Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality

Background: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. Results: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. Conclusions: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain

Copper and iron homeostasis in plants: the challenges of oxidative stress.

International audienceSIGNIFICANCE: Photosynthesis, the process that drives life on earth, relies on transition metal (e.g., Fe and Cu) containing proteins that participate in electron transfer in the chloroplast. However, the light reactions also generate high levels of reactive oxygen species (ROS), which makes metal use in plants a challenge. RECENT ADVANCES: Sophisticated regulatory networks govern Fe and Cu homeostasis in response to metal ion availability according to cellular needs and priorities. Molecular remodeling in response to Fe or Cu limitation leads to its economy to benefit photosynthesis. Fe toxicity is prevented by ferritin, a chloroplastic Fe-storage protein in plants. Recent studies on ferritin function and regulation revealed the interplay between iron homeostasis and the redox balance in the chloroplast. CRITICAL ISSUES: Although the connections between metal excess and ROS in the chloroplast are established at the molecular level, the mechanistic details and physiological significance remain to be defined. The causality/effect relationship between transition metals, redox signals, and responses is difficult to establish. FUTURE DIRECTIONS: Integrated approaches have led to a comprehensive understanding of Cu homeostasis in plants. However, the biological functions of several major families of Cu proteins remain unclear. The cellular priorities for Fe use under deficiency remain largely to be determined. A number of transcription factors that function to regulate Cu and Fe homeostasis under deficiency have been characterized, but we have not identified regulators that mediate responses to excess. Importantly, details of metal sensing mechanisms and cross talk to ROS-sensing mechanisms are so far poorly documented in plants

Les Ferritines chez A. thaliana (fonction et régulation)

Fe is essential for all cells because it is the cofactor of numerous proteins, however, excess free Fe is potentially deleterious for the cell. Ferritins are multimeric proteins, present in all the kingdoms of life that can store iron in a safe and bioavailable form. In mammals, ferritins are the main Fe store. They have been predicted to fulfil the same function in plants, but direct evidences are lacking. In plants, ferritin synthesis in response to iron overload is mainly regulated at the transcriptional level, whereas, in animals, it is mainly regulated at the post-transcriptional level by the aconitase dependent IRP/IRE system. The aims of my PhD project were: (i) to elucidate ferritin function in plant physiology and (ii) to decipher the signaling pathway leading to ferritin accumulation in response to iron overload. (i) To directly study ferritin function in plants, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. The loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporter genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, I showed that in the absence of ferritin, plants had higher levels of ROS, and increased activity of enzymes involved in their detoxification. Ferritins are also involved in iron-detoxification during senescence to avoid ROS accumulation. Seeds ferritins are also involved in the protection against oxidative stress during germination and appear to take part in the integrated iron homeostasis establishment. Taken together, my work showed that Arabidopsis ferritins are essential factors that integrate iron and redox homeostasis, while they do not constitute a major iron source for development. (ii) To study ferritin regulation in A. thaliana, the characterization of mutants in the three genes encoding aconitase permitted us to demonstrate that the IRP/IRE system does not occur in the regulation of iron metabolism in plants. Nevertheless, AtFer1 mRNA stability studies have revealed that iron treatment leads to the destabilization of the AtFer1 mRNA. We identified the presence of DST sequences, characterized as mRNA stability determinant, in the 3'-UTR of AtFer1 mRNA. Using chimeric constructs in which the AtFer1 3'-UTR or the AtFer1 3'-UTR with a mutated DST sequence were fused downstream of reporter genes, we have shown that the DST sequence in the 3'-UTR of AtFer1 is functional and sufficient for the iron-dependent mRNA degradation. Using dst1 and dst2 mutants, which are unable to destabilize transcript via DST sequences, we have shown that the DST1 and DST2 gene products, acting in trans in the DST-dependent degradation pathway, are involved in the degradation of AtFer1. Therefore, in addition to the transcriptional regulation described so far, iron is also involved in DST-dependent post-transcriptional regulation of AtFer1 expression. In conclusion, my work has shown that Arabidopsis ferritins are essential elements, which prevent iron toxicity by maintaining a proper labile iron level into the cell, and that sophisticated mechanisms, involving transcriptional and post-transcriptional regulations, permit the tight adjustment of the ferritin accumulation required for the optimal effectiveness of this systemMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Ferritins and iron storage in plants.

International audienceIron is essential for both plant productivity and nutritional quality. Improving plant iron content was attempted through genetic engineering of plants overexpressing ferritins. However, both the roles of these proteins in the plant physiology, and the mechanisms involved in the regulation of their expression are largely unknown. Although the structure of ferritins is highly conserved between plants and animals, their cellular localization differ. Furthermore, regulation of ferritin gene expression in response to iron excess occurs at the transcriptional level in plants, in contrast to animals which regulate ferritin expression at the translational level. In this review, our knowledge of the specific features of plant ferritins is presented, at the level of their (i) structure/function relationships, (ii) cellular localization, and (iii) synthesis regulation during development and in response to various environmental cues. A special emphasis is given to their function in plant physiology, in particular concerning their respective roles in iron storage and in protection against oxidative stress. Indeed, the use of reverse genetics in Arabidopsis recently enabled to produce various knock-out ferritin mutants, revealing strong links between these proteins and protection against oxidative stress. In contrast, their putative iron storage function to furnish iron during various development processes is unlikely to be essential. Ferritins, by buffering iron, exert a fine tuning of the quantity of metal required for metabolic purposes, and help plants to cope with adverse situations, the deleterious effects of which would be amplified if no system had evolved to take care of free reactive iron

Arabidopsis ferritins as an integrative model linking iron metabolism to light, clock and oxidative stress signalings

Arabidopsis ferritins as an integrative model linking iron metabolism to light, clock and oxidative stress signalings. POSTECH International Conference on Plant Scienc

Spatiotemporal Analysis of Copper Homeostasis in Populus trichocarpa Reveals an Integrated Molecular Remodeling for a Preferential Allocation of Copper to Plastocyanin in the Chloroplasts of Developing Leaves1[C][W][OA]

Plastocyanin, which requires copper (Cu) as a cofactor, is an electron carrier in the thylakoid lumen and essential for photoautotrophic growth of plants. The Cu microRNAs, which are expressed during Cu deprivation, down-regulate several transcripts that encode for Cu proteins. Since plastocyanin is not targeted by the Cu microRNAs, a cofactor economy model has been proposed in which plants prioritize Cu for use in photosynthetic electron transport. However, defects in photosynthesis are classic symptoms of Cu deprivation, and priorities in Cu cofactor delivery have not been determined experimentally. Using hydroponically grown Populus trichocarpa (clone Nisqually-1), we have established a physiological and molecular baseline for the response to Cu deficiency. An integrated analysis showed that Cu depletion strongly reduces the activity of several Cu proteins including plastocyanin, and consequently, photosynthesis and growth are decreased. Whereas plastocyanin mRNA levels were only mildly affected by Cu depletion, this treatment strongly affected the expression of other Cu proteins via Cu microRNA-mediated transcript down-regulation. Polyphenol oxidase was newly identified as Cu regulated and targeted by a novel Cu microRNA, miR1444. Importantly, a spatiotemporal analysis after Cu resupply to previously depleted plants revealed that this micronutrient is preferentially allocated to developing photosynthetic tissues. Plastocyanin and photosynthetic electron transport efficiency were the first to recover after Cu addition, whereas recovery of the other Cu-dependent activities was delayed. Our findings lend new support to the hypothesis that the Cu microRNAs serve to mediate a prioritization of Cu cofactor use. These studies also highlight poplar as an alternative sequenced model for spatiotemporal analyses of nutritional homeostasis

Knock-out of ferritin AtFer1 causes earlier onset of age-dependent leaf senescence in Arabidopsis.

Ferritins are iron-storage proteins involved in the regulation of free iron levels in the cells. Arabidopsis thaliana AtFer1 ferritin, one of the best characterized plant ferritin isoforms to date, strongly accumulates upon treatment with excess iron, via a nitric oxide-mediated pathway. However other environmental factors, such as exposure to oxidative stress or to pathogen attack, as well as developmental factors regulate AtFer1 transcript levels. In particular, recent findings have highlighted an accumulation of the ferritin transcript during senescence. To investigate the physiological relevance of AtFer1 ferritin during senescence we isolated an Arabidopsis mutant knock-out in the AtFer1 gene, which we named atfer1-2. We analyzed it together with a second, independent AtFer1 KO mutant, the atfer1-1 mutant. Interestingly, both atfer1-1 and atfer1-2 mutants show symptoms of accelerated natural senescence; the precocious leaf yellowing is accompanied by accelerated decrease of maximal photochemical efficiency and chlorophyll degradation. However, no accelerated senescence upon dark treatment was observed in the atfer1 mutants with respect to their wt. These results suggest that AtFer1 ferritin isoform is functionally involved in events leading to the onset of age-dependent senescence in Arabidopsis and that its iron-detoxification function during senescence is required when reactive oxygen species accumulate

Bayesian inference of visibility in fog and smoke artificial conditions from 3D-LiDAR point clouds

3D-LiDARs are heavily impacted by a degraded visual environment (DVE) like rain, fog and smoke which limits their use for perception algorithms. The capacity to retrieve information about the environmental conditions from an embedded sensor can be an asset to improve autonomous driving performances. False positive artifacts in the point clouds caused by aerosols and hydrometeors particles tend to cause perception issues and thus need filtered out. However, those artifacts can also be used as valuable information to infer weather properties and maybe improve filters. This article proposes a Bayesian inference model which can classify discrete values of visibility using 3D-LiDAR point clouds. Gamma and Log-normal distributions are used to model the distance distributions of the noise points and the model is extended using the Random Finite Set (RFS) formalism with the Poisson and Binomial RFS models. Experiments in artificial fog and smoke conditions are presented and the classification model is trained and tested independently for each experiment. The used point clouds are extracted from specific parts of the field-of-view that can be used to generalize the proposed method to any outdoor scenario. The model shows good classification results with increased performances when the RFS extension is used

Iron uptake and storage in plants

International audienc