When less is more - Endogenous tagging with TurboID increases the sensitivity of proximity labelling-based experiments

In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Generally, protein fusions with labelling enzymes are transiently overexpressed to perform these experiments. Using a pipeline for the rapid generation CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. We found TurboID and its shorter variant miniTurboID to be superior above other labelling enzymes at physiological expression levels. Endogenous tagging of the μ subunit of the AP-1 complex increased the sensitivity for detection of interactors in a proximity labelling experiment and resulted in a more comprehensive mass spectrometry data set. We were able to identify several known interactors of the complex and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry data in just over a month.

Additional carbon stabilization in temperate subsoils impeded by biogeochemical and hydraulic constraints

<p>Formation of mineral-associated organic matter (MAOM) is a decisive process in the stabilization of OM against rapid microbial decomposition and thus in the soils’ role as global carbon (C) sink. Sorption experiments of dissolved OM (DOM) repeatedly showed that particularly mineral subsoils have a large sorption capacity to retain more C. However, there is also an increasing body of literature, revealing an increasing output of dissolved organic C (DOC) from soils. Here, we investigated into this paradox in forest soil under beech by a combination of a field labelling experiment with <sup>13</sup>C-enriched litter with a unique DO<sup>13</sup>C and <sup>13</sup>CO<sub>2</sub> monitoring, an in-situ C exchange experiment with <sup>13</sup>C-coated minerals, and batch sorption experiments.</p><p>Within two years of <sup>13</sup>C monitoring, only 0.5% of litter-derived DO<sup>13</sup>C entered the subsoil, where it was only short-term stabilized by formation of MAOM but prone to fast microbial mineralization. The <sup>13</sup>C monitoring, sorption/desorption experiments in the laboratory, and also the in-situ C exchange on buried soil minerals revealed that there is a frequent exchange of DOM with native OM and a preferential desorption of recently retained OM. Hence, there appeared to be a steady-state equilibrium between C input and output, facilitated by exchange and microbial mineralization of an adopted microbial community. The remobilized OM was also richer in less sorptive carbohydrates. Along with transport of most of DOM along preferential paths, this further increased the discrepancy between laboratory-measured sorption capacities of subsoil and the actual C loading of minerals. Finally, the <sup>13</sup>C labeling experiments revealed that input of fresh litter-derived OM into subsoil may even mobilize old-soil derived OM. Hence, in the field different biogeochemical constraints are acting that prevent that the laboratory-based C sink can be reached in the field.  We conclude, that forest subsoils can hardly be considered as additional C sink, even at management options that increase DOC input to subsoil.</p>

Vertical partitioning of CO₂ production in a forest soil

Large amounts of total organic carbon are temporarily stored in soils, which makes soil respiration one of the major sources of terrestrial CO2 fluxes within the global carbon cycle. More than half of global soil organic carbon (SOC) is stored in subsoils (below 30 cm), which represent a significant carbon (C) pool. Although several studies and models have investigated soil respiration, little is known about the quantitative contribution of subsoils to total soil respiration or about the sources of CO2 production in subsoils. In a 2-year field study in a European beech forest in northern Germany, vertical CO2 concentration profiles were continuously measured at three locations, and CO2 production was quantified in the topsoil and the subsoil. To determine the contribution of fresh litter-derived C to CO2 production in the three soil profiles, an isotopic labelling experiment, using 13C-enriched leaf litter, was performed. Additionally, radiocarbon measurements of CO2 in the soil atmosphere were used to obtain information about the age of the C source in the CO2 production. At the study site, it was found that 90 % of total soil respiration was produced in the first 30 cm of the soil profile, where 53 % of the SOC stock is stored. Freshly labelled litter inputs in the form of dissolved organic matter were only a minor source for CO2 production below a depth of 10 cm. In the first 2 months after litter application, fresh litter-derived C contributed, on average, 1 % at 10 cm depth and 0.1 % at 150 cm depth to CO2 in the soil profile. Thereafter, its contribution was less than 0.3 % and 0.05 % at 10 and 150 cm depths, respectively. Furthermore CO2 in the soil profile had the same modern radiocarbon signature at all depths, indicating that CO2 in the subsoil originated from young C sources despite a radiocarbon age bulk SOC in the subsoil. This suggests that fresh C inputs in subsoils, in the form of roots and root exudates, are rapidly respired, and that other subsoil SOC seems to be relatively stable. The field labelling experiment also revealed a downward diffusion of 13CO2 in the soil profile against the total CO2 gradient. This isotopic dependency should be taken into account when using labelled 13C and 14C isotope data as an age proxy for CO2 sources in the soil.

Soil nutrient availability alters tree carbon allocation dynamics during drought

Drought alters allocation patterns of carbon (C) and nutrients in trees and eventually impairs tree functioning. Elevated soil nutrient availability might alter the response of trees to drought. We hypothesize that increased soil nutrient availability stimulates root metabolism and carbon allocation to belowground tissues under drought stress. To test this hypothesis, we subjected three-year-old Pinus sylvestris saplings in open-top cambers during two subsequent years to drought using three different water treatments (100%, 20% and 0% plant available water in the soil) and two soil nutrient regimes (ambient and nitrogen-phosphorus-potassium (N-P-K) fertilization corresponding to 5 g N/m2/yr) and released drought thereafter. We conducted a 15N and 13C labelling experiment during the peak of the first-year drought by injecting 15N labelled fertilizer in the soil and exposing the tree canopies to 13C labelled CO2. The abundance of the N and C isotopes in the roots, stem and needles was assessed during the following year. C uptake was slightly lower in drought stressed trees, and extreme drought inhibited largely the N uptake and transport. Carbon allocation to belowground tissues was decreased under drought, but not in combination with fertilization. Our results indicate a potential positive feedback loop, where fertilization improved the metabolism and functioning of the roots, stimulating C allocation to belowground tissues. This way, soil nutrients compensated for drought-induced loss of root functioning, mitigating drought stress of trees.

Soil nutrient availability alters tree carbon allocation dynamics during drought

Drought alters allocation patterns of carbon (C) and nutrients in trees and eventually impairs tree functioning. Elevated soil nutrient availability might alter the response of trees to drought. We hypothesize that increased soil nutrient availability stimulates root metabolism and carbon allocation to belowground tissues under drought stress. To test this hypothesis, we subjected three-year-old Pinus sylvestris saplings in open-top cambers during two subsequent years to drought using three different water treatments (100%, 20% and 0% plant available water in the soil) and two soil nutrient regimes (ambient and nitrogen-phosphorus-potassium (N-P-K) fertilization corresponding to 5 g N/m2/yr) and released drought thereafter. We conducted a 15N and 13C labelling experiment during the peak of the first-year drought by injecting 15N labelled fertilizer in the soil and exposing the tree canopies to 13C labelled CO2. The abundance of the N and C isotopes in the roots, stem and needles was assessed during the following year. C uptake was slightly lower in drought stressed trees, and extreme drought inhibited largely the N uptake and transport. Carbon allocation to belowground tissues was decreased under drought, but not in combination with fertilization. Our results indicate a potential positive feedback loop, where fertilization improved the metabolism and functioning of the roots, stimulating the source activity and hence C allocation to belowground tissues. This way, soil nutrients compensated for drought-induced loss of root functioning, mitigating drought stress of trees.

Contribution of the carbon sources involved in latex regeneration in rubber trees (Hevea brasiliensis): an in situ 13CO2 labelling experiment

<p>Rubber trees (<em>Hevea brasiliensis</em>) are the main source of natural rubber, extracted from latex, which exudes from the trunk after tapping. Tapped trees require large amounts of carbon (C) to regenerate the latex after its collection. Knowing the contribution of C sources involved in latex biosynthesis will help understand how rubber trees face this additional C demand. Whole crown<sup> 13</sup>CO<sub>2 </sub>pulse labelling was performed on 4-year-old rubber trees in June when latex production was low and in October, when it was high. <sup>13</sup>C contents were quantified in the foliage, phloem sap, wood and latex. In both labelling periods, <sup>13</sup>C was recovered in latex just after labelling, indicating that part of the carbohydrates was directly allocated to latex. However, significant <sup>13</sup>C amounts were still recovered in latex after 100 days and the peak was reached significantly later than in phloem sap, demonstrating the contribution of a reserve pool as a source of latex C. The contribution of new photosynthates to latex regeneration was faster and higher when latex metabolism was well established, in October than in June. An improved understanding of C dynamics and source-sink relationship in rubber tree is crucial to adapt tapping system practices and ensure sustainable latex production.</p>