An integrated approach using M. truncatula to identify loci/genes controlling nutritional and physiological quality of legume seeds

An integrated approach using M. truncatula to identify loci/genes controlling nutritional and physiological quality of legume seeds

Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

Background: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. [br/] Methodology/Principal Findings: To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. [br/] Conclusions/Significance: We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

<p>Abstract</p> <p>Background</p> <p>Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in <it>Medicago truncatula</it>.</p> <p>Results</p> <p>This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in <it>M. truncatula</it>. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene.</p> <p>Conclusions</p> <p>This study shows that <it>Medicago truncatula </it>contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.</p

The dynamic cilium in human diseases

Cilia are specialized organelles protruding from the cell surface of almost all mammalian cells. They consist of a basal body, composed of two centrioles, and a protruding body, named the axoneme. Although the basic structure of all cilia is the same, numerous differences emerge in different cell types, suggesting diverse functions. In recent years many studies have elucidated the function of 9+0 primary cilia. The primary cilium acts as an antenna for the cell, and several important pathways such as Hedgehog, Wnt and planar cell polarity (PCP) are transduced through it. Many studies on animal models have revealed that during embryogenesis the primary cilium has an essential role in defining the correct patterning of the body. Cilia are composed of hundreds of proteins and the impairment or dysfunction of one protein alone can cause complete loss of cilia or the formation of abnormal cilia. Mutations in ciliary proteins cause ciliopathies which can affect many organs at different levels of severity and are characterized by a wide spectrum of phenotypes. Ciliary proteins can be mutated in more than one ciliopathy, suggesting an interaction between proteins. To date, little is known about the role of primary cilia in adult life and it is tempting to speculate about their role in the maintenance of adult organs. The state of the art in primary cilia studies reveals a very intricate role. Analysis of cilia-related pathways and of the different clinical phenotypes of ciliopathies helps to shed light on the function of these sophisticated organelles. The aim of this review is to evaluate the recent advances in cilia function and the molecular mechanisms at the basis of their activity

Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation

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Medicago truncatula genotype drives the plant nutritional strategy and its associated rhizosphere bacterial communities

International audienceWith the growing concern of developing a more sustainable agriculture, decreasing the use of inputs, and promoting biological diversity, harnessing plant microbiome through plant genetics is gaining of interest to improve plant growth, nutrition, and health. While genome-wide association studies have been conducted to identify plant genes driving the plant microbiome, more multidisciplinary studies are required to assess the relationships among plant the genetic effects, the plant microbiome and plant fitness. Using a metabarcoding approach, we characterized the rhizosphere bacterial communities of a core collection of 155 Medicago truncatula genotypes together with the plant phenotype, using an ecophysiological framework, and investigate the plant genetic effects through genome-wide association studies. The different genotypes within the M. truncatula core collection showed contrasted growth and carbon and nitrogen nutritional strategies but few loci were associated to these ecophysiological traits. To go further, we described its associated rhizosphere bacterial communities, dominated by Proteobacteria, Actinobacteria and Bacteroidetes, and defined acore rhizosphere bacterial community. Next, occurrence of bacterial candidates predicting plant ecophysiological traits of interest were identified using random forest analyzes. Some of them were heritable and plant loci were identified, pinpointing genes related to response to hormone stimulus, systemic acquired resistance, response to stress, nutrient starvation ortransport, and root development.Together, these results suggest that plant genetic can affect the plant growth and nutritional strategies harnessing keystones bacteria in a well-connected network community

Interpretation of genotype X environment interactions for early maize hybrids over 12 years

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