Aims: Root hairs are important for uptake, especially for nutrients with low mobility in soils with high sorption capacity. Mutants with defective root hairs are expected to have lower nutrient uptake, unless they compensate with more root growth. Since root hairs can also contribute to the plant's water uptake their importance could change over the course of a growing season. It was our objective to investigate the role of root hairs under field conditions. Methods: The root hair mutant rth3 of Zea mays and the corresponding wild-type were grown for two years under field conditions on sand and loam. Results: Shoot growth and P and K uptake of the plants were promoted by the presence of hairs at all growth stages. Differences between genotypes were greater on loam than on sand until tassel emergence, presumably as additional exploitation by hairs is more relevant in loam. Compensation for the absence of root hairs by increased root growth was not observed in absolute terms. The root to shoot ratio was higher for rth3 than for wild-type. Root traits showed high plasticity in response to texture, the most salient being a greater mean root diameter in sand, irrespective of genotype. The mechanism causing the increase in mean root diameter is still unknown. Root length density was higher in sand, which can be explained by a greater need for exploration than exploitation in this substrate. Conclusion: The role of hairs for nutrient uptake could be confirmed under field conditions. The large impact of texture on root growth and consequences for carbon balance require further investigations.

Soil salinity is considered one of the most severe abiotic stresses in plants; plant acclimation to salinity could be a tool to improve salt tolerance even in a sensitive genotype. In this work we investigated the physiological mechanisms underneath the response to gradual and prolonged exposure to sodium chloride in cultivars of Brassica napus L. Fifteen days old seedlings of the cultivars Dynastie (salt tolerant) and SY Saveo (salt sensitive) were progressively exposed to increasing soil salinity conditions for 60 days. Salt exposed plants of both cultivars showed reductions of biomass, size and number of leaves. However, after 60 days the relative reduction in biomass was lower in sensitive cultivar as compared to tolerant ones. An increase of chlorophylls content was detected in both cultivars; the values of the quantum efficiency of PSII photochemistry (ΦPSII) and those of the electron transport rate (ETR) indicated that the photochemical activity was only partially reduced by NaCl treatments in both cultivars. Ascorbate peroxidase (APX) activity was higher in treated samples with respect to the controls, indicating its activation following salt exposure, and confirming its involvement in salt stress response. A gradual exposure to salt could elicit different salt stress responses, thus preserving plant vitality and conferring a certain degree of tolerance, even though the genotype was salt sensitive at the seed germination stage. An improvement of salt tolerance in B. napus could be obtained by acclimation to saline conditions.

Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value < 0.05). All approaches revealed the same trend between genotypes, namely up-regulation of most metabolites in the root hair defective mutant (rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20–79 features, 13–31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula. Graphical Abstract

Purpose: Root exudates are key components driving belowground interaction between plant, microbes and soil. High-end analytical approaches provide advanced insights into exudate metabolite diversity, however, the amount of total carbon (C) released by roots should always be determined as the most basic parameter when characterizing root exudation as it (i) provides quantitative information of C exuded into the surrounding soil and (ii) allows to relate the abundance of individual exudate compounds to total C released. Here we propose a simple and quick, spectrophotometry-based method to quantify total dissolved organic carbon (DOC) concentration in exudation samples that is based on measuring the absorption of a pre-filtered but otherwise untreated exudate sample at 260 nm (DOC₂₆₀). Method: Exudate samples collected from different grass genotypes (Zea mays, Oryza sativa, Hordeum vulgare) grown in various experimental settings (soil, hydroponic) were analysed with the DOC₂₆₀ assay and results were compared with C concentrations obtained by liquid TOC-analyser. Conclusion: We demonstrated that the DOC₂₆₀ method allowed for quick and inexpensive measurements of total dissolved organic carbon concentrations in exudate samples from grass species grown under nutrient sufficient as well as under P deficient conditions. Interestingly, DOC₂₆₀ failed to predict DOC concentrations in exudate samples from plants grown under Zn and Fe deficiency suggesting a strong shift in metabolite composition under micronutrient deficiency. Even though the applicability of the DOC₂₆₀ method remains to be tested on exudate samples originating from dicots and plants exposed to other environmental stresses (e.g. pathogen attack, heavy metal stress, etc), it will help to increase our understanding of root exudation and related rhizosphere processes in the future.

Summary: Deciphering the root exudate‐driven interplay between plants and the rhizosphere microbiota is essential for understanding plant adaptation to the environment and future‐proofing crop production. However, sampling root exudates and rhizosphere soil remains challenging due to the low throughput and destructive nature of the process. We used the staple crop barley ( Hordeum vulgare L.) as a model to benchmark different sampling approaches for simultaneous exudation and microbiota profiling of soil‐grown plants. Exudate profiles and total dissolved organic carbon exudation rates were consistent across different sampling approaches, whereas root biomass, root morphology measurements, and organic nitrogen exudation varied. High‐throughput amplicon sequencing and quantitative PCR (qPCR) of phylogenetic markers and nitrogen cycle‐selected genes revealed a protocol‐specific footprint in the composition and abundance of rhizosphere bacterial and fungal microbiota. Yet, on average, 75% of microbes enriched in and differentiating between barley rhizosphere and unplanted soil controls were recovered across all the sampling approaches evaluated. Our results demonstrated that, under the tested conditions, different sampling approaches produced comparable microbiota and exudation patterns, enabling the integrated study of root exudation and microbial profiles from the same plant. The observed differences across sampling approaches must be considered according to the experimental scope.

Background and aims: A major challenge in root exudation research is obtaining exudates samples that accurately reflect the exudation processes under natural soil growth conditions. Both growth environment and experimental setup can significantly influence root exudation dynamics. This study investigated how different experimental systems and growth conditions affect carbon exudation in maize ( Zea mays L.) roots and whether these factors could influence the detection of genotypic differences between the wild type (B73) and its hairless mutant, rth3. Methods: Maize plants were grown under various experimental conditions, including soil-based and hydroponic systems. Root exudates were collected using a combination of traditional and innovative sampling approaches. Carbon exudation rates were compared across experimental setups and genotypes. Laboratory results were further compared with data from a separate field experiment. Results: Exudation rates obtained from soil-based laboratory experiments were comparable to those observed in the field under similar growth temperatures. The contribution of root hairs to total carbon exudation was negligible compared to the effect of growth conditions and experimental setup. Large differences in root biomass introduced bias into exudation measurements, particularly when root to sampling volume ratio (RSVR) varied substantially. Conclusions: Experimental setup and environmental conditions have a strong influence on root exudation measurement. Soil-based laboratory systems that closely replicate field conditions, particularly temperature, can serve as reliable proxies for field experiments, providing ecologically meaningful data. Maintaining a consistent RSVR is also essential for obtaining accurate and comparable results. These findings offer important methodological guidance for reliably quantifying root carbon exudation in maize