Silicon (Si) is known to improve plant tolerance to abiotic stresses. However, its action at the molecular levels in Brassica napus L., during seed germination and seedling growth, has yet to be fully elucidated. Therefore, this study aimed at investigating the effect of exogenous calcium silicate (Si) on seed germination and seedlings growth of B. napus under salinity and to explore the tolerance mechanisms using physio-biochemical and proteomic approaches. Results showed that salinity increased Na+/K+ ratio and induced an oxidative stress in B. napus seedlings. However, the Si supply increased the ability of B. napus seedlings to withstand salinity through increased Si content, osmoregulation process and antioxidant system, while decreased Na+/K+ and oxidative stress markers. Moreover, the proteome analysis identified a total of 1059 differentially accumulated proteins (DAPs) in response to Si, of which 864 under salinity. These DAPs are associated with varied biological processes, including protein targeting and import in chloroplast and endoplasmic reticulum, photosynthesis and light-harvesting complexes, translation and protein biosynthesis and transport and homeostasis, contributing as a result to the resilience of B. napus seedlings to salinity. Together, these results show that Si confers multifactorial tolerance to salinity in B. napus, acting simultaneously on physiological parameters, biochemical protection and proteomic regulation. The beneficial effects of Si on B. napus seedlings subjected to salt stress suggest that calcium silicate could be alternative way to promote the establishment of this crop under salt stress conditions.

Root senescence remains largely unexplored. In this study, the time-course of the morphological, metabolic, and proteomic changes occurring with root aging were investigated, providing a comprehensive picture of the root senescence program. We found novel senescence-related markers for the characterization of the developmental stage of root tissues. The rapeseed root system is unique in that it consists of the taproot and lateral roots. Our study confirmed that the taproot, which transiently accumulates large quantities of starch and proteins, is specifically dedicated to nutrient storage and remobilization, while the lateral roots are mainly dedicated to nutrient uptake. Proteomic data from the taproot and lateral roots highlighted the different senescence-related events that control nutrient remobilization and nutrient uptake capacities. Both the proteome and enzyme activities revealed senescence-induced proteases and nucleotide catabolic enzymes that deserve attention as they may play important roles in nutrient remobilization efficiency in rapeseed roots. Taking advantage of publicly available transcriptomic and proteomic data on senescent Arabidopsis leaves, we provide a novel lists of senescence-related proteins specific or common to root organs and/ or leaves.

The projects’goal was to develop a method for evaluating biostimulants on winter rapeseed. Several approaches were deployed. First, a group of experts was formed. The objective was above all to share expertise and then propose a list of the most relevant indicators. Secondly, the project partners worked on developing a method under controlled conditions specifically aimed at evaluating the response to biostimulants under stress conditions. This approach was then applied to the development of a protocol under field conditions. Finally, in addition to existing methods, the project focused on the development of a tool to evaluate the stimulation efficiency of biostimulants, based on the identification of candidate genes associated with the biostimulant activity of the products tested. The experimentations set up made it possible to select a pool of 13 candidate genes.

Autophagy is essential for homeostasis and nutrient recycling. Its activity increases with aging and in response to deficiencies. The effects of defective autophagy on root metabolism have not yet been described. Addressing this question through root proteome analyses, we found that most V-ATPases were less abundant in the roots of autophagy mutants than in wild type. V-ATPases deficit, associated with lower root water contents and lower nitrate, magnesium, and potassium concentrations, indicated that the disturbance of cellular ion and water management in autophagy mutants was likely related to vacuole function. Isotopic δ13C analyses and leaf temperature measurements using thermography showed that water deficit in autophagy mutants was not due to excess transpiration, as the conductance of stomata was reduced in mutants compared to wild type. Many proteins related to the catabolism of amino acids and lipids and the tricarboxylic acid (TCA) cycle were over-abundant in atg mutants. The increase in several proteases that paralleled amino acid catabolism suggested that in the absence of autophagic flux, compensatory processes could be established to degrade proteins, recycle amino acids, and fuel TCA. Whether the V-ATPases defect affects energy metabolism and promotes lipid and amino acid catabolism to compensate and fuel TCA remains to be explored. In conclusion, this report establishes for the first time a correlation between autophagy and vacuole function through V-ATPases, particularly with regard to water and ion management. Additionally, this report shows the exacerbation of amino acid catabolism in relation to the stimulation of the TCA cycle in autophagy mutants.

The association of legumes with other non-legume plants, such as Brassica napus L., has been reported as an agro-ecological alternative for reducing the nitrogen (N) inputs required for B. napus growth, thanks in particular to the transfer of N compounds from the legume to B. napus. Moreover, recent studies have evidenced that silicon (Si) supply can improve either N uptake by B. napus or the dinitrogen fixation capacity of T. incarnatum. However, the effect of Si supply on the N nutrition of both B. napus and T. incarnatum, especially when growing in association, has not been assessed so far. The aim of this study was to assess the effect of Si supply on the growth of B. napus and T. incarnatum cultivated in association by focusing particularly on N rhizodeposition by T. incarnatum and its transfer to B. napus. The experiment was performed for 10 weeks under a split-root system combined with an 15N labeling method. The results showed that the Si supply increased the amount of rhizo-deposited N by T. incarnatum by over 40% and enhanced its transfer to B. napus. The transferred N was allocated mainly to pods (17%), as their biomass increased under Si supply. For the first time, this study demonstrates that the association with legume plants together with the Si supply could be an effective approach to improve the agro-ecological balance of B. napus.

In winter oilseed rape (Brassica napus L.), vernalization, prolonged cold exposure, is essential for spring flowering. Although transcriptomic changes in leaves during vernalization are studied, the taproot, a key storage organ, remains unexplored. Recently, high nitrogen (N) and carbon (C) compound levels were observed in the taproot post-vernalization, suggesting potential metabolic activities in this organ during this period. To decipher this, an integrative study combining morphological, ionomic, proteomic, and targeted biochemical analysis was conducted. This study revealed that the taproot is the only compartment that shows net gain in biomass during vernalization and confirmed its role in storing C and N reserves. A comparative proteomic analysis between the beginning and the end of the vernalization period showed that this storage is the result of a strong modulation of proteins involved in N and C metabolisms. Additionally, the up-accumulation of proteins involved in the starch and amino acid metabolisms is consistent with the increase in the starch and amino acid amounts in the taproot during vernalization. Amino acids from the glutamine family are especially accumulated, with proline being the most over-accumulated (127-fold), highlighting the initiation of a protective metabolism in the taproot during the cold stress period related to vernalization. This study also reveals the storage of macro- and microelements, notably iron, copper, and zinc. These findings provide a deeper understanding of the development and maintenance of specific metabolic activities in the taproot of B. napus during vernalization, ensuring the accumulation of essential N and C reserves for subsequent growth and development.

This preliminary study shows that Brassica napus WSCP1 delays chlorophyll degradation and inhibits serine proteases during dark-induced leaf senescence in Arabidopsis. In Brassica napus L., one of the levers for improving Nitrogen Remobilization Efficiency (NRE) consists to delay senescence onset, which prolongs leaf lifespan and reduces the asynchronism between the nitrogen emptying period in these source organs and the filling period of seeds. Water soluble chlorophyll binding proteins (WSCPs) may have a dual function in chlorophyll protection and protease inhibition. As such they are excellent candidates to propose a technical solution to delay leaf senescence. Several isoforms of WSCPs have been identified in the leaves of rapeseed. Among them, WSCP1 presents two motifs in its protein sequence that are associated to the putative dual function. To test if WSCP1 can actually delay leaf senescence, the overexpression of WSCP1 under the control of the SAG12 senescence promoter (pSAG12::WSCP1) was developed in an Arabidopsis thaliana accession that was previously described as early senescent (RIL232). During dark-induced senescence, our main results reveal a lower chlorophyll degradation and a reduction of the serine proteases (SPs) activity in leaves of pSAG12::WSCP1 lines compared to RIL232. Although SP inhibition was strong in pSAG12::WSCP1 leaves compared with RIL232, no difference in leaf protein content was observed. This result suggests either the recruitment of a compensatory proteolytic system in WSCP1-overexpressing lines or that SPs are not essential for protein nitrogen remobilization during dark-induced leaf senescence.

BACKGROUND AND AIMS:Macroautophagy is essential for the degradation and recycling of various macromolecules in eukaryote cells. In plants, autophagy is involved in the degradation of damaged chloroplasts in response to stress. Autophagy is a key player in nitrogen management at the whole-plant level, and autophagy mutants display strong defects in nitrogen remobilization and early leaf senescence phenotypes especially under nitrogen source limitation. It is known that leaf senescence is associated to nutrient remobilization processes and is induced by nitrate limitation. However, it remains to be determined which actors are involved in this interplay and whether nutrient remobilization acts as a signal that enhances leaf senescence or whether senescence-associated chloroplast degradation promotes nutrient remobilization. In this context, our aim is to demine whether the level of autophagy activity controls leaf longevity and influences the progress of leaf senescence balancing resources and waste management. METHODS:In this study, we used Arabidopsis autophagy knock-out mutants (atg) and plants overexpressing autophagy associated genes (ATG8-OE) to compare their phenotypes to wild type under both sufficient nitrate condition and nitrate starvation. KEY RESULTS:The transfer from nitrate-sufficient conditions to nitrate starvation accelerated leaf senescence in all the genotypes. Unexpectedly, under both sufficient and nitrate-starved conditions, both atg mutants and ATG8 over-expressors exhibited earlier leaf senescence phenotypes compared to wild type. Given that autophagy is a longevity factor, the more severe leaf senescence phenotype of over-expressors was puzzling. This study highlights a relationship between autophagy, nitrogen remobilization and leaf senescence and shows how the fine tuning of nutrient management can influence senescence onset. CONCLUSIONS:The fine-tuning of autophagy is necessary to control leaf senescence.

Background and Aims: Numerous studies have reported the beneficial effects of silicon (Si) in alleviating biotic or abiotic stresses in many plant species. However, the role of Si in Fabaceae facing environmental stress is poorly documented. The aim of this study is to investigate the effect of Si on physiological traits and nodulation efficiency in Trifolium incarnatum L. subjected to N-deprivation. Methods Si was supplied (1.7 mM) to N-deprived plants inoculated with Rhizobium leguminosarum bv trifolii and plant physiological traits and nodule ionomic and molecular traits were monitored over 25 days. Results Si supply promoted shoot biomass, the quantity of both Si and N in roots and shoots, and the number, biomass and density of nodules and their nitrogenase abundance which contribute to better dinitrogen (N2) fixation. Ionomic analysis of nodules revealed that Si supply increased the amount of several macroelements (potassium, phosphorus and sulfur) and microelements (copper, zinc and molybdenum) known to improve nodulation efficiency and N2 fixation. Finally, comparative proteomic analysis (+ Si versus -Si) of nodules highlighted that Si modulated the proteome of both symbionts with 989 and 212 differentially accumulated proteins (DAPs) in the infected host root cells and their symbiont bacteria, respectively. Among the DAPs, the roles of those involved in nodulation and N2 fixation are discussed. Conclusion For the first time, this study provides new insights into the effects of Si on both nodular partners and paves the way for a better understanding of the impact of Si on improving nodule function, and more specifically, on the nodules’ N2-fixing capacity.

IntroductionNumerous studies have reported the beneficial effects of silicon (Si) in alleviating biotic or abiotic stresses in many plant species. However, the role of Si in Fabaceae facing environmental stress is poorly documented. The aim of this study is to investigate the effect of Si on physiological traits and nodulation efficiency in Trifolium incarnatum L.MethodsSi was supplied (1.7 mM in the form of Na2SiO3) plants inoculated with Rhizobium leguminosarum bv trifolii and plant physiological traits and nodule ionomic and molecular traits were monitored over 25 days.ResultsSi supply promoted shoot biomass, the quantity of both Si and N in roots and shoots, and the number, biomass and density of nodules and their nitrogenase abundance which contribute to better dinitrogen (N2) fixation. Ionomic analysis of nodules revealed that Si supply increased the amount of several macroelements (potassium, phosphorus and sulfur) and microelements (copper, zinc and molybdenum) known to improve nodulation efficiency and N2 fixation. Finally, comparative proteomic analysis (+Si versus -Si) of nodules highlighted that Si modulated the proteome of both symbionts with 989 and 212 differentially accumulated proteins (DAPs) in the infected host root cells and their symbiont bacteria, respectively.DiscussionAmong the DAPs, the roles of those involved in nodulation and N2 fixation are discussed. For the first time, this study provides new insights into the effects of Si on both nodular partners and paves the way for a better understanding of the impact of Si on improving nodule function, and more specifically, on the nodules’ N2-fixing capacity.

ABSTRACTSenescence related markers have been widely studied in leaves in many plant species. Root senescence is more difficult to characterize. The existence of two different root organs inB. napus,with a taproot that appear to be specifically dedicated to the storage and remobilization of nutrients, offered the possibility of analysing the temporality of the changes linked to aging, based on the degradation of the taproot reserves. Microscopic and biochemical analyses showed that taproot plays an important role in carbon and nitrogen storage as reflected by the large quantities of starch and proteins present at early development stages. The proteomic study associated to the description of biochemical, morphological and anatomic changes provides a comprehensive picture of the main events occurring in the taproot and in the lateral roots with aging. Master modifications as protein and cell wall degradation, amino acid catabolism versus synthesis, nucleic acid degradation are presented and senescence related markers specific or not of the root types were identified. Comparison with Arabidopsis public data facilitated the identification of markers common to root and leaf senescence. The analysis of protease changes provides a list of candidates that may play a role in nitrogen and carbohydrate remobilization from taproot to the shoot and flowering organs and that would deserve attention for further functional analyses.

ABSTRACT Macroautophagy is known for long as essential for the degradation and the recycling of different macromolecules in eukaryotes. However how important is autophagy for nitrogen management at the whole plant level and for plant biomass and yield productivity in unstressed and well feed plants needed further investigation. In this study, we used both autophagy knock-out mutants and autophagy over-expressors that constitutively produce numerous autophagosomes. These mutants and over-expressors were cultivated using hydroponic system to observe and compare their phenotypes under sufficient nitrate supply, and when submitted after a while to strict nitrate starvation. The shift from nitrate sufficient condition to nitrate starvation allowed us to determine how autophagy defective or stimulated lines can use their own nitrogen resources to complete their cycle. Unexpectedly we observed that irrespective of the nitrate conditions, both mutants and over-expressors exhibited early leaf senescence phenotypes relative to wild type. While autophagy mutants exhibited strong defect for N remobilisation and seed production irrespective of nitrate condition, the better performance of autophagy-over expressors for N remobilisation and seeds production was only significant under sufficient nitrate supply, i.e. when autophagy was not naturally stimulated by nitrate limitation. Interestingly, comparisons of genotypes showed that the nitrogen pool used for seed filling originated from rosette leaves, as if rosette and seeds were used as communicating vessels independently of the stem and pod connecting organs. Altogether, results show that autophagy is a master player in nitrogen management at the whole plant level that controls yield production and leaf senescence.

In many crops species, sulfur (S) deprivation negatively affects growth, seed yield quality and plant health. Furthermore, silicon (Si) is known to alleviate many nutritional stresses but the effects of Si supply on plants subjected to S deficiency remain unclear and poorly documented. The objective of this study was to evaluate whether Si supply would alleviate the negative effects of S deprivation on root nodulation and atmospheric dinitrogen (N2) fixation capacity in Trifolium incarnatum subjected (or not) to long-term S deficiency. For this, plants were grown for 63 days in hydroponic conditions with (500 µM) or without S and supplied (1.7 mM) or not with Si. The effects of Si on growth, root nodulation and N2 fixation and nitrogenase abundance in nodules have been measured. The most important beneficial effect of Si was observed after 63 days. Indeed, at this harvest time, a Si supply increased growth, the nitrogenase abundance in nodules and N2 fixation in S-fed and S-deprived plants while a beneficial effect on the number and total biomass of nodules was only observed in S-deprived plants. This study shows clearly for the first time that a Si supply alleviates negative effects of S deprivation in Trifolium incarnatum.

Silicon (Si) is known to alleviate many nutritional stresses. However, in Brassica napus, which is a highly S-demanding species, the Si effect on S deficiency remains undocumented. The aim of this study was to assess whether Si alleviates the negative effects of S deficiency on Brassica napus and modulates root sulfate uptake capacity and S accumulation. For this, Brassica napus plants were cultivated with or without S and supplied or not supplied with Si. The effects of Si on S content, growth, expression of sulfate transporter genes (BnaSultr1.1; BnaSultr1.2) and sulfate transporters activity in roots were monitored. Si supply did not mitigate growth or S status alterations due to S deprivation but moderated the expression of BnaSultr1.1 in S-deprived plants without affecting the activity of root sulfate transporters. The effects of Si on the amount of S taken-up and on S transporter gene expression were also evaluated after 72 h of S resupply. In S-deprived plants, S re-feeding led to a strong decrease in the expression of both S transporter genes as expected, except in Si-treated plants where BnaSultr1.1 expression was maintained over time. This result is discussed in relation to the similar amount of S accumulated regardless of the Si treatment.

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.

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.

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.

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.

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.

While the benefit of silicon (Si) is often reported in cultivated plants, and particularly in those experiencing stress conditions, the underlying mechanisms are poorly described and controversial [ 1 ]. For a long time the mechanical role of Si in the cell wall has been considered as the main explanation for its beneficial effects in alleviating plant stresses. Another assumption is that a low proportion (around 1%) of the Si taken up by plants might modulate some metabolic pathways. Investigating this concept, Haddad et al. [ 2 ] demonstrated that in roots of Brassica napus plants, the modulation of a large range of genes by Si could explain an increased resistance to nitrogen (N) deficiency. However, since the Si benefit is mainly associated with maintaining photosynthetic activity and a delay in leaf senescence in the mature leaves of plants cultivated under N- deficiency [ 3 ], the aim of the present study was to identify (using a RNA sequencing approach) the transcriptomic modifications in the shoot compartment of +Si plants. Our results showed that there were 296 genes differentially expressed genes (DEGs)) in shoots of Brassica napus treated with Si (root supply 1.7 mM for 7 days). Among these genes, 19 and 31 upregulated genes were related to ribosomes and photosynthetic pathways, respectively. From these results, the assumption that a Si supply can facilitate efficient metabolic reinforcement and alleviate the biotic and abiotic stresses experienced by plants is discussed.