Metaproteomic Identification of Diazotrophic Methanotrophs and Their Localization in Root Tissues of Field-Grown Rice Plants

In a previous study by our group, CH₄ oxidation and N₂ fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH₄ oxidation and N₂ fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH₄ oxidation and N₂ fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH₄ oxidation and N₂ fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.     

Effects of Winter Cover Crops Straws Incorporation on CH4 and N2O Emission from Double-Cropping Paddy Fields in Southern China

Residue management in cropping systems is believed to improve soil quality. However, the effects of residue management on methane (CH₄) and nitrous oxide (N₂O) emissions from paddy field in Southern China have not been well researched. The emissions of CH₄ and N₂O were investigated in double cropping rice (Oryza sativa L.) systems with straw returning of different winter cover crops by using the static chamber-gas chromatography technique. A randomized block experiment with three replications was established in 2004 in Hunan Province, China, including rice–rice–ryegrass (Lolium multiflorum L.) (Ry-R-R), rice–rice–Chinese milk vetch (Astragalus sinicus L.) (Mv-R-R) and rice–rice with winter fallow (Fa-R-R). The results showed that straw returning of winter crops significantly increased the CH₄ emission during both rice growing seasons when compared with Fa-R-R. Ry-R-R plots had the largest CH₄ emissions during the early rice growing season with 14.235 and 15.906 g m⁻² in 2012 and 2013, respectively, when Ry-R-R plots had the largest CH₄ emission during the later rice growing season with 35.673 and 38.606 g m⁻² in 2012 and 2013, respectively. The Ry-R-R and Mv-R-R also had larger N₂O emissions than Fa-R-R in both rice seasons. When compared to Fa-R-R, total N₂O emissions in the early rice growing season were increased by 0.05 g m⁻² in Ry-R-R and 0.063 g m⁻² in Mv-R-R in 2012, and by 0.058 g m⁻² in Ry-R-R and 0.068 g m⁻² in Mv-R-R in 2013, respectively. Similar result were obtained in the late rice growing season, and the total N₂O emissions were increased by 0.104 g m⁻² in Ry-R-R and 0.073 g m⁻² in Mv-R-R in 2012, and by 0.108 g m⁻² in Ry-R-R and 0.076 g m⁻² in Mv-R-R in 2013, respectively. The global warming potentials (GWPs) from paddy fields were ranked as Ry-R-R>Mv-R-R>Fa-R-R. As a result, straw returning of winter cover crops has significant effects on increase of CH₄ and N₂O emission from paddy field in double cropping rice system.     

Impacts of maize hybrids with different nitrogen use efficiency on root-associated microbiota based on distinct rhizosphere soil metabolites

Plant genotypes shape root-associated microbiota that affect plant nutrient acquisition and productivity. It is unclear how maize hybrids modify root-associated microbiota and their functions and relationship with nitrogen use efficiency (NUE) by regulating rhizosphere soil metabolites. Here, two N-efficient (NE) (ZD958, DMY3) and two N-inefficient (NIE) maize hybrids (YD9953, LY99) were used to investigate this issue under low N (60 kg N ha⁻¹, LN) and high N (180 kg N ha⁻¹, HN) field conditions. NE hybrids had higher yield than NIE hybrids under LN but not HN. NE and NIE hybrids recruited only distinct root-associated bacterial microbiota in LN. The bacterial network stability was stronger in NE than NIE hybrids. Compared with NIE hybrids, NE hybrids recruited more bacterial taxa that have been described as plant growth-promoting rhizobacteria (PGPR), and less related to denitrification and N competition; this resulted in low N₂O emission and high rhizosphere NO₃⁻-N accumulation. NE and NIE hybrids had distinct rhizosphere soil metabolite patterns, and their specific metabolites were closely related to microbiota and specific genera under LN. Our findings reveal the relationships among plant NUE, rhizosphere soil metabolites, root-associated microbiota, and soil nutrient cycling, and this information is informative for breeding NE crops.     

Effect of Substrate Concentration on Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosarcina barkeri and M. acetivorans and in Rice Field Soil

Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH₄ production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (ɛ) associated with consumption of total acetate (ɛ_ac), consumption of acetate-methyl (ɛ_ac-methyl) and production of CH₄ (ɛ_CH4) were an ɛ_ac of −30.5‰, an ɛ_ac-methyl of −25.6‰, and an ɛ_CH4 of −27.4‰ for M. barkeri and an ɛ_ac of −35.3‰, an ɛ_ac-methyl of −24.8‰, and an ɛ_CH4 of −23.8‰ for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an ɛ_ac of −18.7‰, an ɛ_ac-methyl of −16.9‰, and an ɛ_CH4 of −20.8‰. However, in rice field soil as well as in the pure cultures, values of ɛ_ac and ɛ_ac-methyl decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The ɛ values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.Methane (CH₄) is the most abundant reduced gas in the earth''s atmosphere and is an important greenhouse gas with a high global-warming potential (7). It is presently a matter of discussion whether the contribution of CH₄ to the greenhouse effect will increase in the future (3, 23). This has made it necessary and more urgent to understand natural processes that lead to the production of CH₄.Methanogenesis, the microbial formation of CH₄, is the final step in the degradation of organic matter in anoxic environments like natural wetlands, lake sediments, and flooded rice fields. The most important precursors for the production of CH₄ are acetate (equation 1) and CO₂ (equation 2) with the following reactions (8): (1) (2)Acetate is the most important substrate since it contributes more than 67% to microbial methanogenesis during anoxic degradation of polysaccharides. In methanogenic environments only two genera of archaea, Methanosaeta and Methanosarcina, are capable of using acetate (2). While Methanosaeta can be considered a specialist that uses only acetate, Methanosarcina can use a wide range of substrates besides acetate, for example, H₂/CO₂, methanol, methylamines, and methylated sulfides. Among methanogens, Methanosarcinaceae also display the largest environmental distribution. They can be found in freshwater sediments and soil, marine habitats, landfills, and animal gastrointestinal tracts (46).Additionally, differences between Methanosarcina and Methanosaeta were found for isotope fractionation of stable carbon. The fractionation factor (α) or, equivalently, the enrichment factor (ɛ) during acetoclastic methanogenesis in Methanosarcina barkeri strains typically ranges from an α of 1.021 to 1.027 or an ɛ of −27‰ to −21‰ (14, 27, 48), whereas isotope fractionation in Methanosaeta spp. is weaker, i.e., an α of 1.007 (ɛ = −7‰) for Methanosaeta thermophila (43) and an α of 1.010 (ɛ = −10‰) for Methanosaeta concilii (34). It is suggested that the two archaeal genera differ in isotope fractionation due to differences in their biochemical activation of acetate to acetyl-coenzyme A (acetyl-CoA) (34). However, isotopic data for acetoclastic methanogens are rare. For instance, all data for Methanosarcina refer to only one species, namely M. barkeri.Hence, in this study we investigated whether differences in carbon isotope fractionation within the genus Methanosarcina occur. Therefore, we determined isotope ratios of stable carbon in cultures of the acetoclastic species M. barkeri and Methanosarcina acetivorans. Second, we were interested if these data, obtained from pure cultures, could also be applied to understand natural environments. For that reason, we determined isotope fractionation during acetoclastic methanogenesis in the model system rice field soil. Furthermore, we discuss the effect of substrate concentration on carbon isotope fractionation and the importance of monitoring isotope fractionation during the course of acetate consumption.     

Annual emissions of dissolved CO2, CH4, and N2O in the subsurface drainage from three cropping systems

Indirect emission of nitrous oxide (N₂O), associated with nitrogen (N) leaching and runoff from agricultural lands is a major source of atmospheric N₂O. Recent studies have shown that carbon dioxide (CO₂) and methane (CH₄) are also emitted via these pathways. We measured the concentrations of three dissolved greenhouse gases (GHGs) in the subsurface drainage from field lysimeter that had a shallow groundwater table. Aboveground fluxes of CH₄ and N₂O were monitored using an automated closed‐chamber system. The annual total emissions of dissolved and aboveground GHGs were compared among three cropping systems; paddy rice, soybean and wheat, and upland rice. The annual drainage in the paddy rice, the soybean and wheat, and the upland rice plots was 1435, 782, and 1010 mm yr^?1, respectively. Dissolved CO₂ emissions were highest in the paddy rice plots, and were equivalent to 1.05–1.16% of the carbon storage in the topsoil. Dissolved CH₄ emissions were also higher in the paddy rice plots, but were only 0.03–0.05% of the aboveground emissions. Dissolved N₂O emissions were highest in the upland rice plots, where leached N was greatest due to small crop biomass. In the soybean and wheat plots, large crop biomass, due to double cropping, decreased the drainage volume, and thus decreased dissolved GHG emissions. Dissolved N₂O emissions from both the soybean and wheat plots and the upland rice plots were equivalent to 50.3–67.3% of the aboveground emissions. The results indicate that crop type and rotation are important factors in determining dissolved GHG emissions in the drainage from a crop field.     

Methane-Derived Carbon in the Benthic Food Web in Stream Impoundments

Methane gas (CH₄) has been identified as an important alternative source of carbon and energy in some freshwater food webs. CH₄ is oxidized by methane oxidizing bacteria (MOB), and subsequently utilized by chironomid larvae, which may exhibit low δ¹³C values. This has been shown for chironomid larvae collected from lakes, streams and backwater pools. However, the relationship between CH₄ concentrations and δ¹³C values of chironomid larvae for in-stream impoundments is unknown. CH₄ concentrations were measured in eleven in-stream impoundments located in the Queich River catchment area, South-western Germany. Furthermore, the δ¹³C values of two subfamilies of chironomid larvae (i.e. Chironomini and Tanypodinae) were determined and correlated with CH₄ concentrations. Chironomini larvae had lower mean δ¹³C values (−29.2 to −25.5 ‰), than Tanypodinae larvae (−26.9 to −25.3 ‰). No significant relationships were established between CH₄ concentrations and δ¹³C values of chironomids (p>0.05). Mean δ¹³C values of chironomid larvae (mean: −26.8‰, range: −29.2‰ to −25.3‰) were similar to those of sedimentary organic matter (SOM) (mean: −28.4‰, range: −29.3‰ to −27.1‰) and tree leaf litter (mean: −29.8 ‰, range: −30.5‰ to −29.1‰). We suggest that CH₄ concentration has limited influence on the benthic food web in stream impoundments.     

Promotion of photosynthesis in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene by nitric oxide donors

We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C₄ pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca²⁺ chelator, a Ca²⁺ channel inhibitor, and a hydrogen peroxide (H₂O₂) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (P_N) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the P_N of rice plants. The treatments with phospholipase inhibitors and a Ca²⁺ chelator decreased the P_N of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca²⁺ both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca²⁺ level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca²⁺.     

施硅对增温稻田CH4和N2O排放的影响

夜间增温幅度大于白天是气候变暖的显著特征。夜间增温影响水稻生产及CH4和N2O排放。硅是作物有益元素,施硅可提高产量,减少稻田CH4排放。增温或施硅单因子对稻田CH4和N2O排放影响已有报道,但二者耦合如何影响水稻生产及稻田CH4和N2O排放,尚不清楚。通过田间模拟试验,研究了夜间增温下施硅对水稻生长、产量及温室气体持续增温/冷却潜势和排放强度的影响。采用铝箔反光膜夜间(19:00-6:00)覆盖水稻冠层进行模拟夜间增温试验。增温设2水平,即常温对照(CK)和夜间增温(NW);施硅量设2水平,即Si0(不施硅)和Si1(钢渣硅肥,200 kgSiO2/ha)。结果表明,施硅可缓解夜间增温对水稻根系活力的抑制作用,降低夜间增温对水稻地上部、地下部干重和产量的抑制作用。夜间增温显著提高CH4累计排放量,而施硅显著降低CH4累计排放量。夜间增温下施硅处理稻田CH4累计排放量在分蘖期、拔节期、抽穗-扬花期和灌浆成熟期比未施硅处理分别低48.12%、49.16%、61.59%和39.13%。夜间增温或施硅均促进稻田N2O排放,夜间增温下施硅在上述生育期以及全生育期的累计排放量依次比对照高78.17%、51.45%、52.01%、26.14%和40.70%。研究认为,施硅可缓解夜间增温对稻田综合增温潜势和排放强度的促进作用。     

Methane and nitrous oxide fluxes in annual and perennial land-use systems of the irrigated areas in the Aral Sea Basin

Land use and agricultural practices can result in important contributions to the global source strength of atmospheric nitrous oxide (N₂O) and methane (CH₄). However, knowledge of gas flux from irrigated agriculture is very limited. From April 2005 to October 2006, a study was conducted in the Aral Sea Basin, Uzbekistan, to quantify and compare emissions of N₂O and CH₄ in various annual and perennial land-use systems: irrigated cotton, winter wheat and rice crops, a poplar plantation and a natural Tugai (floodplain) forest. In the annual systems, average N₂O emissions ranged from 10 to 150 μg N₂O-N m⁻² h⁻¹ with highest N₂O emissions in the cotton fields, covering a similar range of previous studies from irrigated cropping systems. Emission factors (uncorrected for background emission), used to determine the fertilizer-induced N₂O emission as a percentage of N fertilizer applied, ranged from 0.2% to 2.6%. Seasonal variations in N₂O emissions were principally controlled by fertilization and irrigation management. Pulses of N₂O emissions occurred after concomitant N-fertilizer application and irrigation. The unfertilized poplar plantation showed high N₂O emissions over the entire study period (30 μg N₂O-N m⁻² h⁻¹), whereas only negligible fluxes of N₂O (<2 μg N₂O-N m⁻² h⁻¹) occurred in the Tugai. Significant CH₄ fluxes only were determined from the flooded rice field: Fluxes were low with mean flux rates of 32 mg CH₄ m⁻² day⁻¹ and a low seasonal total of 35.2 kg CH₄ ha⁻¹. The global warming potential (GWP) of the N₂O and CH₄ fluxes was highest under rice and cotton, with seasonal changes between 500 and 3000 kg CO₂ eq. ha⁻¹. The biennial cotton–wheat–rice crop rotation commonly practiced in the region would average a GWP of 2500 kg CO₂ eq. ha⁻¹ yr⁻¹. The analyses point out opportunities for reducing the GWP of these irrigated agricultural systems by (i) optimization of fertilization and irrigation practices and (ii) conversion of annual cropping systems into perennial forest plantations, especially on less profitable, marginal lands.     

Changes of methane and nitrous oxide emissions in a transition bog in central Germany (German National Park Harz Mountains) after rewetting

During the last decades, various renaturation programmes have been initialized to recover nutrient sink and ecological functions of peatlands by rewetting. Rewetting, however, often results in the formation of hotspots for methane (CH₄) emissions and in temporal dieback of local vegetation. The present study aimed at quantifying changes of CH₄ and nitrous oxide (N₂O) emissions in a peatland currently under continuous rewetting conditions. Emissions where studied at a permanently flooded site and a non-flooded peat site with fluctuating water tables by using common closed chamber method. The permanently flooded site revealed extremely high CH₄ emissions (up to 1195 mg C m^?2 d^?1) which were positively correlated with temperature, nutrient content, dissolved organic carbon and nitrogen concentration of the peat soil water. In contrast, the non-flooded peat site, with lower and fluctuating water tables (WT), showed significantly lower CH₄ emissions and an increasing trend of CH₄ release associated with a generally increasing WT caused by the progressing rewetting process. Lower N₂O emissions (<24 µg N m^?2 d^?1) were observed at the flooded site. By contrast, the non-flooded peat site with fluctuating WT showed significantly higher N₂O emissions (up to 4178 µg N m^?2 d^?1), in particular at high temperatures during summer time. The present results indicate that permanently flooded conditions during rewetting processes might cause higher CH₄ emissions compared to fluctuating WT which in contrast might enhance N₂O emissions. In total, however, no decreasing trend for CH₄ emissions throughout the five-year renaturation period could be found. At least for N₂O we observed a decreasing trend during rewetting.     

Effects of N-fertilisation on CH4 oxidation and production, and consequences for CH4 emissions from microcosms and rice fields

The world's growing human population causes an increasing demand for food, of which rice is one of the most important sources. In rice production nitrogen is often a limiting factor. As a consequence increasing amounts of fertiliser will have to be applied to maximise yields. There is an ongoing discussion on the possible effects of fertilisation on CH₄ emissions. We therefore investigated the effects of N‐fertiliser (urea) on CH₄ emission, production and oxidation in rice microcosms and field experiments. In the microcosms, a substantial but short‐lived reduction of CH₄ emission was observed after N‐addition to 43‐d‐old rice plants. Methane oxidation increased by 45%, demonstrated with inhibitor measurements and model calculations based on stable carbon isotope data (δ¹³CH₄). A second fertilisation applied to 92‐d‐old plants had no effect on CH₄ emission rates. The positive effect of additional N on methanotrophic bacteria was also found in vitro for potential CH₄ oxidation rates in soil and root samples from the microcosm and field experiments, indicated by elevated initial oxidation rates and reduced lag‐phases. Fertilisation did not affect methane production in the microcosms. In the field, the effects were diverse: methane production was inhibited in the topsoil, but stimulated instead in the bulk soil. Stimulation occurred probably in the anaerobic food chain at the level of hydrolytic or fermenting bacteria, because acetate, a methanogenic precursor, increased simultaneously. Combining field, microcosm and laboratory experiments we conclude that any agricultural treatment improving the N‐supply to the rice plants will also be favourable for the CH₄ oxidising bacteria. However, N‐fertilisation had only a transient influence and was counter‐balanced in the field by an elevated CH₄ production. A negative effect of the fertilisation was a transient increase of N₂O emissions from the microcosms. However, integrating over the season the global warming potential (GWP) of N₂O emitted after fertilisation was still negligible compared to the GWP of emitted CH₄.     

A record of N2O and CH4 emissions and underlying soil processes of Korean rice paddies as affected by different water management practices

Rice is staple food of half of mankind and paddy soils account for the largest anthropogenic wetlands on earth. Ample of research is being done to find cultivation methods under which the integrative greenhouse effect caused by emitted CH₄ and N₂O would be mitigated. Whereas most of the research focuses on quantifying such emissions, there is a lack of studies on the biogeochemistry of paddy soils. In order to deepen our mechanistic understanding of N₂O and CH₄ fluxes in rice paddies, we also determined NO₃ ^? and N₂O concentrations as well as N₂O isotope abundances and presence of O₂ along soil profiles of paddies which underwent three different water managements during the rice growing season(s) in (2010 and) 2011 in Korea. Largest amounts of N₂O (2 mmol m^?2) and CH₄ (14.5 mol m^?2) degassed from the continuously flooded paddy, while paddies with less flooding showed 30–60 % less CH₄ emissions and very low to negative N₂O balances. In accordance, the global warming potential (GWP) was lowest for the Intermittent Irrigation paddy and highest for the Traditional Irrigation paddy. The N₂O emissions could the best be explained (*P < 0.05) with the δ¹⁵N values and N₂O concentrations in 40–50 cm soil depth, implying that major N₂O production/consumption occurs there. No significant effect of NO₃ ^? on N₂O production has been found. Our study gives insight into the soil of a rice paddy and reveals areas along the soil profile where N₂O is being produced. Thereby it contributes to our understanding of subsoil processes of paddy soils.     

Methane and nitrous oxide emissions from direct-seeded and seedling-transplanted rice paddies in southeast China

Background and aims

The rice production is experiencing a shift from conventionally seedling-transplanted (TPR) to direct-seeded (DSR) cropping systems in Southeast Asia. Besides the difference in rice crop establishment, water regime is typically characterized as water-saving moist irrigation for DSR and flooding-midseason drainage-reflooding and moist irrigation for TPR fields, respectively. A field experiment was conducted to quantify methane (CH₄) and nitrous oxide (N₂O) emissions from the DSR and TPR rice paddies in southeast China.

Methods

Seasonal measurements of CH₄ and N₂O fluxes from the DSR and TPR plots were simultaneously taken by static chamber-GC technique.

Results

Seasonal fluxes of CH₄ averaged 1.58 mg m^?2 h^?1 and 1.02 mg m^?2 h^?1 across treatments in TPR and DSR rice paddies, respectively. Compared with TPR cropping systems, seasonal N₂O emissions from DSR cropping systems were increased by 49 % and 46 % for the plots with or without N application, respectively. The emission factors of N₂O were estimated to be 0.45 % and 0.69 % of N application, with a background emission of 0.65 and 0.95 kg N₂O-N ha^?1 under the TPR and DSR cropping regimes, respectively. Rice biomass and grain yield were significantly greater in the DSR than in the TPR cropping systems. The net global warming potential (GWP) of CH₄ and N₂O emissions were comparable between the two cropping systems, while the greenhouse gas intensity (GHGI) was significantly lower in the DSR than in the TPR cropping systems.

Conclusions

Higher grain yield, comparable GWP, and lower GHGI suggest that the DSR instead of conventional TPR rice cropping regime would weaken the radiative forcing of rice production in terms of per unit of rice grain yield in China, and DSR rice cropping regime could be a promising rice development alternative in mainland China.     

Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L.)

Overexpression of NHX genes has been previously shown to improve salt tolerance of transgenic plants. In this study, transgenic rice plants overexpressing AtNHX5 showed not only high salt tolerance, but also high drought tolerance. Measurements of ion levels indicated that Na⁺ and K⁺ contents were all higher in AtNHX5 overexpressing shoots than in wild type (WT) shoots in high saline conditions. After exposure to water deficiency and salt stress, the WT plants all died, while the AtNHX5 overexpressing rice plants had a higher survival rate, dry weight, leaf water content, and leaf chlorophyll contents, accumulated more proline, and had less membrane damage than the WT plants. In addition, seeds of both transgenic and WT plants germinated on 1/2 MS medium supplemented with 250 mM mannitol, but overexpression of AtNHX5 improved the shoot growth of the seedlings. Taken together, the results indicate that AtNHX5 gene could enhance the tolerance of rice plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions or proline) to counter the osmotic stress caused by abiotic factors.     

Assessing the performance of the photo‐acoustic infrared gas monitor for measuring CO2, N2O,and CH4 fluxes in two major cereal rotations

Rapid, precise, and globally comparable methods for monitoring greenhouse gas (GHG) fluxes are required for accurate GHG inventories from different cropping systems and management practices. Manual gas sampling followed by gas chromatography (GC) is widely used for measuring GHG fluxes in agricultural fields, but is laborious and time‐consuming. The photo‐acoustic infrared gas monitoring system (PAS) with on‐line gas sampling is an attractive option, although it has not been evaluated for measuring GHG fluxes in cereals in general and rice in particular. We compared N₂O, CO_2, and CH₄ fluxes measured by GC and PAS from agricultural fields under the rice–wheat and maize–wheat systems during the wheat (winter), and maize/rice (monsoon) seasons in Haryana, India. All the PAS readings were corrected for baseline drifts over time and PAS‐CH₄ (PCH₄) readings in flooded rice were corrected for water vapor interferences. The PCH₄ readings in ambient air increased by 2.3 ppm for every 1000 mg cm^?3 increase in water vapor. The daily CO₂, N₂O, and CH₄ fluxes measured by GC and PAS from the same chamber were not different in 93–98% of all the measurements made but the PAS exhibited greater precision for estimates of CO₂ and N₂O fluxes in wheat and maize, and lower precision for CH₄ flux in rice, than GC. The seasonal GC‐ and PAS‐N₂O (PN₂O) fluxes in wheat and maize were not different but the PAS‐CO₂ (PCO₂) flux in wheat was 14–39% higher than that of GC. In flooded rice, the seasonal PCH₄ and PN₂O fluxes across N levels were higher than those of GC‐CH₄ and GC‐N₂O fluxes by about 2‐ and 4fold, respectively. The PAS (i) proved to be a suitable alternative to GC for N₂O and CO₂ flux measurements in wheat, and (ii) showed potential for obtaining accurate measurements of CH₄ fluxes in flooded rice after making correction for changes in humidity.     

On the identification of ionic species of neutral halogen dimers, monomers and pincer type palladacycles in solution by electrospray mass and tandem mass spectrometry

Electrospray (ESI) mass spectra analysis of acetonitrile solutions of a series of neutral chloro dimers, pincer type, and monomeric palladacycles has enabled the detection of several of their derived ionic species. The monometallic cationic complexes Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (1a) and [Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)]⁺ (1b) and the bimetallic cationic complex [κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]Pd-Cl-Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (1c) were detected from an acetonitrile solution of the pincer palladacycles Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](Cl) 1. For the dimeric compounds {Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](μ-Cl)}₂ (2, Y=H and 3, CF₃), highly electronically unsaturated palladacycles [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (2d, 3d) and their mono and di-acetonitrile adducts, namely, [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)]⁺ (2e, 3e) and [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)₂]⁺ (2f and 3f) were detected together with the bimetallic complex [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]-Cl-Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N](CH₃)₂]⁺ (2a, 3a) and its acetonitrile adducts [κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)Pd-Cl-Pd[ κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (2b, 3b) and [κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)Pd-Cl-Pd[κ¹-C, κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂(CH₃CN)]⁺ (2c, 3c). The dimeric palladacycle {Pd[κ¹-C,κ¹-N-C(CH₃O-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](μ-Cl)}₂ (4) is unique as it behaves as a pincer type compound with the OCH₃ substituent acting as an intramolecular coordinating group which prevents acetonitrile full coordination, thus forming the cationic complexes [(C₆H₄(o-CH₃O)CC(Cl)CH₂N(CH₃)₂-κO,κC,κN)Pd]⁺ (4b), [(C₆H₄(o-CH₃O)CC(Cl)CH₂N(CH₃)₂- κO,κC,κN)Pd(CH₃CN)]⁺ (4c) and [(C₆H₄ (o-MeO)CC(Cl)CH₂N(CH₃)₂-κO, κC,κN)Pd-Cl-Pd(C₆H₄(o-CH₃O)CC(Cl)CH₂N(CH₃)₂-κO,κC,κN)]⁺ (4a). ESI-MS spectra analysis of acetonitrile solutions of the monomeric palladacycles Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](Cl)(Py) (5, Y=H and 6, Y=CF₃) allows the detection of some of the same species observed in the spectra of the dimeric palladacycles, i.e., monometallic cationic 2d-3d, 2e-3e and {Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](Py)}⁺ (5a, 6a) and {Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)(Py)}⁺ (5b, 6b) and the bimetallic 2a, 3a, 2b, 3b, 2c and 3c. In all cationic complexes detected by ESI-MS, the cyclometallated moiety was intact indicating the high stability of the four or six electron anionic chelate ligands. The anionic (chloride) or neutral (pyridine) ligands are, however, easily replaced by the acetonitrile solvent.     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

UV-B辐射增强对抗除草剂转基因水稻 CH4排放的影响

在大田条件下,研究了UV-B(ultraviolet-B)辐射增强对抗除草剂转基因水稻及亲本常规水稻甲烷(CH4)排放的影响。UV-B辐射设2水平,即对照(CK,自然光),增强(Elevated,14.4 kJ·m-·2d-1),相当于南京地区大气臭氧耗损25%的辐射剂量。结果表明,UV-B辐射增强并没有改变稻田CH4排放通量的季节性变化规律。与对照相比,UV-B辐射增强显著提高CH4排放通量和累积排放量。水稻分蘖期CH4累积排放量最高,占全生育累积排放量的51.55%—61.01%;其次是拔节至孕穗期,占20.00%—26.64%。抗除草剂转基因水稻的CH4排放通量和累积排放量显著低于亲本常规水稻。研究说明,UV-B辐射增强下种植抗除草剂转基因水稻对于减缓稻田甲烷排放有积极意义。     

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

The intramolecular distribution of nitrogen isotopes in N₂O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N₂O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N₂O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N₂O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N₂O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ~33‰ and ~0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N₂O.     

Emissions of CH4 and N2O under Different Tillage Systems from Double-Cropped Paddy Fields in Southern China

Understanding greenhouse gases (GHG) emissions is becoming increasingly important with the climate change. Most previous studies have focused on the assessment of soil organic carbon (SOC) sequestration potential and GHG emissions from agriculture. However, specific experiments assessing tillage impacts on GHG emission from double-cropped paddy fields in Southern China are relatively scarce. Therefore, the objective of this study was to assess the effects of tillage systems on methane (CH₄) and nitrous oxide (N₂O) emission in a double rice (Oryza sativa L.) cropping system. The experiment was established in 2005 in Hunan Province, China. Three tillage treatments were laid out in a randomized complete block design: conventional tillage (CT), rotary tillage (RT) and no-till (NT). Fluxes of CH₄ from different tillage treatments followed a similar trend during the two years, with a single peak emission for the early rice season and a double peak emission for the late rice season. Compared with other treatments, NT significantly reduced CH₄ emission among the rice growing seasons (P<0.05). However, much higher variations in N₂O emission were observed across the rice growing seasons due to the vulnerability of N₂O to external influences. The amount of CH₄ emission in paddy fields was much higher relative to N₂O emission. Conversion of CT to NT significantly reduced the cumulative CH₄ emission for both rice seasons compared with other treatments (P<0.05). The mean value of global warming potentials (GWPs) of CH₄ and N₂O emissions over 100 years was in the order of NT<RT<CT, which indicated NT was significantly lower than both CT and RT (P<0.05). This suggests that adoption of NT would be beneficial for GHG mitigation and could be a good option for carbon-smart agriculture in double rice cropped regions.