Identification of key regulators of legume seed adaptation to environmental fluctuations – REGULEG

In the REGULEG project, we made use of the genetic diversity that exists for the model legume barrel medic (Medicago truncatula) to identify genes that are responsible for the changes in seed composition and vigor traits when submitted to different environments. Plants of 200 accessions that were collected from different geographic regions and for which the genomes are sequenced were grown in four different environments (drought, different times of sowing) in Angers and Montpellier, and seeds were collected at maturity. A wide range of seed traits related to seed weight, composition and vigour were determined on the seeds of the four environments. For each seed trait, we calculated the plasticity indices for each genotype, which represents the variation of each seed trait between production environments for a given accession. Next, these plasticity indices were used to perform association genetics, which focuses on the identification of correlations between phenotypic traits and genetic variants in the genomes of the 200 accessions with the aim to identify and locate the underlying genes in the genome. The knowledge on plasticity genes was transferred from barrel medic to cultivated pea via phylogenetic studies and expression studies. The role of some candidate genes was verified using pea and barrel medic mutants in which the genes were no longer functional.

A number of putative plasticity genes were identified that are involved in the modulation of seed composition and seed vigour and lifespan when plants are grown in different environmental conditions. Among the major discoveries were the identification of a homocysteine S-methyltransferase 3 gene that acts as a plasticity gene by contributing to stabilizing the 7S to 11S globulin ratio under sulfur-limiting conditions, and the role of a bHLH transcription factor that is linked to the flowering pathway in the plasticity of seed vigor upon drought. These candidate genes can be targeted to mitigate negative impacts of environmental stresses on seed proteins and seed vigour, thereby enhancing homogeneity and yield stability via the transfer of knowledge to cultivated pea.

The REGULEG project has contributed to the fundamental understanding of the mechanisms behind molecular plasticity of seed traits, providing a mechanistic explanation behind the phenotypic plasticity and revealing some of the main genes and pathways that explain variations in seed quality by changing environment during seed production. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed proteins. For instance, to improve seed protein stability, the identification of HMT3 and stress responsive regulators expressed during the seed filling stage are interesting candidates. Likewise, genes like ABI4, bHLH49, SMP and MIEL1 are interesting candidate genes to improve stability of seed vigor. A selection of the most promising genes to improve stability has been targeted by Tilling in pea and functional analysis on these mutants is currently underway. If validated, they are interesting genes to be targeted in prebreeding programs to improve stability to environment in other valuable legume crops (bean, pea, soybean, faba bean).
It is expected that the identification of regulators of the plasticity of seed quality will find their applicability beyond legume species, as we have shown for MIEL1, which was discovered in M. truncatula, but shows a similar phenotype in Arabidopsis and thus a conserved plasticity regulator.

Cartelier, K., Aimé, D., Ly Vu, J., Combes-Soia, L., Labas, V., Prosperi, J.-M., Buitink, J., Gallardo, K. and Le Signor, C. (2021), Genetic determinants of seed protein plasticity in response to the environment in Medicago truncatula. The Plant Journal. Accepted Author Manuscript. doi.org/10.1111/tpj.15236
Chen Z, Ly Vu J, Ly Vu B, Buitink J, Leprince O, Verdier J (2021) Genome-wide association studies of seed performance traits in response to heat stress in Medicago truncatula reveal MYB30-Interacting E3 Ligase1 as a regulator of seed germination plasticity. Accepted in Frontiers in Plant Science, doi: 10.3389/fpls.2021.673072
Zinsmeister J, Leprince O, Buitink J (2020) Molecular and environmental factors regulating seed longevity Biochemical Journal 477: 305–323, doi.org/10.1042/BCJ20190165
Pecrix et al. (2018) Whole-genome landscape of Medicago truncatula symbiotic genes Medicago genome, Nature Plants 4: 1017-1025 doi.org/10.1038/s41477-018-0286-7
Buitink J, Leprince O (2018) «Letters to the twenty-first century botanist. Second series: “What is a seed? – 2. Regulation. Botany Letters 165, 181-185, doi.org/10.1080/23818107.2018.1476177
Leprince O, Pellizzaro A, Berriri S, Buitink J (2016) Late seed maturation: drying without dying. Journal of Experimental Botany doi: 10.1093/jxb/erw363

A major part of human food and animal feed supply is based on seeds. There is an increasing need for plant proteins to sustain the increase in meat consumption world-wide. Production of grain legumes, which are an important source of proteins is therefore strategically and economically important for food security. Given their ability to fix atmospheric nitrogen, they are pivotal to the development of European sustainable agriculture. Despite these advantages, European countries are increasingly dependent on protein imports, mostly from South American soybean. Instability of legume seed yield and protein content due to environmental fluctuations, in particular drought and high temperature limits their wide adoption in Europe, and breeding for stabilized nutritional quality and yield is needed. Producing highly vigorous seeds is an additional key lever to increase crop production efficiency. The desirable characteristics of high seed vigor are the seed storability in the dry state (longevity), high and synchronous germination and seedling establishment, even under suboptimal conditions. Seed vigor is a complex trait determined by gene by environment interactions that remain poorly understood. Therefore, stable production of high vigor seeds is very difficult to achieve. Also, improvement of nutritional quality through breeding for seed size and seed composition should not be achieved at the expense of seed vigor and vice-versa. Mitigation of climate change effects on legume seed physiological quality and composition represents thus a major and urgent challenge for the seed industry. Phenotypic plasticity is the ability of a genotype to produce distinct phenotypes in different environments and has been widely studied in relation to (a)biotic stress and nutrition in vegetative tissues. Phenotypic plasticity is expected to play an increasing role in efforts for crop improvement. However, we are remarkably ignorant about the underlying factors that are responsible for the dramatic effects that environment can have on physiological and nutritional seed quality. How precisely does a developing seed customize its size, composition and vigor to specific environments? The objective of this project is to identify key genes regulating the plasticity of nutritional and physiological seed quality of Medicago truncatula using GxE genome wide association studies (GWAS). A multilayer regulatory network will be generated that describes the molecular processes related to seed filling and the acquisition of vigor that are modulated by altered environment. Significant GWAS loci will be integrated in the network to obtain the key regulatory genes that play a role in the molecular plasticity of seed quality traits. Using the close synteny of the M. truncatula genome with that of legume crops, we will transfer knowledge into pea to test for gene-to-field applicability. This project will highlight the mechanisms governing plasticity in legume seed quality traits that are related to adaptation to changing environments, thereby providing new cues towards the improvement of the grain legume sector.