Rice G protein γ subunit qPE9-1 modulates root elongation for phosphorus uptake by involving 14-3-3 protein OsGF14b and plasma membrane H+-ATPase

Heterotrimeric G protein is involved in plant growth and development, while the role of rice (Oryza sativa) G protein γ subunit qPE9-1 in response to low-phosphorus (LP) conditions remains unclear. The gene expression of qPE9-1 was significantly induced in rice roots under LP conditions. Rice varieties carrying the qPE9-1 allele showed a stronger primary root response to LP than the varieties carrying the qpe9-1 allele (mutant of the qPE9-1 allele). Transgenic rice plants with the qPE9-1 allele had longer primary roots and higher P concentrations than those with the qpe9-1 allele under LP conditions. The plasma membrane (PM) H⁺-ATPase was important for the qPE9-1-mediated response to LP. Furthermore, OsGF14b, a 14-3-3 protein that acts as a key component in activating PM H⁺-ATPase for root elongation, is also involved in the qPE9-1 mediation. Moreover, the overexpression of OsGF14b in WYJ8 (carrying the qpe9-1 allele) partially increased primary root length under LP conditions. Experiments using R18 peptide (a 14-3-3 protein inhibitor) showed that qPE9-1 is important for primary root elongation and H⁺ efflux under LP conditions by involving the 14-3-3 protein. In addition, rhizosheath weight, total P content, and the rhizosheath soil Olsen-P concentration of qPE9-1 lines were higher than those of qpe9-1 lines under soil drying and LP conditions. These results suggest that the G protein γ subunit qPE9-1 in rice plants modulates root elongation for phosphorus uptake by involving the 14-3-3 protein OsGF14b and PM H⁺-ATPase, which is required for rice P use.     

Contrasting responses of heterotrophic and autotrophic respiration to experimental warming in a winter annual‐dominated prairie

Understanding how soil respiration (Rs) and its source components respond to climate warming is crucial to improve model prediction of climate‐carbon (C) feedback. We conducted a manipulation experiment by warming and clipping in a prairie dominated by invasive winter annual Bromus japonicas in Southern Great Plains, USA. Infrared radiators were used to simulate climate warming by 3 °C and clipping was used to mimic yearly hay mowing. Heterotrophic respiration (Rh) was measured inside deep collars (70 cm deep) that excluded root growth, while total soil respiration (Rs) was measured inside surface collars (2–3 cm deep). Autotrophic respiration (Ra) was calculated by subtracting Rh from Rs. During 3 years of experiment from January 2010 to December 2012, warming had no significant effect on Rs. The neutral response of Rs to warming was due to compensatory effects of warming on Rh and Ra. Warming significantly (P < 0.05) stimulated Rh but decreased Ra. Clipping only marginally (P < 0.1) increased Ra in 2010 but had no effect on Rh. There were no significant interactive effects of warming and clipping on Rs or its components. Warming stimulated annual Rh by 22.0%, but decreased annual Ra by 29.0% across the 3 years. The decreased Ra was primarily associated with the warming‐induced decline of the winter annual productivity. Across the 3 years, warming increased Rh/Rs by 29.1% but clipping did not affect Rh/Rs. Our study highlights that climate warming may have contrasting effects on Rh and Ra in association with responses of plant productivity to warming.     

Utilization of milk energy by suckling mink kits

A total of 36 mink dams and their litters of 3, 6 or 9 kits were used for determination of milk intake of the suckling young by means of deuterium dilution technique, and chemical composition of milk and of kit bodies. Measurements were performed during lactation weeks 1?–?4, each week with 3 dams with each litter size. Milk intake was determined over a 48?h measurement period, and by the end of this milk samples were collected and 2 kits (litters of 6 and 9) or 1 kit per litter (litters of 3) were killed for body chemical composition. Based on the results, different models were applied for calculation of the energetic efficiency of milk. Dam milk yield increased steadily from week 1 until week 3 but only slightly from week 3 to 4. The increase declined with increasing litter size, and for dams suckling 9 kits the increment from week 3 to week 4 was only 2?g. The dry matter content of milk increased significantly as lactation progressed, being reflected in crude protein increasing from 6.9% in lactation week 1 to 8.1% in week 4. Milk fat increased concomitantly from 5.6% to 8.0%. In kit bodies, crude protein content increased from 9.4% in week 1 to about 12% in weeks 3 and 4. Body fat content increased from week 1 (4.1%) to week 3 (8.4%) and then declined in week 4 (7.1%). Animals suckled in litters of 3 kits had the highest milk intake and live weight and kits suckled in litters of 9 had the lowest milk intake, live weight and daily gain. In terms of milk intake per g gain kits in litters of 6 were the most efficient, with 4.1?g milk per g body gain. The metabolizable energy requirement for maintenance (MEm) was estimated to 448 kJ/kg^0.75 and the efficiency of utilization of ME for body gain (k_g) to 0.67, the estimates being higher (MEm) or in good agreement with previous findings (k_g) in suckling mink kits.     

Plant–microbe networks in soil are weakened by century-long use of inorganic fertilizers

Understanding the changes in plant–microbe interactions is critically important for predicting ecosystem functioning in response to human-induced environmental changes such as nitrogen (N) addition. In this study, the effects of a century-long fertilization treatment (> 150 years) on the networks between plants and soil microbial functional communities, detected by GeoChip, in grassland were determined in the Park Grass Experiment at Rothamsted Research, UK. Our results showed that plants and soil microbes have a consistent response to long-term fertilization—both richness and diversity of plants and soil microbes are significantly decreased, as well as microbial functional genes involved in soil carbon (C), nitrogen (N) and phosphorus (P) cycling. The network-based analyses showed that long-term fertilization decreased the complexity of networks between plant and microbial functional communities in terms of node numbers, connectivity, network density and the clustering coefficient. Similarly, within the soil microbial community, the strength of microbial associations was also weakened in response to long-term fertilization. Mantel path analysis showed that soil C and N contents were the main factors affecting the network between plants and microbes. Our results indicate that century-long fertilization weakens the plant–microbe networks, which is important in improving our understanding of grassland ecosystem functions and stability under long-term agriculture management.     

Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.     

Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase

The sulfonation of estrogens by human estrogen sulfotransferase (humSULT1E1) plays a vital role in controlling the active levels of these hormones in the body. To understand more fully the structural and functional characteristics of humSULT1E1, we have carried out site-directed mutagenesis of critical amino acids found in the substrate-binding cleft. Three single amino acid mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this study. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety of substrates. Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3alpha-phenol group of beta-estradiol, thereby stabilising the substrate in the active site. However, substitution to a neutral alanine at this position improved substrate specificity of humSULT1E1 for beta-estradiol, estrone, and dehydroepiandrosterone (DHEA). The exchange of valine 145 for negatively charged glutamic acid markedly improved the ability of humSULT1E1 to sulfonate dopamine, but caused a reduction in specificity constants toward steroids tested, in particular DHEA. The presence of a histidine residue at position 107 was shown to be essential for the production of a functional protein, as substitution of this amino acid to alanine resulted in complete loss of activity of humSULT1E1 towards all substrates tested.     

Localisation and functional studies on the 5′ -nucleotidase of the cattle tick Boophilus microplus

The ecto-5-nucleotidase from the cattle tick Boophilus microplus is an unusual enzyme, hydrolysing a variety of nucleoside mono-, di- and triphosphates to release the free nucleoside. The gene has been sequenced and the recombinant protein expressed as a functional, active enzyme. Nevertheless, the function of the enzyme in the tick remains obscure. The enzyme is present throughout the life cycle, but in largest amounts in unfed larvae and adult ticks. The tissue location has been studied in adult female ticks by Western blotting, RT-PCR and immunofluorescence. All methods show the enzyme to be principally in the Malpighian tubules, though significant amounts are also present on the surface of ovaries and in detectible amounts in other tissues. This, together with the known specificity of the enzyme, suggests a role in purine salvage pathways. Sensitivity of ticks to allopurinol, an inhibitor of hypoxanthine-guanine-phosphoribosyltransferase, supports the importance of purine salvage in this tick and the potential role of nucleotidase in this pathway.