Identification of histone demethylases in Saccharomyces cerevisiae

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.     

Investigation of alpha-synuclein fibril structure by site-directed spin labeling

The misfolding and fibril formation of alpha-synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of alpha-synuclein fibrils. We generated fibrils from a total of 83 different spin-labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of alpha-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of alpha-synuclein fibrils must therefore be arranged in a parallel, in-register structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62-67 located at the beginning of the NAC region, as well as the N- and C-terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential beta-sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.     

Reproduction strategy of Trias verrucosa (Orchidaceae) from China

Trias, an orchid genus, was reported in this paper from China as a newly recorded genus for the first time. The genus and its new species, T. verrucosa Z. J. Liu, L. J. Chen et S. P. Lei, were described and discussed. Trias consists of 19 species ranging from Assam of India to Indonesia and Australia in the southeast via Myanmar to Deccan of India in the southwest. Its occurrence in West Yunnan of China is a further indication of phytogeographical relations between this region and the tropical Asia. This new species grows on shady rocks in forests. By observing of its biological characteristics such as phenology and blooming biology, lots of cloned ramets of generation overlap were found, but there were no fruited plants, and that clonal reproduction could repeat had nothing to do with whether the ramet bloomed or not. The flowering season of this species is from early April to early May. The opening of flowers on plants or inflorescence was irregular and the florescence of single flowers was rather short, only lasting 4–5 d, and no fruited flowers were found. Based on the detection of mating systems, no flowers of artificial self-pollination and artificial cross-pollination have fruited. Since the flowering period is just before the rainy season in this region, the ecological conditions of dryness, strong wind, low temperature and weak light during that period of time are evidently unfavorable to plant blooming, pollinating and fruiting. Apparently, the short duration and sterility of each flower would avoid the invalid energy waste in unfavorable circumstances and save the limited energy for more valid asexual reproduction so that the opportunity of multiplication in an uncertain environment would be increased to ensure the progenitive success. The P/O value of T. verrucosa is 187.4 ± 22.4, which is obviously related with the highly valid usage of its conglutinated and hard tuberous pollinarium during pollination, indicating that this species could hold the characteristic of sexual reproduction. This is an adaptation of this type of plants to the rather atrocious ecological circumstance there by its reproduction strategy of strengthening asexual reproduction and weakening sexual reproduction, and so it enables the plants to survive in this region, long-distanced from the places where its relatives dwell.     

Impact of allelopathic rice seedlings on rhizospheric microbial populations and their functional diversity

Field performance of rice allelopathic potential is indirectly regulated by the microflora in the rhizosphere. The present study aimed to investigate the dynamics of microbial populations and their functional diversities in the seedling rhizospheres of rice cultivars with varied allelopathic activities by employing agar plate bioassay, fumigation and BIOLOG analysis. Rice cultivars significantly affected the microbial carbon content in their associated rhizospheric soil. The microbial carbon contents were ranked in a decreasing order as Iguape Cateto (441.0 mg·kg^–1) > IAC47 (389.7 mg·kg^–1) > PI312777 (333.2 mg·kg^–1) > Lemont (283.8 mg·kg^–1) with the nil-rice control soil of 129.3 mg·kg^–1. Similarly, the respiration rate of the soils was 1.404, 1.019, 0.671 and 0.488 μgC·g^–1· h^–1 for PI312777, Iguape Cateto, IAC47 and Lemont, respectively. The respiration rate was only 0.304 μ gC·g^–1·h^–1 for the control soil. The microbial flora in the rhizospheric soil of different rice cultivars was dominated by bacteria (58.4%–65.6%), followed by actinomycete (32.2%–39.4%) and fungi (2.2%–2.8%). BIOLOG analysis showed that the value of Average Well Color Development (AWCD) differed significantly among rice cultivars. It was always the highest in the rhizospheric soil of the strongly allelopathic rice cv. PI312777, and the lowest in the rhizospheric soil of the poorly allelopathic rice cv. Lemont. The AWCD value reached the maximum in all the sampled soils after 144 hours of incubation. The AWCD values from the rhizospheric soils of PI312777, IAC47, Iguape Cateto and Lemont were 1.89, 1.79, 1.60 and 1.43 times higher than that of the control soil. Principal Component Analysis (PCA) identified 3 principal component factors (PCF) in relation to carbon sources, accounting for 70.1%, 11.3% and 7.0% of the variation, respectively. 19 categories of carbon sources were significantly positively correlated to the 3 principal components. Phenolic acids, carbohydrates, amino acids and amides were significantly correlated to the principal component 1, phenolic acids, carbohydrates and fatty acids to the principal component 2, and carbohydrates and hydroxylic acids to the principal component 3. Amino acids and amides were the two main carbon sources separating the 3 principal component factors. In addition, the total microbial population in the rhizospheric soil was significantly positively correlated with AWCD, microbial biomass carbon, microbial respiration and Shannon index. There was a significantly positive correlation between the total microbial population and the inhibition rate (IR) on the root length of lettuce owing to the different allelopathic activities of the rice cultivars. These results suggest that changes in microbial population, activity and functional diversity in the rhizospheres are highly cultivar-dependent. These changes might play an important role in governing the rice allelopathic activity in the field.     

Physiological adaptability of three mangrove species to salt stress

The impact of salinity on three arboreal mangrove plants, Sonneratia apetala (Sa), S. caseolaris (Sc) and Rhizophora stylosa (Rs), was studied. The three mangrove species were treated with different salinity levels over a three-month period. The response and adaptation of these three mangrove species to salinity were shown to be different. Net photosynthesis rate, stomata conductance and transpiration rate of leaves decreased and soluble sugar content in leaves increased, with salt concentration in all three mangrove species. The malondial dehyde (MDA) content in stems and leaves of Sa and Sc somewhat decreased when the salinity was lower than 10, but rapidly increased with increasing salt concentration. The MDA content in stems and leaves of Rs increased only when salinity was greater than 40. No changes were observed in the MDA content of roots in the three mangrove species. The adaptabilities of Sa and Sc to salt tolerance were limited. The more salt tolerant the mangrove Rs, the more likely the free oxygen radicals were eliminated through the increase in activity of superoxide dismutase (SOD). Results of this experiment identified salinity levels best suited for the growth and metabolism of the species, which provides information necessary for maintaining mangrove forestation along the South China coast.     

Drought-resistant physiological characteristics of four shrub species in arid valley of Minjiang River, China

The formation of 161 km-long arid valley (170 km²) of Mingjiang River is attributed to the effect of foehn. The arid valley presents harsh conditions for the growth of vegetation, which include higher annual evaporation and transpiration rate (1400–2000 mm), lower annual precipitation rate (400–700 mm), steep slope, and infertile soils. Although the vegetation is dominated by xerophilous bushes and grasses, it plays a significant role in soil and water conservation in watershed and provides important references in the arid valley as it is very difficult to grow trees. Drought-resistant physiological characteristics of four native species, i.e., Sophora davidiana, Bauhinia faberi var. microphylla, Convolvulus tragacanthoides, and Artemisia gmelinii, have been measured. It was found that the content of chlorophyll, free proline and soluble sugars showed an increase, whereas evaporation ratio, WSD, RWC and cytolemma osmosis showed a decrease under drought stress. Integrated assessment of the four species indicated that the integrated drought resistance index was the highest in July (0.507), followed by September, August and June, whereas it was the lowest in May (0.442). The result provided a basis for vegetation restoration, especially it improved survival of planted species in May and June when plant species exhibit the weakest drought resistance.     

Determination of species-area relationships and minimum sampling area for the shrub communities in the arid valley in the upper reach of the Minjiang River, China

The minimum sampling areas (MSAs) for the shrub communities in the arid valley in the upper reach of the Minjiang River, China, were studied by fitting community species-area relationships using 3 types of equations. The MSAs were determined at the proportional factor (ρ) 0.6, 0.7, 0.8 and 0.9. The proportional factors represent the proportion of the number of species within a sampling plot in the total number of species. The MSAs of the shrub communities at different elevations and on different slope faces for ρ = 0.6, 0.7 and 0.8 were all around 100 m². Hence, the MSAs could be set to be 100 m² (10 m × 10 m) at 60%–80% precision levels. For ρ = 0.9, that is, for a 90% precision level, the MSAs were less than 200 m² (10 m × 20 m). The MSAs and species richness increased gradually with the rising elevation. At the elevation below 2000 m, the MSAs and species richness on the north-facing slope were larger than those on the south-facing slope. However, at the elevation around 2200 m, there was no difference amongst different facing slopes. For the shrub communities in the arid valley in the upper reach of the Minjiang River, the species-area curves by fitting the first two equations are better than that by fitting the third equation.     

Effects of plant hedgerows on soil erosion and soil fertility on sloping farmland in the purple soil area

Effect of using plant hedgerows on controlling soil and water losses has received wide recognition and this technology has been applied in many areas in the world. Yet, studies on the effect of using plant hedgerows on soil fertility on sloping lands are rare. Carrying out an eight-year fixed field experiment, the authors investigated the effect of two different hedgerows against the control treatment on soil fertility. Results showed that clay particles tended to accumulate in front of the plant hedgerows and began to erode downward below the hedgerows along the contour lines across the field. Distribution of soil organic matter and all plant nutrients except potassium (K) showed the same pattern as the clay particles. Potassium, however, was evenly distributed in the field without any noticeable influence from the hedgerows. Since the fixed experiment started, soil phosphorus (P) kept accumulating, while soil organic matter and K were in depletion. The results accordingly suggested better nutrient management practices on the sloping lands by using properly reduced rates of P and increased rates of farm manure and K. Taking the sloping field as a whole, special attention in nutrient management should be given to the soil strips —the portions below the plant hedgerows suffering from more serious soil erosion.