A conditional marker gene allowing both positive and negative selection in plants

Selectable markers enable transgenic plants or cells to be identified after transformation. They can be divided into positive and negative markers conferring a selective advantage or disadvantage, respectively. We present a marker gene, dao1, encoding D-amino acid oxidase (DAAO, EC 1.4.3.3) that can be used for either positive or negative selection, depending on the substrate. DAAO catalyzes the oxidative deamination of a range of D-amino acids. Selection is based on differences in the toxicity of different D-amino acids and their metabolites to plants. Thus, D-alanine and D-serine are toxic to plants, but are metabolized by DAAO into nontoxic products, whereas D-isoleucine and D-valine have low toxicity, but are metabolized by DAAO into the toxic keto acids 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively. Hence, both positive and negative selection is possible with the same marker gene. The marker has been successfully established in Arabidopsis thaliana, and proven to be versatile, rapidly yielding unambiguous results, and allowing selection immediately after germination

Symbiosis constraints : strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa-a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N2-fixing cyanobacteria (Nostoc sp.)-as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N-fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ13C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO2 among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth

Decadal nitrogen addition alters chemical composition of soil organic matter in a boreal forest

Boreal forests store approximately 470 Pg of carbon (C) in the soil, and rates of soil C accumulation are significantly enhanced by long-term nitrogen (N) enrichment. Dissecting the compositional profile of soils could help better understand the potential mechanisms driving changes in C cycling under enriched N conditions.We examined the impacts of long-term N addition on the chemical composition of soil organic matter (SOM) in a mature boreal forest. Two large experimental plots (15 ha each) were established: a control and a fertilised plot. The latter received NH4NO3 fertilizer at an average rate of 75 kg N ha(-1) year for 12 years. While the centre of this plot received the prescribed amounts of fertilizer, the year-to-year variation in distribution of fertilizer around the designated edges of the plot created a gradient in N-loading. Along this gradient, a compositional shift in SOM in the organic horizon was assessed using two methods: pyrolysis-gas chromatography/mass spectrometry (GC/MS) and solid-state C-13 nuclear magnetic resonance spectroscopy (C-13 NMR).Both of these methods revealed that the chemical composition of SOM changed with increasing N loading, with an increased fraction of lignin derivatives (i.e., aromatic, methoxy/N-alkyl C) relative to that of carbohydrate (i.e., 0-alkyl C), accompanied by increased soil C mass (kg m(-2)) at the fertilised plot. Also, the relative abundance of N compounds in the pyrolysis products increased with the N loading, mainly due to increased methyl N-acetyl-alpha(D)-glucosaminide in the F/H horizon, plausibly of microbial origin. Microbial N processing likely contributed to soil accumulation of fertilizer-derived N.Our results corroborate the hypothesis that addition of inorganic N suppresses enzymatic white-rot decomposition relative to non-enzymatic brown-rot oxidation. Taken together, our study suggests that N enrichment leads to a selective accumulation of lignin-derived compounds and points to a key role of such compounds for N-induced SOM accumulation

Swedish forest growth decline: A consequence of climate warming?

Following an almost century-long increase, forest growth in Sweden has abruptly decreased during the last decade. Lower than expected forest biomass trajectories threaten national targets for carbon sequestration and bioeconomy. While climate-related drought is the most likely cause, the critical question is whether this recent growth decline is transient, or the beginning of a new normal where conventional management actions may risk further losses of resilience to water stress. We argue that improved mechanistic insights through better integrated research are urgently needed to avoid worsening the situation and further delaying necessary actions

Organic nitrogen enhances nitrogen nutrition and early growth of Pinus sylvestris seedlings

Boreal trees are capable of taking up organic nitrogen (N) as effectively as inorganic N. Depending on the abundance of soil N forms, plants may adjust physiological and morphological traits to optimize N uptake. However, the link between these traits and N uptake in response to soil N sources is poorly understood. We examined Pinus sylvestris L. seedlings' biomass growth and allocation, transpiration and N uptake in response to additions of organic N (the amino acid arginine) or inorganic N (ammonium nitrate). We also monitored in situ soil N fluxes in the pots following an addition of N, using a microdialysis system. Supplying organic N resulted in a stable soil N flux, whereas the inorganic N resulted in a sharp increase of nitrate flux followed by a rapid decline, demonstrating a fluctuating N supply and a risk for loss of nitrate from the growth medium. Seedlings supplied with organic N achieved a greater biomass with a higher N content, thus reaching a higher N recovery compared with those supplied inorganic N. In spite of a higher N concentration in organic N seedlings, root-to-shoot ratio and transpiration per unit leaf area were similar to those of inorganic N seedlings. We conclude that enhanced seedlings' nutrition and growth under the organic N source may be attributed to a stable supply of N, owing to a strong retention rate in the soil medium

The carbon bonus of organic nitrogen enhances nitrogen use efficiency of plants

The importance of organic nitrogen (N) for plant nutrition and productivity is increasingly being recognized. Here we show that it is not only the availability in the soil that matters, but also the effects on plant growth. The chemical form of N taken up, whether inorganic (such as nitrate) or organic (such as amino acids), may significantly influence plant shoot and root growth, and nitrogen use efficiency (NUE). We analysed these effects by synthesizing results from multiple laboratory experiments on small seedlings (Arabidopsis, poplar, pine and spruce) based on a tractable plant growth model. A key point is that the carbon cost of assimilating organic N into proteins is lower than that of inorganic N, mainly because of its carbon content. This carbon bonus makes it more beneficial for plants to take up organic than inorganic N, even when its availability to the roots is much lower - up to 70% lower for Arabidopsis seedlings. At equal growth rate, root:shoot ratio was up to three times higher and nitrogen productivity up to 20% higher for organic than inorganic N, which both are factors that may contribute to higher NUE in crop production

Using microdialysis with a deuterium oxide tracer to estimate water exchange, water content and active surface area of the probe

Microdialysis is a useful tool for measuring in situ fluxes of soil compounds with minimal disturbance of soil structure and function. Fluxes of sampled compounds are commonly calculated per unit of membrane surface area, assuming that the entire membrane surface is capable of exchange - which is unlikely given varying soil moisture and the occlusion of membrane pores by the soil solid phase. We present a method to quantify the degree of connectivity of the microdialysis probe membrane to the surrounding soil by means of water exchange between a microdialysis perfusate and soil solution using deuterium (2H2O; equilibrated to DHO) as an internal standard. We applied the method to a range of probe membrane surface areas and soil moisture conditions to generate empirical models that estimate membrane surface area active in exchange. Our results suggest that even in a saturated sandy soil, active membrane surface areas reach only 40.3% of the probe surface area, perhaps due to occlusion by soil particles. However, when accounting for volumetric water content of the soil, active surface areas approached 80-90% of the area likely in contact with water, indicating that sampling efficiency of waterfilled pores may still be high, particularly at slow flow rates. Furthermore, our method enables assessment of local soil water content around the probe. Models estimating soil water content were applied to field measurements of DHO exchange in three soil horizons (Organic, B1, B2) at two boreal sites, and in situ estimates were similar to those from conventional soil moisture methods when models were calibrated with the same soil type. We present DHO exchange as a powerful method for improving microdialysis flux interpretations in future studies, and for exploring small-scale water variability in relatively undisturbed soils