Assay of proteins by Lowry's method in the presence of high concentrations of β-mercaptoethanol

β-Mercaptoethanol interferes in the determination of protein by the Lowry method (1–6). The interference can be overcome by the precipitation of proteins by trichloroacetic acid or acetone or by the use of H₂O₂ which oxidizes the sulfhydryl groups of β-mercaptoethanol (5). Both these methods have inherent disadvantages. Ross and Schatz (5) described a procedure for protein determination in the presence of high concentrations of β-mercaptoethanol where they removed the interference by the addition of iodoacetate. But addition of iodoacetate decreased the sensitivity of the reaction. The removal of interference by β-mercaptoethanol by heating has also been reported (3), but we observed that this procedure is not feasible when a large amount of β-mercaptoethanol is present in the protein samples. In the method reported in this communication, we made use of vacuum drying for the removal of interference by β-merceptoethanol. This method is simple, sensitive, takes less time, and can be used for the determination of protein in the presence of β-mercaptoethanol at levels as high as 10% in a sample volume of 1.0 ml (1.43 mmol) without using any additional chemical steps.     

Increased Cuticle Permeability Caused by a New Allele of ACETYL-COA CARBOXYLASE1 Enhances CO2 Uptake

Carbon dioxide (CO₂) is an essential substrate for photosynthesis in plants. CO₂ is absorbed mainly through the stomata in land plants because all other aerial surfaces are covered by a waxy layer called the cuticle. The cuticle is an important barrier that protects against extreme water loss; however, this anaerobic layer limits CO₂ uptake. Simply, in the process of adapting to a terrestrial environment, plants have acquired drought tolerance in exchange for reduced CO₂ uptake efficiency. To evaluate the extent to which increased cuticle permeability enhances CO₂ uptake efficiency, we investigated the CO₂ assimilation rate, carbon content, and dry weight of the Arabidopsis (Arabidopsis thaliana) mutant excessive transpiration1 (extra1), whose cuticle is remarkably permeable to water vapor. We isolated the mutant as a new allele of ACETYL-COA CARBOXYLASE1, encoding a critical enzyme for fatty acid synthesis, thereby affecting cuticle wax synthesis. Under saturated water vapor conditions, the extra1 mutant demonstrated a higher CO₂ assimilation rate, carbon content, and greater dry weight than did the wild-type plant. On the other hand, the stomatal mutant slow-type anion channel-associated1, whose stomata are continuously open, also exhibited a higher CO₂ assimilation rate than the wild-type plant; however, the increase was only half of the amount exhibited by extra1. These results indicate that the efficiency of CO₂ uptake via a permeable cuticle is greater than the efficiency via stomata and confirm that land plants suffer a greater loss of CO₂ uptake efficiency by developing a cuticle barrier.

To absorb carbon dioxide (CO₂) for photosynthesis, land plants expose their wet surfaces to a dry atmosphere and suffer evaporative water loss as a consequence (Hall et al., 1993). As too much water loss would result in dehydration, plants cover most of their aerial surfaces with a relatively impermeable layer, called the cuticle, and take in CO₂ mainly through stomatal pores, which make up only about 2% per a leaf area (Willmer and Fricker, 1996). In other words, the cuticle provides drought tolerance to plants in exchange for reduced efficiency in CO₂ uptake.The cuticle is a continuous membrane consisting of a polymer matrix (cutin), polysaccharides, and organic solvent‐soluble lipids (cuticular waxes; Holloway, 1982; Jeffree, 1996; Riederer and Schreiber, 2001). The cuticle is an important structure to protect plants against excess drought, high temperature, strong UV radiation, pathogens, and harmful insects (Kerstiens, 1996a, 1996b; Burghardt and Riederer, 2006; Riederer and Müller, 2006; Domínguez et al., 2011; Yeats and Rose, 2013). The cuticle limits the transpiration through plant surfaces other than through the stomatal pores to <10% of the total (Mohr and Schopfer, 1995). On the other hand, this impermeable layer also strongly restricts CO₂ influx. Boyer et al. (1997) and Boyer (2015a, 2015b) reported a lower conductance for CO₂ than for water vapor in cuticles of intact leaves of grape (Vitis vinifera) and sunflower (Helianthus annuus) due to the differences in molecular size and diffusion paths between the two gases. However, although many studies have explored the water permeability of cuticles in various conditions and species (Kerstiens, 1996a; Riederer and Müller, 2006; Kosma et al., 2009; Schreiber and Schönherr, 2009), much less attention has been directed to CO₂, despite its substantial role in photosynthesis.In this study, we verified the hypothesis that plants could absorb CO₂ more efficiently under non-drought stress conditions if their cuticles are more permeable. In addition, we also investigated the extent to which a permeable cuticle can enhance CO₂ uptake efficiency. To verify the hypothesis, we investigated whether the CO₂ uptake efficiency is increased in a mutant with a high cuticle permeability. For this research, we isolated an Arabidopsis (Arabidopsis thaliana) mutant named excessive transpiration1 (extra1), which exhibited marked evaporative water loss due to an increased cuticle permeability caused by a new allele of ACETYL-COA CARBOXYLASE1 (ACC1). ACC1 encodes a critical enzyme for the synthesis of malonyl-CoA, an essential substrate for fatty acid synthesis (Baud et al., 2003). To evaluate CO₂ uptake efficiency, we investigated CO₂ assimilation rate, carbon content, and dry weight of the extra1 mutant and compared them to that of wild-type plants as well as that of another mutant, slow-type anion channel-associated1 (slac1) with continuously open stomata (Negi et al., 2008; Vahisalu et al., 2008). Our results reveal that the increased cuticle permeability strongly and constantly enhances CO₂ uptake efficiency under non-drought stress conditions.     

16S rRNA PCR followed by restriction endonuclease digestion: A rapid approach for genus level identification of important enteric bacterial pathogens

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated.     

Responses to drought induced oxidative stress in five finger millet varieties differing in their geographical distribution

The study presents the impact of drought stress on five finger millet varieties (PR202, VL146, VL315, PES400 and VR708), representing contrasting areas of Indian sub-continent. Drought stress induced increase in the activity of superoxide dismutase, ascorbate peroxidase and glutathione reductase was higher in PR202 and VL315, while the activity was lower in the varieties PES400 and VR708. Ascorbate peroxidase : superoxide dismutase ratio, which is a crucial factor in alleviating drought stress, was higher in varieties PR202 and VL315, whilst the varieties PES400 and VR708 exhibited a lower ratio under stress. The variety PES400 recorded maximum stress induced damage, as indicated by higher accumulation of malondialdehyde and hydrogen peroxide; whereas the variety PR202 recorded least stress induced cytotoxic damage. The results clearly indicate that better drought tolerance of the variety PR202 is positively related to the capacity of its antioxidant system to scavenge reactive oxygen species, resulting in a reduced incidence of oxidative damage. Ascorbate peroxidase : superoxide dismutase ratio is found to be a critical factor governing the stress tolerance potential of different varieties. Therefore, varieties PR202 and VL315 were found to be tolerant while PES400 was susceptible to drought stress.     

Leaf and bud demography and shoot growth in evergreen and deciduous trees of central Himalaya, India

Leaf and bud demography and shoot growth were studied in 10 evergreen (ES) and 15 deciduous (DS) tree species occurring between 600 and 2200 m elevation in the central Himalayan mountains in India. Results were analyzed to help explain why ES prevail in the vegetation of this region, even though the number of ES is no greater than for DS. Although each species had its own pattern with regard to leaf and bud demography and seasonality of shoot extension and radial growth, it was possible to group the species on the basis of shoot growth phenology. In most species, leaves emerged during March-April, at the onset of warm and dry summer season. The ES recruit leaves in shoots more rapidly than the DS. Across all species, peak number of leaves per shoot (5.8–20.7), peak leaf area per shoot (116.2–1559.2 cm²), peak number of vegetative buds per shoot (1.9–14.5), bud survival per shoot (23–84%), shoot extension growth (6.4–40.8 cm) and shoot extension period (13–30 weeks) varied considerably. The peak leaf area per shoot (587.7 vs. 246.7 cm²) and shoot extension growth (19.3 vs. 11.2 cm) were significantly greater for DS than for ES, and these two functional groups of species were clearly separable with regard to shoot growth characteristics.Results indicate that rapid recruitment of leaf crop in the shoots, longer leaf life-span, and access to ground water due to deep roots were some of the advantages, the ES had over the DS, that may have likely enable them to maintain growth for a longer period in this region of warm winters and longer winter day length as compared to temperate climates. In the shallow rooted DS, shoot growth seems to be much affected by a seasonal drought in winter and they are likely to be affected more in the event of failure of monsoon rains in this region.