Identification and validation of cold responsive microRNAs of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.     

Formation of Biogenic Amines and Associated Biochemical and Microbial Attributes of Whole Sutchi Catfish (Pangasianodon hypophthalmus) during Iced Storage

Sutchi catfish (Pangasianodon hypophthalmus) is a highly preferred farmed species that is produced in huge quantities. Meat from ice-stored (4 ± 2°C) whole Sutchi catfish was evaluated for formation of biogenic amines, such as putrescine, cadaverine, histamine (HIM), agmatine, tyramine, spermine, and spermidine, and compared with biochemical, microbial, and sensory attributes during 22 days. Analysis of content of biogenic amines in the meat was carried out by liquid chromatography with tandem mass spectrometry using a without derivatization method. Three amines, namely, tyramine, spermidine, and spermine, were only present on 0^th day of storage. Presence of cadaverine was noticed from 9^th day onwards, where the aerobic plate count (APC) reached 4.85 log CFU/g. Putrescine was detected on the 22^nd day of storage, where the APC crossed 7 log CFU/g. HIM was detected in lower quantities from 3^rd day onwards. A shelf life of 15 days was determined based on sensory and microbiological evaluation. Although the samples were biochemically acceptable throughout the storage period, APC exceeded the limit on day 19, and the gradual increase of H₂S-producing bacteria, Brochothrix thermosphacta, Aeromonas, and Enterobacteriaceae, was observed during storage.     

Effect of biochar amendment on soil carbon balance and soil microbial activity

We investigated the behavior of biochars in arable and forest soil in a greenhouse experiment in order to prove that these amendments can increase carbon storage in soils. Two qualities of biochar were produced by hydrothermal pyrolysis from ¹³C labeled glucose (0% N) and yeast (5% N), respectively. We quantified respiratory losses of soil and biochar carbon and calculated mean residence times of the biochars using the isotopic label. Extraction of phospholipid fatty acids from soil at the beginning and after 4 months of incubation was used to quantify changes in microbial biomass and to identify microbial groups utilizing the biochars. Mean residence times varied between 4 and 29 years, depending on soil type and quality of biochar. Yeast-derived biochar promoted fungi in the soil, while glucose-derived biochar was utilized by Gram-negative bacteria. Our results suggest that residence times of biochar in soils can be manipulated with the aim to “design” the best possible biochar for a given soil type.     

Overstory-specific effects of litter fall on the microbial carbon turnover in a mature deciduous forest

Mature deciduous forests can serve as important carbon (C) sinks, but the C storage differs significantly in dependency on the tree species. To specify the significance of overstory-specific effects of litter fall on the soil microbial C turnover, we have investigated the ¹³C isotopic signature of microbial biomarker phospholipid fatty acids (PLFAs). Samples were taken under pure Fagus sylvatica and mixed overstory (F. sylvatica and Fraxinus excelsior or F. excelsior, Acer spp. and F. sylvatica) in a mature temperate deciduous forest in Central Germany 4 weeks prior to and 3 weeks after litter fall. Accordingly, the CO₂ emission from soil was measured before, during and after the litter fall to investigate the response of decomposition. At all sites and at both sampling dates the fungal biomarker PLFA 18:2ω6,9 had predominantly lower δ¹³C values (from −32 to −43‰) than the bacterial biomarker PLFAs (δ¹³C values from −23 to −39‰). This difference indicated that fungi generally used preferentially plant derived C, whereas the bacterial populations include groups which used SOM derived C, independent on the overstory trees. Under pure F. sylvatica overstory the δ¹³C values of microbial biomarker PLFAs were slightly decreased (up to 2‰ for 17:0br) or unchanged after litter fall. By contrast, under both variants of mixed overstory the δ¹³C values of biomarker PLFAs of fungi (18:2ω6,9) were increased after litter fall (+3.5 and +3.8‰). This might be explained partly by a faster initial decomposition of foliar litter from mixed overstory already during litter fall as confirmed by higher CO₂ emission under mixed F. excelsior, Acer spp. and F. sylvatica than under pure F. sylvatica in this period. However, the involved microbial populations differed overstory-specific. Bacterial biomarker PLFAs with strongest overstory-specific differences in the response on litter fall were 17:0br (Gram-positive bacteria), 18:1 and 19:0cy (Gram-negative bacteria). The present results indicate that a tree species conversion even exclusively between deciduous tree species might alter the soil microbial C turnover during litter decomposition and suggest that it would in the long-term change the SOM stability and C storage.     

Expression of kit ligand and insulin‐like growth factor binding protein 3 during in vivo or in vitro development of ovarian follicles in sheep

Expression of Kit ligand (KL) and insulin‐like growth factor binding protein (IGFBP3) genes was studied at different in vivo and corresponding in vitro stages of development of the ovarian follicles in sheep. The expression of both KL and IGFBP3 was significantly higher in the primordial follicles relative to any other stage of development. Compared to the other stages, the KL expression in the cumulus cells from in vivo grown large antral follicles and that of IGFBP3 in COCs’ isolated from large antral follicles matured in vitro for 24 hr were significantly higher. In the oocytes from in vivo grown ovarian follicles, the expression of KL was the same at all the stages of development. Insulin‐like growth factor binding protein 3 expression was also the same in the oocytes at all the stages of the development except for a significantly lower expression in those from antral follicles. The expression of KL in the cumulus cells decreased significantly in the in vitro grown early antral follicles but did not change further as the development progressed. The expression of IGFBP3 in the cumulus cells from in vitro grown ovarian follicles appeared to increase as the development progressed although the increase was not significant between any two consecutive stages of development. In the oocytes in in vitro grown ovarian follicles, the expression levels of KL and IGFBP3 genes did not change with development. It is concluded that (i) KL and IGFBP3 genes follow specific patterns of expression during ovarian folliculogenesis and (ii) in vitro culture of preantral follicles compromises the development potential through alterations in the stage‐specific patterns of expression of these and other developmentally important genes.     

Organic carbon sequestration in earthworm burrows

Earthworms strongly affect soil organic carbon cycling. The aim of this study was to determine whether deep burrowing anecic earthworms enhance carbon storage in soils and decrease C turnover. Earthworm burrow linings were separated into thin cylindrical sections with different distances from the burrow wall to determine gradients from the burrow wall to the surrounding soil. Organic C, total N, radiocarbon (¹⁴C) concentration, stable isotope values (δ¹³C, δ¹⁵N) and extracellular enzyme activities were measured in these samples. Anecic earthworms increased C stocks by 270 and 310 g m^?2 accumulated in the vertical burrows. C-enrichment of the burrow linings was spatially highly variable within a distance of millimetres around the burrow walls. It was shown that C accumulation in burrows can be fast with C sequestration rates of about 22 g C m^?2 yr^?1 in the burrow linings, but accumulated C in the burrows may be mineralised fast with turnover times of only 3–5 years. Carbon stocks in earthworm burrows strongly depended on the earthworm activity which maintains continuous C input into the burrows. The enhanced extracellular enzyme activity of fresh casts was not persistent, but was 47% lower in inhabited burrows and 62% lower in abandoned burrows. Enzyme activities followed the C concentrations in the burrows and were not further suppressed due to earthworms. Radiocarbon concentrations and stable isotopes in the burrow linings showed an exponential gradient with the youngest and less degraded organic matter in the innermost part of the burrow wall. Carbon accumulation by anecic earthworm is restricted to distinct burrows with less influence to the surrounding soil. Contrary to the initial hypothesis, that organic C is stabilised due to earthworms, relaxation time experiments with nuclear magnetic resonance spectroscopy (NMR) did not reveal any enhanced adsorption of C on iron oxides with C stabilising effect. Our results suggest that earthworm activity does not substantially increase subsoil C stocks but burrows serve as fast ways for fresh C transport into deep soil horizons.     

Mechanisms of short-term soil carbon storage in experimental grasslands

We investigated the fate of root and litter derived carbon in soil organic matter and dissolved organic matter in soil profiles, in order to explain mechanisms of short-term soil carbon storage. A time series of soil and soil solution samples was investigated at the field site of The Jena Experiment between 2002 and 2004. In addition to the main experiment with C3 plants, a C4 species (Amaranthus retroflexus L.) naturally labeled with ¹³C was grown on an extra plot. Changes in organic carbon concentration in soil and soil solution were combined with stable isotope measurements to follow the fate of plant carbon into the soil and soil solution. A split plot design with plant litter removal versus double litter input simulated differences in biomass input. After 2 years, the no litter and double litter treatment, respectively, showed an increase of 381 g C m^?2 and 263 g C m^?2 to 20 cm depth, while 71 g C m^?2 and 393 g C m^?2 were lost between 20 and 30 cm depth. The isotopic label in the top 5 cm indicated that 115 g C m^?2 and 156 g C m^?2 of soil organic carbon were derived from C4 plant material on the no litter and the double litter treatment, respectively. Without litter, this equals the total amount of 97 g C m^?2 that was newly stored in the same soil depth, whereas with double litter this clearly exceeded the stored amount of 75 g C m^?2. Our results indicate that litter input resulted in lower carbon storage and larger carbon losses and consequently accelerated turnover of soil organic carbon. Isotopic evidence showed that inherited soil organic carbon was replaced by fresh plant carbon near the soil surface. Our results suggest that primarily carbon released from soil organic matter, not newly introduced plant organic matter, was transported in the soil solution. However, the total flow of dissolved organic carbon was not sufficient to explain the observed carbon storage in deeper soil layers, and the existence of additional carbon uptake mechanisms is discussed.     

Storage and stability of organic matter and fossil carbon in a Luvisol and Phaeozem with continuous maize cropping: A synthesis

Quantitative information about the amount and stability of organic carbon (OC) in different soil organic‐matter (OM) fractions and in specific organic compounds and compound‐classes is needed to improve our understanding of organic‐matter sequestration in soils. In the present paper, we summarize and integrate results performed on two different arable soils with continuous maize cropping (a) Stagnic Luvisol with maize cropping for 24 y, b) Luvic Phaeozem with maize cropping for 39 y) to identify (1) the storage of OC in different soil organic‐matter fractions, (2) the function of these fractions with respect to soil‐OC stabilization, (3) the importance and partitioning of fossil‐C deposits, and (4) the rates of soil‐OC stabilization as assessed by compound‐specific isotope analyses. The fractionation procedures included particle‐size fractionation, density fractionation, aggregate fractionation, acid hydrolysis, different oxidation procedures, isolation of extractable lipids and phospholipid fatty acids, pyrolysis, and the determination of black C. Stability of OC was determined by ¹³C and ¹⁴C analyses. The main inputs of OC were plant litter (both sites) and deposition of fossil C likely from coal combustion and lignite dust (only Phaeozem).