Multi-omics analyses of sid2 mutant reflect the need of isochorismate synthase ICS1 to cope with sulfur limitation in Arabidopsis thaliana.

The SID2 (SA INDUCTION-DEFICIENT2) gene that encodes ICS1 (isochorismate synthase), plays a central role in salicylic acid biosynthesis in Arabidopsis. The sid2 and NahG (encoding a bacterial SA hydroxylase) overexpressing mutants (NahG-OE) have currently been shown to outperform wild type, presenting delayed leaf senescence, higher plant biomass and better seed yield. When grown under sulfate-limited conditions (low-S), sid2 mutants exhibited early leaf yellowing compared to the NahG-OE, the npr1 mutant affected in SA signaling pathway, and WT. This indicated that the hypersensitivity of sid2 to sulfate limitation was independent of the canonical npr1 SA-signaling pathway. Transcriptomic and proteomic analyses revealed that major changes occurred in sid2 when cultivated under low-S, changes that were in good accordance with early senescence phenotype and showed the exacerbation of stress responses. The sid2 mutants displayed a lower sulfate uptake capacity when cultivated under low-S and lower S concentrations in their rosettes. Higher glutathione concentrations in sid2 rosettes under low-S were in good accordance with the higher abundance of proteins involved in glutathione and ascorbate redox metabolism. Amino acid and lipid metabolisms were also strongly modified in sid2 under low-S. Depletion of total fatty acids in sid2 under low-S was consistent with the fact that S-metabolism plays a central role in lipid synthesis. Altogether, our results show that functional ICS1 is important for plants to cope with S limiting conditions.

Improving crop yield potential: Underlying biological processes and future prospects.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.

Carbon and nitrogen remobilization during seed filling in Arabidopsis is strongly impaired in the pyrroline-5-carboxylate dehydrogenase mutant.

Proline is an amino acid that is degraded in the mitochondria by the sequential action of proline dehydrogenase (ProDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) to form glutamate. We investigated the phenotypes of Arabidopsis wild-type plants, the knockout prodh1 prodh2 double-mutant, and knockout p5cdh allelic mutants grown at low and high nitrate supplies. Surprisingly, only p5cdh presented lower seed yield and produced lighter seeds. Analyses of elements in above-ground organs revealed lower C concentrations in the p5cdh seeds. Determination of C, N, and dry matter partitioning among the above-ground organs revealed a major defect in stem-to-seed resource allocations in this mutant. Again surprisingly, defects in C, N, and biomass allocation to seeds dramatically increased in high-N conditions. 15N-labelling consistently confirmed the defect in N remobilization from the rosette and stem to seeds in p5cdh. Consequently, the p5cdh mutants produced morphologically abnormal, C-depleted seeds that displayed very low germination rates. The most striking result was the strong amplification of the N-remobilization defects in p5cdh under high nitrate supply, and interestingly this phenotype was not observed in the prodh1 prodh2 double-mutant irrespective of nitrate supply. This study reveals an essential role of P5CDH in carbon and nitrogen remobilization for reserve accumulation during seed development in Arabidopsis.

Discovery of the biostimulant effect of asparagine and glutamine on plant growth in Arabidopsis thaliana.

Protein hydrolysates have gained interest as plant biostimulants due to their positive effects on plant performances. They are mainly composed of amino acids, but there is no evidence of the role of individual of amino acids as biostimulants. In this study we carried out in vitro experiments to monitor the development of Arabidopsis seedlings on amino acid containing media in order to analyze the biostimulant properties of the twenty individual proteinogenic amino acids. We demonstrated that proteinogenic amino acids are not good nitrogen sources as compared to nitrate for plant growth. Biostimulant analyses were based on leaf area measurements as a proxy of plant growth. We developed the Amino Acid Use Efficiency index to quantify the biostimulating effect of individual amino acids in the presence of nitrate. This index allowed us to classify amino acids into three groups, characterized by their inhibiting, neutral, and beneficial effects regarding leaf area. Glutamine and asparagine demonstrated the most significant effects in promoting leaf area in the presence of nitrate supply. The stimulating effect was confirmed by using the L and D enantiomeric forms. Both L-glutamine and L-asparagine stimulated leaf area at low concentrations, emphasizing their biostimulating properties. Our plant growth design and AAUE index pave the way for the identification of other bioactive molecules in protein hydrolysates and for the comparison of biostimulant performances.

Modulation of plant nitrogen remobilization and postflowering nitrogen uptake under environmental stresses.

Plants are sessile organisms that take up nitrogen (N) from the soil for growth and development. At the postflowering stage, N that plants require for seed growth and filling derives from either root uptake or shoot remobilization. The balance between N uptake and N remobilization determines the final carbon (C) and N composition of the seed. The N uptake and N remobilization mechanisms are regulated by endogenous signals, including hormones, developmental stage, and carbon/nitrogen ratio, and by environmental factors. The cellular responses to the environment are relatively well known. However, the effects of environmental stresses on the balance between N uptake and N remobilization are still poorly understood. Thus, this study aims to analyze the impact of environmental stresses (drought, heat, darkness, triggered defense, and low nitrate) on N fluxes within plants during seed filling. Using publicly available Arabidopsis transcriptome data, expression of several marker genes involved in N assimilation, transport, and recycling was analyzed in relation to stress. Results showed that the responses of genes encoding inorganic N transporters, N assimilation, and N recycling are mainly regulated by N limitation, the genes encoding housekeeping proteases are principally sensitive to C limitation, and the response of genes involved in the transport of organic N is controlled by both C and N limitations. In addition, 15N data were used to examine the effects of severe environmental stresses on N remobilization and N uptake, and a schematic representation of the major factors that regulate the balance between N remobilization and N uptake under the stress and control conditions was provided.

Corrigendum: Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

[This corrects the article DOI: 10.3389/fpls.2021.807798.].

How Lipids Contribute to Autophagosome Biogenesis, a Critical Process in Plant Responses to Stresses.

Throughout their life cycle, plants face a tremendous number of environmental and developmental stresses. To respond to these different constraints, they have developed a set of refined intracellular systems including autophagy. This pathway, highly conserved among eukaryotes, is induced by a wide range of biotic and abiotic stresses upon which it mediates the degradation and recycling of cytoplasmic material. Central to autophagy is the formation of highly specialized double membrane vesicles called autophagosomes which select, engulf, and traffic cargo to the lytic vacuole for degradation. The biogenesis of these structures requires a series of membrane remodeling events during which both the quantity and quality of lipids are critical to sustain autophagy activity. This review highlights our knowledge, and raises current questions, regarding the mechanism of autophagy, and its induction and regulation upon environmental stresses with a particular focus on the fundamental contribution of lipids. How autophagy regulates metabolism and the recycling of resources, including lipids, to promote plant acclimation and resistance to stresses is further discussed.

A proposed role for endomembrane trafficking processes in regulating tonoplast content and vacuole dynamics under ammonium stress conditions in Arabidopsis root cells.

Ammonium (NH4+) stress has multiple effects on plant physiology, therefore, plant responses are complex, and multiple mechanisms are involved in NH4+ sensitivity and tolerance in plants. Root growth inhibition is an important quantitative readout of the effects of NH4+ stress on plant physiology, and cell elongation appear as the principal growth inhibition target. We recently proposed autophagy as a relevant physiological mechanisms underlying NH4+ sensitivity response in Arabidopsis. In a brief overview, the impaired macro-autophagic flux observed under NH4+ stress conditions has a detrimental impact on the cellular energetic balance, and therefore on the energy-demanding plant growth. In contrast to its inhibitory effect on the autophagosomes flux to vacuole, NH4+ toxicity induced a micro-autophagy-like process. Consistent with the reduced membrane flux to the vacuole related to macro-autophagy inhibition and the increased tonoplast degradation due to enhanced micro-autophagy, the vacuoles of the root cells of the NH4+-stressed plants showed lower tonoplast content and a decreased perimeter/area ratio. As the endosome-to-vacuole trafficking is another important process that contributes to membrane flux toward the vacuole, we evaluated the effects of NH4+ stress on this process. This allows us to propose that autophagy could contribute to vacuole development as well as possible avenues to follow for future studies.

Current Understanding of Leaf Senescence in Rice.

Leaf senescence, which is the last developmental phase of plant growth, is controlled by multiple genetic and environmental factors. Leaf yellowing is a visual indicator of senescence due to the loss of the green pigment chlorophyll. During senescence, the methodical disassembly of macromolecules occurs, facilitating nutrient recycling and translocation from the sink to the source organs, which is critical for plant fitness and productivity. Leaf senescence is a complex and tightly regulated process, with coordinated actions of multiple pathways, responding to a sophisticated integration of leaf age and various environmental signals. Many studies have been carried out to understand the leaf senescence-associated molecular mechanisms including the chlorophyll breakdown, phytohormonal and transcriptional regulation, interaction with environmental signals, and associated metabolic changes. The metabolic reprogramming and nutrient recycling occurring during leaf senescence highlight the fundamental role of this developmental stage for the nutrient economy at the whole plant level. The strong impact of the senescence-associated nutrient remobilization on cereal productivity and grain quality is of interest in many breeding programs. This review summarizes our current knowledge in rice on (i) the actors of chlorophyll degradation, (ii) the identification of stay-green genotypes, (iii) the identification of transcription factors involved in the regulation of leaf senescence, (iv) the roles of leaf-senescence-associated nitrogen enzymes on plant performance, and (v) stress-induced senescence. Compiling the different advances obtained on rice leaf senescence will provide a framework for future rice breeding strategies to improve grain yield.

Salicylic acid is a key player of Arabidopsis autophagy mutant susceptibility to the necrotrophic bacterium Dickeya dadantii.

Autophagy is a ubiquitous vesicular process for protein and organelle recycling in eukaryotes. In plant, autophagy is reported to play pivotal roles in nutrient recycling, adaptation to biotic and abiotic stresses. The role of autophagy in plant immunity remains poorly understood. Several reports showed enhanced susceptibility of different Arabidopsis autophagy mutants (atg) to necrotrophic fungal pathogens. Interaction of necrotrophic bacterial pathogens with autophagy is overlooked. We then investigated such interaction by inoculating the necrotrophic enterobacterium Dickeya dadantii in leaves of the atg2 and atg5 mutants and an ATG8a overexpressing line. Overexpressing ATG8a enhances plant tolerance to D. dadantii. While atg5 mutant displayed similar susceptibility to the WT, the atg2 mutant exhibited accelerated leaf senescence and enhanced susceptibility upon infection. Both phenotypes were reversed when the sid2 mutation, abolishing SA signaling, was introduced in the atg2 mutant. High levels of SA signaling in atg2 mutant resulted in repression of the jasmonic acid (JA) defense pathway known to limit D. dadantii progression in A. thaliana. We provide evidence that in atg2 mutant, the disturbed hormonal balance leading to higher SA signaling is the main factor causing increased susceptibility to the D. dadantii necrotroph by repressing the JA pathway and accelerating developmental senescence.

Advances in Plant Autophagy.

Ubiquitin-proteasome and lysosome-autophagy are the two main cellular degradation systems controlling cellular homeostasis in eukaryotes [...].

Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

Owing to the large genetic diversity of barley and its resilience under harsh environments, this crop is of great value for agroecological transition and the need for reduction of nitrogen (N) fertilizers inputs. In the present work, we investigated the diversity of a North African barley genotype collection in terms of growth under limiting N (LN) or ample N (HN) supply and in terms of physiological traits including amino acid content in young seedlings. We identified a Moroccan variety, Laanaceur, accumulating five times more lysine in its leaves than the others under both N nutritional regimes. Physiological characterization of the barley collection showed the genetic diversity of barley adaptation strategies to LN and highlighted a genotype x environment interaction. In all genotypes, N limitation resulted in global biomass reduction, an increase in C concentration, and a higher resource allocation to the roots, indicating that this organ undergoes important adaptive metabolic activity. The most important diversity concerned leaf nitrogen use efficiency (LNUE), root nitrogen use efficiency (RNUE), root nitrogen uptake efficiency (RNUpE), and leaf nitrogen uptake efficiency (LNUpE). Using LNUE as a target trait reflecting barley capacity to deal with N limitation, this trait was positively correlated with plant nitrogen uptake efficiency (PNUpE) and RNUpE. Based on the LNUE trait, we determined three classes showing high, moderate, or low tolerance to N limitation. The transcriptomic approach showed that signaling, ionic transport, immunity, and stress response were the major functions affected by N supply. A candidate gene encoding the HvNRT2.10 transporter was commonly up-regulated under LN in the three barley genotypes investigated. Genes encoding key enzymes required for lysine biosynthesis in plants, dihydrodipicolinate synthase (DHPS) and the catabolic enzyme, the bifunctional Lys-ketoglutarate reductase/saccharopine dehydrogenase are up-regulated in Laanaceur and likely account for a hyperaccumulation of lysine in this genotype. Our work provides key physiological markers of North African barley response to low N availability in the early developmental stages.

Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90.1 and ROF1.

In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein.

Ammonium stress increases microautophagic activity while impairing macroautophagic flux in Arabidopsis roots.

Plant responses to NH4+ stress are complex, and multiple mechanisms underlying NH4+ sensitivity and tolerance in plants may be involved. Here, we demonstrate that macro- and microautophagic activities are oppositely affected in plants grown under NH4+ toxicity conditions. When grown under NH4+ stress conditions, macroautophagic activity was impaired in roots. Root cells accumulated autophagosomes in the cytoplasm, but showed less autophagic flux, indicating that late steps of the macroautophagy process are affected under NH4+ stress conditions. Under this scenario, we also found that the CCZ1-MON1 complex, a critical factor for vacuole delivery pathways, functions in the late step of the macroautophagic pathway in Arabidopsis. In contrast, an accumulation of tonoplast-derived vesicles was observed in vacuolar lumens of root cells of NH4+ -stressed plants, suggesting the induction of a microautophagy-like process. In this sense, some SYP22-, but mainly VAMP711-positive vesicles were observed inside vacuole in roots of NH4+ -stressed plants. Consistent with the increased tonoplast degradation and the reduced membrane flow to the vacuole due to the impaired macroautophagic flux, the vacuoles of root cells of NH4+ -stressed plants showed a simplified structure and lower tonoplast content. Taken together, this study presents evidence that postulates late steps of the macroautophagic process as a relevant physiological mechanism underlying the NH4+ sensitivity response in Arabidopsis, and additionally provides insights into the molecular tools for studying microautophagy in plants.

Integrating multiple omics to identify common and specific molecular changes occurring in Arabidopsis under chronic nitrate and sulfate limitations.

Plants have fundamental dependences on nitrogen and sulfur and frequently have to cope with chronic limitations when their supply is sub-optimal. This study aimed at characterizing the metabolomic, proteomic, and transcriptomic changes occurring in Arabidopsis leaves under chronic nitrate (Low-N) and chronic sulfate (Low-S) limitations in order to compare their effects, determine interconnections, and examine strategies of adaptation. Metabolite profiling globally revealed opposite effects of Low-S and Low-N on carbohydrate and amino acid accumulations, whilst proteomic data showed that both treatments resulted in increases in catabolic processes, stimulation of mitochondrial and cytosolic metabolism, and decreases in chloroplast metabolism. Lower abundances of ribosomal proteins and translation factors under Low-N and Low-S corresponded with growth limitation. At the transcript level, the major and specific effect of Low-N was the enhancement of expression of defence and immunity genes. The main effect of chronic Low-S was a decrease in transcripts of genes involved in cell division, DNA replication, and cytoskeleton, and an increase in the expression of autophagy genes. This was consistent with a role of target-of-rapamycin kinase in the control of plant metabolism and cell growth and division under chronic Low-S. In addition, Low-S decreased the expression of several NLP transcription factors, which are master actors in nitrate sensing. Finally, both the transcriptome and proteome data indicated that Low-S repressed glucosinolate synthesis, and that Low-N exacerbated glucosinolate degradation. This showed the importance of glucosinolate as buffering molecules for N and S management.

Concurrent activation of OsAMT1;2 and OsGOGAT1 in rice leads to enhanced nitrogen use efficiency under nitrogen limitation.

Nitrogen (N) is a major factor for plant development and productivity. However, the application of nitrogenous fertilizers generates environmental and economic problems. To cope with the increasing global food demand, the development of rice varieties with high nitrogen use efficiency (NUE) is indispensable for reducing environmental issues and achieving sustainable agriculture. Here, we report that the concomitant activation of the rice (Oryza sativa) Ammonium transporter 1;2 (OsAMT1;2) and Glutamate synthetase 1 (OsGOGAT1) genes leads to increased tolerance to nitrogen limitation and to better ammonium uptake and N remobilization at the whole plant level. We show that the double activation of OsAMT1;2 and OsGOGAT1 increases plant performance in agriculture, providing better N grain filling without yield penalty under paddy field conditions, as well as better grain yield and N content when plants are grown under N llimitations in field conditions. Combining OsAMT1;2 and OsGOGAT1 activation provides a good breeding strategy for improving plant growth, nitrogen use efficiency and grain productivity, especially under nitrogen limitation, through the enhancement of both nitrogen uptake and assimilation.

OsASN1 Overexpression in Rice Increases Grain Protein Content and Yield under Nitrogen-Limiting Conditions.

Nitrogen (N) is a major limiting factor affecting crop yield in unfertilized soil. Thus, cultivars with a high N use efficiency (NUE) and good grain protein content (GPC) are needed to fulfill the growing food demand and to reduce environmental burden. This is especially true for rice (Oryza sativa L.) that is cultivated with a high input of N fertilizer and is a primary staple food crop for more than half of the global population. Here, we report that rice asparagine synthetase 1 (OsASN1) is required for grain yield and grain protein contents under both N-sufficient (conventional paddy fields) and N-limiting conditions from analyses of knockout mutant plants. In addition, we show that overexpression (OX) of OsASN1 results in better nitrogen uptake and assimilation, and increased tolerance to N limitation at the seedling stage. Under field conditions, the OsASN1 OX rice plants produced grains with increased N and protein contents without yield reduction compared to wild-type (WT) rice. Under N-limited conditions, the OX plants displayed increased grain yield and protein content with enhanced photosynthetic activity compared to WT rice. Thus, OsASN1 can be an effective target gene for the development of rice cultivars with higher grain protein content, NUE, and grain yield under N-limiting conditions.

Transcriptional Plasticity of Autophagy-Related Genes Correlates with the Genetic Response to Nitrate Starvation in Arabidopsis Thaliana.

In eukaryotes, autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. For a better understanding of the relationship between autophagy and nitrogen metabolism, we studied the transcriptional plasticity of autophagy genes (ATG) in nine Arabidopsis accessions grown under normal and nitrate starvation conditions. The status of the N metabolism in accessions was monitored by measuring the relative expression of 11 genes related to N metabolism in rosette leaves. The transcriptional variation of the genes coding for enzymes involved in ammonium assimilation characterize the genetic diversity of the response to nitrate starvation. Starvation enhanced the expression of most of the autophagy genes tested, suggesting a control of autophagy at transcriptomic level by nitrogen. The diversity of the gene responses among natural accessions revealed the genetic variation existing for autophagy independently of the nutritive condition, and the degree of response to nitrate starvation. We showed here that the genetic diversity of the expression of N metabolism genes correlates with that of the ATG genes in the two nutritive conditions, suggesting that the basal autophagy activity is part of the integral response of the N metabolism to nitrate availability.

Autophagy Controls Sulphur Metabolism in the Rosette Leaves of Arabidopsis and Facilitates S Remobilization to the Seeds.

Sulphur deficiency in crops became an agricultural concern several decades ago, due to the decrease of S deposition and the atmospheric sulphur dioxide emissions released by industrial plants. Autophagy, which is a conserved mechanism for nutrient recycling in eukaryotes, is involved in nitrogen, iron, zinc and manganese remobilizations from the rosette to the seeds in Arabidopsis thaliana. Here, we have compared the role of autophagy in sulphur and nitrogen management at the whole plant level, performing concurrent labelling with 34S and 15N isotopes on atg5 mutants and control lines. We show that both 34S and 15N remobilizations from the rosette to the seeds are impaired in the atg5 mutants irrespective of salicylic acid accumulation and of sulphur nutrition. The comparison in each genotype of the partitions of 15N and 34S in the seeds (as % of the whole plant) indicates that the remobilization of 34S to the seeds was twice more efficient than that of 15N in both autophagy mutants and control lines under high S conditions, and also in control lines under low S conditions. This was different in the autophagy mutants grown under low S conditions. Under low S, the partition of 34S to their seeds was indeed not twice as high but similar to that of 15N. Such discrepancy shows that when sulphate availability is scarce, autophagy mutants display stronger defects for 34S remobilization relative to 15N remobilization than under high S conditions. It suggests, moreover, that autophagy mainly affects the transport of N-poor S-containing molecules and possibly sulphate.

Reserve lipids and plant autophagy.

Autophagy is a universal mechanism that facilitates the degradation of unwanted cytoplasmic components in eukaryotic cells. In this review, we highlight recent developments in the investigation of the role of autophagy in lipid homeostasis in plants by comparison with algae, yeast, and animals. We consider the storage compartments that form the sources of lipids in plants, and the roles that autophagy plays in the synthesis of triacylglycerols and in the formation and maintenance of lipid droplets. We also consider the relationship between lipids and the biogenesis of autophagosomes, and the role of autophagy in the degradation of lipids in plants.