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1.
Elife ; 122024 Jan 31.
Article in English | MEDLINE | ID: mdl-38294329

ABSTRACT

Seedling root traits impact plant establishment under challenging environments. Pearl millet is one of the most heat and drought tolerant cereal crops that provides a vital food source across the sub-Saharan Sahel region. Pearl millet's early root system features a single fast-growing primary root which we hypothesize is an adaptation to the Sahelian climate. Using crop modeling, we demonstrate that early drought stress is an important constraint in agrosystems in the Sahel where pearl millet was domesticated. Furthermore, we show that increased pearl millet primary root growth is correlated with increased early water stress tolerance in field conditions. Genetics including genome-wide association study and quantitative trait loci (QTL) approaches identify genomic regions controlling this key root trait. Combining gene expression data, re-sequencing and re-annotation of one of these genomic regions identified a glutaredoxin-encoding gene PgGRXC9 as the candidate stress resilience root growth regulator. Functional characterization of its closest Arabidopsis homolog AtROXY19 revealed a novel role for this glutaredoxin (GRX) gene clade in regulating cell elongation. In summary, our study suggests a conserved function for GRX genes in conferring root cell elongation and enhancing resilience of pearl millet to its Sahelian environment.


Pearl millet is a staple food for over 90 million people living in regions of Africa and India that typically experience high temperatures and little rainfall. It was domesticated about 4,500 years ago in the Sahel region of West Africa and is one of the most heat and drought tolerant cereal crops worldwide. In most plants, organs known as roots absorb water and essential nutrients from the soil. Young pearl millet plants develop a fast-growing primary root, but it is unclear how this unique feature helps the crop to grow in hot and dry conditions. Using weather data collected from the Sahel over a 20-year period, Fuente, Grondin et al. predicted by modelling that early drought stress is the major factor limiting pearl millet growth and yield in this region. Field experiments found that plants with primary roots that grow faster within soil were better at tolerating early drought than those with slower growing roots. Further work using genetic approaches revealed that a gene known as PgGRXC9 promotes the growth of the primary root. To better understand how this gene works, the team examined a very similar gene in a well-studied model plant known as Arabidopsis. This suggested that PgGRXC9 helps the primary root to grow by stimulating cell elongation within the root. Since it is well adapted to dry conditions, pearl millet is expected to play an important role in helping agriculture adjust to climate change. The findings of Fuente, Grondin et al. may be used by plant breeders to create more resilient and productive varieties of pearl millet.


Subject(s)
Arabidopsis , Pennisetum , Droughts , Pennisetum/genetics , Glutaredoxins , Genome-Wide Association Study , Crops, Agricultural
2.
Methods Mol Biol ; 2238: 173-191, 2021.
Article in English | MEDLINE | ID: mdl-33471331

ABSTRACT

Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Genome, Plant , Oryza/growth & development , Plants, Genetically Modified/growth & development , Protoplasts/physiology , Gene Transfer Techniques , Genetic Vectors/genetics , Oryza/genetics , Plant Breeding , Plants, Genetically Modified/genetics , Transformation, Genetic , Transgenes/physiology
3.
BMC Mol Cell Biol ; 21(1): 92, 2020 Dec 10.
Article in English | MEDLINE | ID: mdl-33302866

ABSTRACT

BACKGROUND: In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. RESULTS: We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. CONCLUSION: The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Plant , High-Throughput Screening Assays , Laser Capture Microdissection , Oryza/genetics , Plant Roots/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Organ Specificity/genetics , Paraffin Embedding , RNA, Plant/genetics , RNA, Plant/metabolism , Reproducibility of Results
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