (Orchidaceae) is indigenous to montane rain forests of Central and South America. Recently, as habitat has fragmented and wild populations dwindled, the chances for successful cross-pollination within the genus have been reduced. Since cultivated species of Restrepia have been vegetatively propagated, they remain genetically close to those in the wild, making ex situ collections of the genus useful model populations for investigating breeding systems. Restrepia are found in clade B of the Pleurothallidinae, the only clade in which self-incompatibility (SI) has not yet been confirmed. In the current study, private collections of Restrepia were used to study the operation of SI within the genus to assist future ex situ conservation of this and related genera.• METHODS: A variety of self-pollination, intraspecific, and interspecific crosses were performed across the genus, and pollen tube growth was studied.• KEY RESULTS: Individual species exhibited varying degrees of SI. Self-pollinations performed across 26 species in the genus produced few viable seeds, with the exception of R. aberrans. Viable "filled" seeds with embryos were shown to require an intraspecific cross. Primary hybrids between species produced >90% seeds with embryos that germinated well.• CONCLUSIONS: The type of SI operating within the genus was considered to be best explained by gametophytic self-incompatibility (GSI) with interspecific variation in its phenotypic expression. The implications of these findings are discussed in relation to SI in the Pleurothallidinae and conservation strategies for Restrepia and related genera.

Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis.

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.

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.

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

Using 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.

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 beta-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.