Transcriptomic, Metabolomic and Ionomic Analyses Reveal Early Modulation of Leaf Mineral Content in Brassica napus under Mild or Severe Drought.

While it is generally acknowledged that drought is one of the main abiotic factors affecting plant growth, how mineral nutrition is specifically and negatively affected by water deficit has received very little attention, other than being analyzed as a consequence of reduced growth. Therefore, Brassica napus plants were subjected to a gradual onset of water deficits (mild, severe, or severe extended), and leaves were analyzed at the ionomic, transcriptomic and metabolic levels. The number of Differentially Expressed Genes (DEGs) and of the most differentially accumulated metabolites increased from mild (525 DEGs, 57 metabolites) to severe (5454 DEGs, 78 metabolites) and severe extended (9346 DEGs, 95 metabolites) water deficit. Gene ontology enrichment analysis of the 11,747 DEGs identified revealed that ion transport was one of the most significant processes affected, even under mild water deficit, and this was also confirmed by the shift in ionomic composition (mostly micronutrients with a strong decrease in Mo, Fe, Zn, and Mn in leaves) that occurred well before growth reduction. The metabolomic data and most of the transcriptomic data suggested that well-known early leaf responses to drought such as phytohormone metabolism (ABA and JA), proline accumulation, and oxidative stress defense were induced later than repression of genes related to nutrient transport.

Comparative Omics Analysis of Brassica napus Roots Subjected to Six Individual Macronutrient Deprivations Reveals Deficiency-Specific Genes and Metabolomic Profiles.

The early and specific diagnosis of a macronutrient deficiency is challenging when seeking to better manage fertilizer inputs in the context of sustainable agriculture. Consequently, this study explored the potential for transcriptomic and metabolomic analysis of Brassica napus roots to characterize the effects of six individual macronutrient deprivations (N, Mg, P, S, K, and Ca). Our results showed that before any visual phenotypic response, all macronutrient deprivations led to a large modulation of the transcriptome and metabolome involved in various metabolic pathways, and some were common to all macronutrient deprivations. Significantly, comparative transcriptomic analysis allowed the definition of a subset of 3282, 2011, 6325, 1384, 439, and 5157 differentially expressed genes (DEGs) specific to N, Mg, P, S, K, and Ca deprivations, respectively. Surprisingly, gene ontology term enrichment analysis performed on this subset of specific DEGs highlighted biological processes that are common to a number of these macronutrient deprivations, illustrating the complexity of nutrient interactions. In addition, a set of 38 biochemical compounds that discriminated the macronutrient deprivations was identified using a metabolic approach. The opportunity to use these specific DEGs and/or biochemical compounds as potential molecular indicators to diagnose macronutrient deficiency is discussed.

Nutrient deficiencies modify the ionomic composition of plant tissues: a focus on cross-talk between molybdenum and other nutrients in Brassica napus.

The composition of the ionome is closely linked to a plant's nutritional status. Under certain deficiencies, cross-talk induces unavoidable accumulation of some nutrients, which upsets the balance and modifies the ionomic composition of plant tissues. Rapeseed plants (Brassica napus L.) grown under controlled conditions were subject to individual nutrient deficiencies (N, K, P, Ca, S, Mg, Fe, Cu, Zn, Mn, Mo, or B) and analyzed by inductively high-resolution coupled plasma mass spectrometry to determine the impact of deprivation on the plant ionome. Eighteen situations of increased uptake under mineral nutrient deficiency were identified, some of which have already been described (K and Na, S and Mo, Fe, Zn and Cu). Additionally, as Mo uptake was strongly increased under S, Fe, Cu, Zn, Mn, or B deprivation, the mechanisms underlying the accumulation of Mo in these deficient plants were investigated. The results suggest that it could be the consequence of multiple metabolic disturbances, namely: (i) a direct disturbance of Mo metabolism leading to an up-regulation of Mo transporters such as MOT1, as found under Zn or Cu deficiency, which are nutrients required for synthesis of the Mo cofactor; and (ii) a disturbance of S metabolism leading to an up-regulation of root SO42- transporters, causing an indirect increase in the uptake of Mo in S, Fe, Mn, and B deficient plants.

Using plant traits to explain plant-microbe relationships involved in nitrogen acquisition.

It has long been recognized that plant species and soil microorganisms. are tightly linked, but understanding how different species vary in their effects on soil is currently limited. In this study, we identified those. plant characteristics (identity, specific functional traits, or resource acquisition strategy) that were the best predictors of nitrification and denitrification processes. Ten plant populations representing eight species collected from three European grassland sites were chosen for their contrasting plant trait values and resource acquisition strategies. For each individual plant, leaf and root traits and the associated potential microbial activities (i.e., potential denitrification rate [DEA], maximal nitrification rate [NEA], and NH4+ affinity of the microbial community [NHScom]) were measured at two fertilization levels under controlled growth conditions. Plant traits were powerful predictors of plant-microbe interactions, but relevant plant traits differed in relation to the microbial function studied. Whereas denitrification was linked to the relative growth rate of plants, nitrification was strongly correlated to root trait characteristics (specific root length, root nitrogen concentration, and plant affinity for NH4+) linked to plant N cycling. The leaf economics spectrum (LES) that commonly serves as an indicator of resource acquisition strategies was not correlated to microbial activity. These results suggest that the LES alone is not a good predictor of microbial activity, whereas root traits appeared critical in understanding plant-microbe interactions.

How to improve the hygienic quality of forages for horse feeding.

BACKGROUND: Improving the hygienic quality of forages for horse nutrition seems to be a reasonable target for decreasing the prevalence of pulmonary diseases. The aim of the experiment was to study the effects of different agricultural practices on the main aero-allergens contained in forages, including breathable dust, fungi, mycotoxins and pollens. RESULTS: Results showed that the late harvest of hay, a second crop or a haylage production provides a good alternative to increase hygienic quality by reducing fungi contamination and breathable dust content. Barn drying of hay, while having no effect on breathable dust, similarly reduced fungi contamination. In contrast, when hay was harvested at a lower dry mass content (750 g DM kg⁻¹ versus 850 g DM kg⁻¹), both breathable dust and fungi contaminations were increased, which could at least be reversed by adding propionic acid just before baling. Zearalenone was detected in different hays, and even in one case, in breathable dust. CONCLUSION: Overall, our data suggest that different approaches can be used to increase forage hygienic quality for horse feeding and thus reduce their exposure to factors involved in equine pulmonary disease.

Drought specifically downregulates mineral nutrition: Plant ionomic content and associated gene expression.

One of the main limiting factors of plant yield is drought, and while the physiological responses to this environmental stress have been broadly described, research addressing its impact on mineral nutrition is scarce. Brassica napus and Triticum aestivum were subjected to moderate or severe water deficit, and their responses to drought were assessed by functional ionomic analysis, and derived calculation of the net uptake of 20 nutrients. While the uptake of most mineral nutrients decreased, Fe, Zn, Mn, and Mo uptake were impacted earlier and at a larger scale than most physiological parameters assessed (growth, ABA concentration, gas exchanges and photosynthetic activity). Additionally, in B. napus, the patterns of 183 differentially expressed genes in leaves related to the ionome (known ionomic genes, KIGs) or assumed to be involved in transport of a given nutrient were analyzed. This revealed three patterns of gene expression under drought consisting of up (transport of Cl and Co), down (transport of N, P, B, Mo, and Ni), or mixed levels (transport of S, Mg, K, Zn, Fe, Cu, or Mn) of regulation. The three patterns of gene regulations are discussed in relation to specific gene functions, changes of leaf ionomic composition and with consideration of the crosstalks that have been established between elements. It is suggested that the observed reduction in Fe uptake occurred via a specific response to drought, leading indirectly to reduced uptake of Zn and Mn, and these may be taken up by common transporters encoded by genes that were downregulated.

Specificity and Plasticity of the Functional Ionome of Brassica napus and Triticum aestivum Exposed to Micronutrient or Beneficial Nutrient Deprivation and Predictive Sensitivity of the Ionomic Signatures.

The specific variation in the functional ionome was studied in Brassica napus and Triticum aestivum plants subjected to micronutrient or beneficial mineral nutrient deprivation. Effects of these deprivations were compared to those of macronutrient deprivation. In order to identify early events, plants were harvested after 22 days, i.e., before any significant reduction in growth relative to control plants. Root uptake, tissue concentrations and relative root nutrient contents were analyzed revealing numerous interactions with respect to the 20 elements quantified. The assessment of the functional ionome under individual mineral nutrient deficiency allows the identification of a large number of interactions between elements, although it is not totally exhaustive, and gives access to specific ionomic signatures that discriminate among deficiencies in N, P, S, K, Ca, Mn, Fe, Zn, Na, Si, and Se in both species, plus Mg, Cl, Cu, and Mo in wheat. Ionome modifications and components of ionomic signatures are discussed in relation to well-known mechanisms that may explain crosstalks between mineral nutrients, such as between Na and K, V, Se, Mo and S or Fe, Zn and Cu. More surprisingly, when deprived of beneficial nutrients such as Na, Si, Co, or Se, the plant ionome was strongly modified while these beneficial nutrients contributed greatly to the leaf ionomic signature of most mineral deficiencies.

Specificity and Plasticity of the Functional Ionome of Brassica napus and Triticum aestivum Subjected to Macronutrient Deprivation.

The composition of the functional ionome was studied in Brassica napus and Triticum aestivum with respect to the response of 20 elements under macronutrient deprivation. Analysis of relative root contents showed that some nutrients, such as Fe, Ni, Cu, Na, V, and Co, were largely sequestered in roots. After 10 days of deprivation of each one of these 6 macronutrients, plant growth was similar to control plants, and this was probably the result of remobilization from roots (Mg and Ca) or old leaves (N, P, K, S). Some tissue concentrations and net nutrient uptakes into roots were either decreased or increased, revealing multiple interactions (93 in wheat, 66 in oilseed rape) that were common to both species (48) or were species specific. While some interactions have been previously described (increased uptake of Na under K deficiency; or increased uptake of Mo and Se under S deficiency), a number of new interactions were found and some key mechanisms underlying their action have been proposed from analysis of Arabidopsis mutants. For example, nitrate uptake seemed to be functionally linked to Na(influx, while the uptake of vanadium was probably mediated by sulfate transporters whose expression was stimulated during S deprivation.

Rapeseed-legume intercrops: plant growth and nitrogen balance in early stages of growth and development.

In this study we tested whether legumes can improve the growth and N and S nutrition of rapeseed in an intercropping system and compared the effect of mixtures on legume N-fixation and soil N-resources. Rapeseed was cultivated in low N conditions in monocrops using one (R) or two plants (RR) per pot and in mixtures with lupine, clover or vetch. The R monocrop was the most relevant control, intraspecific competition inducing a significant growth delay resulting in a significantly lower leaf number, in RR monocrop compared to R and the three mixtures considered. Plant biomass, and the N and S contents of rapeseed grown in mixtures were the same than those measured in R monocrop. Compared to the monocrop, the proportion of N derived from the atmosphere was increased by 34, 140 and 290% in lupine, clover and vetch, respectively when intercropped with rapeseed. In mixture with clover and lupine, the soil N pool at harvest was higher than in other treatments, while N export by crop was constant. Legumes suffered from competition for soil S resulting in a decrease of 40% in their S content compared to the monocrop. Compared to rapeseeds grown in R monocrop and in mixture with lupine and vetch, rapeseed mixed with clover showed significantly higher SPAD values in old leaves. In our conditions, mixing legumes with rapeseed is relevant to reduce N fertilization and improve nutrition and growth of rapeseed.

Nitrogen transfer from Lupinus albus L., Trifolium incarnatum L. and Vicia sativa L. contribute differently to rapeseed (Brassica napus L.) nitrogen nutrition.

Nitrogen (N) transfer is well documented in legume-cereal intercropping but this is less often reported for legume-Brassica intercrops even though Brassica crops require higher levels of N fertilizers. The present study was carried out to quantify N transfer from legumes (Lupinus albus L., Trifolium incarnatum L. or Vicia sativa L.) to rapeseed (Brassica napus L.) using the split-root (15)N-labelling method. After three months we observed that legumes did not alter the growth of rapeseed. Vetch showed the lowest growth and demonstrated low (15)N shoot to root translocation and no significant N transfer to rapeseed. In contrast, significant (15)N enrichment was found in lupine and clover and (15)N was transferred to the associated rapeseed plants (around 6 and 4 mg N plant(-1), respectively), which contributed 2 to 3% of the rapeseed total N. Additionally, the data revealed that N2 fixation dominated the N nutrition in lupine despite the high N level provided in the donor compartment, suggesting a greater niche segregation between companion plants. Based on the results of this study we suggest that intercropping can be a relevant contributor to rapeseed N nutrition. Among the three legumes tested, clover and lupine seemed to be the best intercropping candidates.

Non-Specific Root Transport of Nutrient Gives Access to an Early Nutritional Indicator: The Case of Sulfate and Molybdate.

Under sulfur (S) deficiency, crosstalk between nutrients induced accumulation of other nutrients, particularly molybdenum (Mo). This disturbed balanced between S and Mo could provide a way to detect S deficiency and therefore avoid losses in yield and seed quality in cultivated species. Under hydroponic conditions, S deprivation was applied to Brassica napus to determine the precise kinetics of S and Mo uptake and whether sulfate transporters were involved in Mo uptake. Leaf contents of S and Mo were also quantified in a field-grown S deficient oilseed rape crop with different S and N fertilization applications to evaluate the [Mo]:[S] ratio, as an indicator of S nutrition. To test genericity of this indicator, the [Mo]:[S] ratio was also assessed with other cultivated species under different controlled conditions. During S deprivation, Mo uptake was strongly increased in B. napus. This accumulation was not a result of the induction of the molybdate transporters, Mot1 and Asy, but could be a direct consequence of Sultr1.1 and Sultr1.2 inductions. However, analysis of single mutants of these transporters in Arabidopsis thaliana suggested that other sulfate deficiency responsive transporters may be involved. Under field conditions, Mo content was also increased in leaves by a reduction in S fertilization. The [Mo]:[S] ratio significantly discriminated between the plots with different rates of S fertilization. Threshold values were estimated for the hierarchical clustering of commercial crops according to S status. The use of the [Mo]:[S] ratio was also reliable to detect S deficiency for other cultivated species under controlled conditions. The analysis of the leaf [Mo]:[S] ratio seems to be a practical indicator to detect early S deficiency under field conditions and thus improve S fertilization management.

Leaf mineral nutrient remobilization during leaf senescence and modulation by nutrient deficiency.

Higher plants have to cope with fluctuating mineral resource availability. However, strategies such as stimulation of root growth, increased transporter activities, and nutrient storage and remobilization have been mostly studied for only a few macronutrients. Leaves of cultivated crops (Zea mays, Brassica napus, Pisum sativum, Triticum aestivum, Hordeum vulgare) and tree species (Quercus robur, Populus nigra, Alnus glutinosa) grown under field conditions were harvested regularly during their life span and analyzed to evaluate the net mobilization of 13 nutrients during leaf senescence. While N was remobilized in all plant species with different efficiencies ranging from 40% (maize) to 90% (wheat), other macronutrients (K-P-S-Mg) were mobilized in most species. Ca and Mn, usually considered as having low phloem mobility were remobilized from leaves in wheat and barley. Leaf content of Cu-Mo-Ni-B-Fe-Zn decreased in some species, as a result of remobilization. Overall, wheat, barley and oak appeared to be the most efficient at remobilization while poplar and maize were the least efficient. Further experiments were performed with rapeseed plants subjected to individual nutrient deficiencies. Compared to field conditions, remobilization from leaves was similar (N-S-Cu) or increased by nutrient deficiency (K-P-Mg) while nutrient deficiency had no effect on Mo-Zn-B-Ca-Mn, which seemed to be non-mobile during leaf senescence under field conditions. However, Ca and Mn were largely mobilized from roots (-97 and -86% of their initial root contents, respectively) to shoots. Differences in remobilization between species and between nutrients are then discussed in relation to a range of putative mechanisms.

Vegetation ecology meets ecosystem science: Permanent grasslands as a functional biogeography case study.

The effect of biodiversity on ecosystem functioning has been widely acknowledged, and the importance of the functional roles of species, as well as their diversity, in the control of ecosystem processes has been emphasised recently. However, bridging biodiversity and ecosystem science to address issues at a biogeographic scale is still in its infancy. Bridging this gap is the primary goal of the emerging field of functional biogeography. While the rise of Big Data has catalysed functional biogeography studies in recent years, comprehensive evidence remains scarce. Here, we present the rationale and the first results of a country-wide initiative focused on the C3 permanent grasslands. We aimed to collate, integrate and process large databases of vegetation relevés, plant traits and environmental layers to provide a country-wide assessment of ecosystem properties and services which can be used to improve regional models of climate and land use changes. We outline the theoretical background, data availability, and ecoinformatics challenges associated with the approach and its feasibility. We provide a case study of upscaling of leaf dry matter content averaged at ecosystem level and country-wide predictions of forage digestibility. Our framework sets milestones for further hypothesis testing in functional biogeography and earth system modelling.