Regulation of N2O emissions from agricultural soil on the transcriptional level

一氧化二氮（N2O）是一种重要的温室气体，也是21世纪唯一最有效的臭氧消耗物质。全球大约62%的N2O排放来自土壤，其中很大一部分是作为细菌反硝化的中间物释放的，即硝酸盐（NO3-）逐步还原为二氮气体（N2）。除了反硝化群落的组成外，介导反硝化四个步骤的基因产物的活性在很大程度上决定了反硝化过程的效率和气态终产物组成（N2O vs . N2）。以往的研究主要集中在群落组成对田间反硝化活性的作用，或通过分离菌株的实验室实验确定pH和O2分压等独立因素对不同反硝化基因表达的影响。然而，阐明环境条件与田间样品中反硝化基因表达之间关系的研究很少，尽管这类研究将为我们提供所需的数据，以便预测哪种土壤管理方式会导致反硝化N2O排放的增加或减少。因此，本项目旨在利用一个超过50年的施肥试验作为田间场地，确定不同施肥制度、反硝化速率和反硝化基因表达之间的相关性。在这个现场，使用了两种不同的矿物肥料（硫酸铵和硝酸钙）和两种不同的有机肥料（污水污泥和牛粪）的土壤样本将与未施肥的土壤样本进行比较。在这些样品中，我们将量化两种不同的亚硝酸盐（NO2-）还原酶基因的基因和mRNA转录数，并与潜在的反硝化速率进行比较。N2O还原酶基因也是如此，其产物是唯一已知的N2O消耗介质。鉴于N2O还原酶的独特作用，个体样品中编码该酶的基因和转录序列的多样性有待进一步分析，因为该基因表达变体之间的差异很可能在N2O降解调控中发挥关键作用。一旦确定了导致反硝化基因表达增加或减少的施肥制度，将利用微观实验确定这种效应发挥作用的速度。这些将进一步用于阐明哪些主要因子增加或降低NO2-和N2O还原酶基因的转录率。因此，通过在遗传水平上揭示不同环境条件下N2O生产和消耗的调控，我们的研究将有助于更可靠地预测如何通过相应的施肥来减少N2O的排放。

Nitrous oxide (N2O) is an important greenhouse gas and the single most potent ozone-depleting substance of the 21st century. About 62 % of total global N2O emissions stem from soils, a great fraction of which is released as an intermediate of bacterial denitrification, the stepwise reduction of nitrate (NO3-) to di-nitrogen gas (N2). Besides the composition of the denitrifying community, the activities of the gene products that mediate the four steps of denitrification largely determine the efficiency and gaseous end product composition (N2O versus N2) of this process. Previous studies have focused on the role of community composition on denitrification activity in the field or identified detached factors such as pH and O2 partial pressure that have an influence on the expression of the different denitrification genes using laboratory experiments with isolated strains. However, studies elucidating the connection between environmental conditions and expression of denitrification genes in field samples are scarce, even though this kind of studies would provide us with the data we need in order to predict which ways of soil management will lead to an increase or decrease of N2O emissions from denitrification. Consequently, this project aims to identify correlations between different fertilization regimes, denitrification rates and expression of denitrification genes using a more than 50 year-old fertilization experiment as a field site. From this field site, samples from soils which have received two different mineral (ammonium sulfate and calcium nitrate) and two different organic (sewage sludge and cattle manure) fertilizers will be compared to samples from unfertilized soil. Gene and mRNA transcript numbers of two different nitrite (NO2-) reductase genes whose products are involved in N2O production will be quantified in these samples and compared to potential denitrification rates. The same will be done for the N2O reductase gene, the product of which is the only known mediator of N2O consumption. Given the unique role of the N2O reductase, the diversity among the gene and transcript sequences encoding this enzyme within the individual samples will be subject to further analysis, since the differences among the expressed variants of this gene are very likely to play a key role in the regulation of N2O degradation. Once fertilization regimes that lead to an increase or decrease of the expression of the denitrification genes have been identified, the speed with which this effect comes into play will be determined using microcosm experiments. These will further be used in order to elucidate which primary factors increase or decrease the transcription rate of NO2-- and N2O reductase genes. Thus, by sheding light on the regulation of the production and consumption of N2O under different environmental conditions on the genetic level, our study will help to predict more reliably how N2O emissions can be reduced by fertilizing arable soil accordingly.