Resource allocation modeling for autonomous prediction of plant cell phenotypes.

Predicting the plant cell response in complex environmental conditions is a challenge in plant biology. Here we developed a resource allocation model of cellular and molecular scale for the leaf photosynthetic cell of Arabidopsis thaliana, based on the Resource Balance Analysis (RBA) constraint-based modeling framework. The RBA model contains the metabolic network and the major macromolecular processes involved in the plant cell growth and survival and localized in cellular compartments. We simulated the model for varying environmental conditions of temperature, irradiance, partial pressure of CO2 and O2, and compared RBA predictions to known resource distributions and quantitative phenotypic traits such as the relative growth rate, the C:N ratio, and finally to the empirical characteristics of CO2 fixation given by the well-established Farquhar model. In comparison to other standard constraint-based modeling methods like Flux Balance Analysis, the RBA model makes accurate quantitative predictions without the need for empirical constraints. Altogether, we show that RBA significantly improves the autonomous prediction of plant cell phenotypes in complex environmental conditions, and provides mechanistic links between the genotype and the phenotype of the plant cell.

An atypical NLR gene confers bacterial wilt susceptibility in Arabidopsis.

Quantitative disease resistance (QDR) remains the most prevalent form of plant resistance in crop fields and wild habitats. Genome-wide association studies (GWAS) have proved to be successful in deciphering the quantitative genetic basis of complex traits such as QDR. To unravel the genetics of QDR to the devastating worldwide bacterial pathogen Ralstonia solanacearum, we performed a GWAS by challenging a highly polymorphic local mapping population of Arabidopsis thaliana with four R. solanacearum type III effector (T3E) mutants, identified as key pathogenicity determinants after a first screen on an A. thaliana core collection of 25 accessions. Although most quantitative trait loci (QTLs) were highly specific to the identity of the T3E mutant (ripAC, ripAG, ripAQ, and ripU), we finely mapped a common QTL located on a cluster of nucleotide-binding domain and leucine-rich repeat (NLR) genes that exhibited structural variation. We functionally validated one of these NLRs as a susceptibility factor in response to R. solanacearum, named it Bacterial Wilt Susceptibility 1 (BWS1), and cloned two alleles that conferred contrasting levels of QDR. Further characterization indicated that expression of BWS1 leads to suppression of immunity triggered by different R. solanacearum effectors. In addition, we showed a direct interaction between BWS1 and RipAC T3E, and BWS1 and SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1b), the latter interaction being suppressed by RipAC. Together, our results highlight a putative role for BWS1 as a quantitative susceptibility factor directly targeted by the T3E RipAC, mediating negative regulation of the SGT1-dependent immune response.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

Proton exchange by the vacuolar nitrate transporter CLCa is required for plant growth and nitrogen use efficiency.

Nitrate is a major nutrient and osmoticum for plants. To deal with fluctuating nitrate availability in soils, plants store this nutrient in their vacuoles. Chloride channel a (CLCa), a 2NO3-/1H+ exchanger localized to the vacuole in Arabidopsis (Arabidopsis thaliana), ensures this storage process. CLCa belongs to the CLC family, which includes anion/proton exchangers and anion channels. A mutation in a glutamate residue conserved across CLC exchangers is likely responsible for the conversion of exchangers to channels. Here, we show that CLCa with a mutation in glutamate 203 (E203) behaves as an anion channel in its native membrane. We introduced the CLCaE203A point mutation to investigate its physiological importance into the Arabidopsis clca knockout mutant. These CLCaE203A mutants displayed a growth deficit linked to the disruption of water homeostasis. Additionally, CLCaE203A expression failed to complement the defect in nitrate accumulation of clca and favored higher N-assimilation at the vegetative stage. Further analyses at the post-flowering stages indicated that CLCaE203A expression results in an increase in N uptake allocation to seeds, leading to a higher nitrogen use efficiency compared to the wild-type. Altogether, these results point to the critical function of the CLCa exchanger on the vacuole for plant metabolism and development.

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.

Environmental justice burden and Black-White disparities in spontaneous preterm birth in Harris County, Texas.

Introduction: Given limited evidence of previous studies, we evaluated the role of environmental justice (EJ) burden (i.e., a neighborhood characterized by both increased environmental burden and socioeconomic deprivation) in Black-White disparities in spontaneous preterm birth (sPTB) in Harris County, Texas and compared results that evaluated neighborhood-level socioeconomic deprivation alone. Methods: We conducted a retrospective analysis using PeriBank, a database and biospecimen repository of gravidae giving birth at two hospitals in the Texas Medical Center. We included 3,703 non-Hispanic Black and 5,475 non-Hispanic white gravidae who were U.S.-born, delivered from August 2011-December 2020, and resided in Harris County, TX. We used data from the U.S. EPA EJScreen to characterize the EJ burden of participant's zip code of residence from fine particulate matter (PM2.5), ozone, and proximity to National Priorities List (NPL) sites and calculated zip-code level Area Deprivation Index (ADI). We assessed the contribution of neighborhood-level variables to the Black-White disparity in sPTB by evaluating attenuation of the odds ratio (OR) representing the effect of race in multivariable logistic regression models, controlling for individual-level characteristics. We also conducted race-stratified analyses between each neighborhood variable and sPTB. Exposure indices were treated as continuous variables; in stratified models, ORs and 95% Confidence Intervals (CIs) are presented per 10-unit increase in the neighborhood variable. Results: Accounting for individual-level variables, Black gravidae had 79% higher odds of sPTB than white gravidae (OR = 1.79, 95%CI = 1.32, 2.44); the disparity was moderately attenuated when accounting for EJ burden or ADI (ORs ranged from 1.58 to 1.69). Though we observed no association between any of the EJ burden indices and sPTB among white gravidae, we found increased risks among Black gravidae, with ORs of similar magnitude for each EJ variable. For example, Black gravidae experienced 17% increased odds of sPTB associated with a 10-unit increase in the EJ burden index for PM2.5 (OR = 1.17, 95%CI = 0.97, 1.40). No racial differences were observed in the association of ADI with sPTB. Discussion: Though we observed limited evidence of the contribution of living in EJ neighborhoods to the Black-White disparity in sPTB, our study suggests living in an EJ neighborhood may differentially impact Black and white gravidae.

Impairment of sugar transport in the vascular system acts on nitrogen remobilization and nitrogen use efficiency in Arabidopsis.

Carbon (C) and nitrogen (N) metabolisms have long been known to be coupled, and this is required for adjusting nitrogen use efficiency (NUE). Despite this intricate relationship, it is still unclear how deregulation of sugar transport impacts N allocation. Here, we investigated in Arabidopsis the consequences of the simultaneous downregulation of the genes coding for the sugar transporters SWEET11, SWEET12, SWEET16, and SWEET17 on various anatomical and physiological traits ranging from the stem's vascular system development to plant biomass production, seed yield, and N remobilization and use efficiency. Our results show that intracellular sugar exchanges mediated by SWEET16 and SWEET17 proteins specifically impact vascular development but do not play a significant role in the distribution of N. Most importantly, we showed that the double mutant swt11 swt12, which has an impacted vascular development, displays an improved NUE and nitrogen remobilization to the seeds. In addition, a significant negative correlation between sugar and amino acids contents and the inflorescence stem radial growth exists, highlighting the complex interaction between the maintenance of C/N homeostasis and the inflorescence stem development. Our results thus deepen the link between sugar transport, C/N allocation, and vascular system development.

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.

Natural variation in the long-distance transport of nutrients and photoassimilates in response to N availability.

Phloem and xylem tissues are necessary for the allocation of nutrients and photoassimilates. However, how the long-distance transport of carbon (C) and nitrogen (N) is coordinated with the central metabolism is largely unknown. To better understand how the genetic and environmental factors influence C and N transport, we analysed the metabolite profiles of phloem exudates and xylem saps of five Arabidopsis thaliana accessions grown in low or non-limiting N supply. We observed that xylem saps were composed of 46 or 56% carbohydrates, 27 or 45% amino acids, and 5 or 13% organic acids in low or non-limiting N supply, respectively. In contrast, phloem exudates were composed of 76 or 86% carbohydrates, 7 or 18% amino acids, and 5 or 6% organic acids. Variation in N supply impacted amino acid, organic acid and sugar contents. When comparing low N and non-limiting N, the most striking differences were variations of glutamine, aspartate, and succinate abundance in the xylem saps and citrate and fumarate abundance in phloem exudates. In addition, we observed a substantial variation of metabolite content between genotypes, particularly under high N. The content of several organic acids, such as malate, citrate, fumarate, and succinate was affected by the genotype alone or by the interaction between genotype and N supply. This study confirmed that the response of the transport of nutrients in the phloem and the xylem to N availability is associated with the regulation of the central metabolism and could be an adaptive trait.

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

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

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.

The Consequences of a Disruption in Cyto-Nuclear Coadaptation on the Molecular Response to a Nitrate Starvation in Arabidopsis.

Mitochondria and chloroplasts are important actors in the plant nutritional efficiency. So, it could be expected that a disruption of the coadaptation between nuclear and organellar genomes impact plant response to nutrient stresses. We addressed this issue using two Arabidopsis accessions, namely Ct1 and Jea, and their reciprocal cytolines possessing the nuclear genome from one parent and the organellar genomes of the other one. We measured gene expression, and quantified proteins and metabolites under N starvation and non-limiting conditions. We observed a typical response to N starvation at the phenotype and molecular levels. The phenotypical response to N starvation was similar in the cytolines compared to the parents. However, we observed an effect of the disruption of genomic coadaptation at the molecular levels, distinct from the previously described responses to organellar stresses. Strikingly, genes differentially expressed in cytolines compared to parents were mainly repressed in the cytolines. These genes encoded more mitochondrial and nuclear proteins than randomly expected, while N starvation responsive ones were enriched in genes for chloroplast and nuclear proteins. In cytolines, the non-coadapted cytonuclear genomic combination tends to modulate the response to N starvation observed in the parental lines on various biological processes.

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.

Post-flowering biotic and abiotic stresses impact nitrogen use efficiency and seed filling in Arabidopsis thaliana.

Nitrogen (N) is an essential nutrient that plants require for the synthesis of amino acids, proteins, and many other important metabolites. Plant metabolism and growth are consequently dependent on the amount of N that is assimilated and distributed from source leaves to developing sinks, such as fruits and seeds. The environmental stresses enhanced by climate change deeply influence seed yield and seed composition, and may disturb N use efficiency (NUE) in pants. We aimed to investigate plant responses to extreme climates with regard to NUE, N remobilization efficiency, and seed composition. By studying a collection of Arabidopsis genotypes showing a range of C:N ratios in seeds, we investigated the impact of different post-flowering growth conditions (control, heat, drought, low nitrate availability, induced senescence, and induced plant defense) on seed yield, N allocation in organs, NUE, and N remobilization efficiency. We analysed how post-flowering stresses could change seed filling and showed that post-flowering stresses change both the range of N and C concentrations and the C:N stoichiometry in seeds. Using a new trait, called delta seed composition, we measured the deviation in C:N stoichiometry of each genotype and revealed the genetic determinism of the C:N stoichiometry. Altogether, the results indicate that extreme climate impacts NUE dramatically in plants and generates different bottlenecks in N fluxes during seed filling.

ACCELERATED CELL DEATH 6 Acts on Natural Leaf Senescence and Nitrogen Fluxes in Arabidopsis.

As the last step of leaf development, senescence is a molecular process involving cell death mechanism. Leaf senescence is trigged by both internal age-dependent factors and environmental stresses. It must be tightly regulated for the plant to adopt a proper response to environmental variation and to allow the plant to recycle nutrients stored in senescing organs. However, little is known about factors that regulate both nutrients fluxes and plant senescence. Taking advantage of variation for natural leaf senescence between Arabidopsis thaliana accessions, Col-0 and Ct-1, we did a fine mapping of a quantitative trait loci for leaf senescence and identified ACCELERATED CELL DEATH 6 (ACD6) as the causal gene. Using two near-isogeneic lines, differing solely around the ACD6 locus, we showed that ACD6 regulates rosette growth, leaf chlorophyll content, as well as leaf nitrogen and carbon percentages. To unravel the role of ACD6 in N remobilization, the two isogenic lines and acd6 mutant were grown and labeled with 15N at the vegetative stage in order to determine 15N partitioning between plant organs at harvest. Results showed that N remobilization efficiency was significantly lower in all the genotypes with lower ACD6 activity irrespective of plant growth and productivity. Measurement of N uptake at vegetative and reproductive stages revealed that ACD6 did not modify N uptake efficiency but enhanced nitrogen translocation from root to silique. In this study, we have evidenced a new role of ACD6 in regulating both sequential and monocarpic senescences and disrupting the balance between N remobilization and N uptake that is required for a good seed filling.

Autophagy and Nutrients Management in Plants.

Nutrient recycling and mobilization from organ to organ all along the plant lifespan is essential for plant survival under changing environments. Nutrient remobilization to the seeds is also essential for good seed production. In this review, we summarize the recent advances made to understand how plants manage nutrient remobilization from senescing organs to sink tissues and what is the contribution of autophagy in this process. Plant engineering manipulating autophagy for better yield and plant tolerance to stresses will be presented.

Proteomic and lipidomic analyses of the Arabidopsis atg5 autophagy mutant reveal major changes in endoplasmic reticulum and peroxisome metabolisms and in lipid composition.

Autophagy is a universal mechanism in eukaryotic cells that facilitates the degradation of unwanted cell constituents and is essential for cell homeostasis and nutrient recycling. The salicylic acid-independent effects of autophagy defects on leaf metabolism were determined through large-scale proteomic and lipidomic analyses of atg5 and atg5/sid2 mutants under different nitrogen and sulfur growth conditions. Results revealed that irrespective of the growth conditions, plants carrying the atg5 mutation presented all the characteristics of endoplasmic reticulum (ER) stress. Increases in peroxisome and ER proteins involved in very long chain fatty acid synthesis and ß-oxidation indicated strong modifications of lipid metabolism. Lipidomic analyses revealed changes in the concentrations of sphingolipids, phospholipids and galactolipids. Significant accumulations of phospholipids and ceramides and changes in GIPCs (glycosyl-inositol-phosphoryl-ceramides) in atg5 mutants indicated large modifications in endomembrane-lipid and especially plasma membrane-lipid composition. Decreases in chloroplast proteins and galactolipids in atg5 under low nutrient conditions, indicated that chloroplasts were used as lipid reservoirs for ß-oxidation in atg5 mutants. In conclusion, this report demonstrates the strong impact of autophagy defect on ER stress and reveals the role of autophagy in the control of plant lipid metabolism and catabolism, influencing both lipid homeostasis and endomembrane composition.

Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis.

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds.

Autophagy is essential for nutrient recycling and plays a fundamental role in seed production and grain filling in plants. Autophagy participates in nitrogen remobilization at the whole-plant level, and the seeds of autophagy mutants present abnormal C and N contents relative to wild-type (WT) plants. It is well known that autophagy (ATG) genes are induced in leaves during senescence; however, expression of such genes in seeds has not yet been reported. In this study we show that most of the ATG genes are induced during seed maturation in Arabidopsis siliques. Promoter-ATG8f::UIDA and promoter-ATG8f::GFP fusions showed the strong expression of ATG8f in the phloem companion cells of pericarps and the funiculus, and in the embryo. Expression was especially strong at the late stages of development. The presence of many GFP-ATG8 pre-autophagosomal structures and autophagosomes confirmed the presence of autophagic activity in WT seed embryos. Seeds of atg5 and WT plants grown under low- or high-nitrate conditions were analysed. Nitrate-independent phenotypes were found with higher seed abortion in atg5 and early browing, higher total protein concentrations in the viable seeds of this mutant as compared to the WT. The higher total protein accumulation in atg5 viable seeds was significant from early developmental stages onwards. In addition, relatively low and early accumulation of 12S globulins were found in atg5 seeds. These features led us to the conclusion that atg5 seed development is accelerated and that the protein storage deposition pathway is somehow abnormal or incomplete.