Spatial and Temporal Variation in Selection of Genes Associated with Pearl Millet Varietal Quantitative Traits In situ.

Ongoing global climate changes imply new challenges for agriculture. Whether plants and crops can adapt to such rapid changes is still a widely debated question. We previously showed adaptation in the form of earlier flowering in pearl millet at the scale of a whole country over three decades. However, this analysis did not deal with variability of year to year selection. To understand and possibly manage plant and crop adaptation, we need more knowledge of how selection acts in situ. Is selection gradual, abrupt, and does it vary in space and over time? In the present study, we tracked the evolution of allele frequency in two genes associated with pearl millet phenotypic variation in situ. We sampled 17 populations of cultivated pearl millet over a period of 2 years. We tracked changes in allele frequencies in these populations by genotyping more than seven thousand individuals. We demonstrate that several allele frequencies changes are compatible with selection, by correcting allele frequency changes associated with genetic drift. We found marked variation in allele frequencies from year to year, suggesting a variable selection effect in space and over time. We estimated the strength of selection associated with variations in allele frequency. Our results suggest that the polymorphism maintained at the genes we studied is partially explained by the spatial and temporal variability of selection. In response to environmental changes, traditional pearl millet varieties could rapidly adapt thanks to this available functional variability.

Genetic basis of pearl millet adaptation along an environmental gradient investigated by a combination of genome scan and association mapping.

Identifying the molecular bases of adaptation is a key issue in evolutionary biology. Genome scan is an efficient approach for identifying important molecular variation involved in adaptation. Association mapping also offers an opportunity to gain insight into genotype-phenotype relationships. Using these two approaches coupled with environmental data should help to come up with a refined picture of the evolutionary process underlying adaptation. In this study, we first conducted a selection scan analysis on a transcription factor gene family. We focused on the MADS-box gene family, a gene family which plays a crucial role in vegetative and flower development. Twenty-one pearl millet populations were sampled along an environmental gradient in West Africa. We identified one gene, i.e. PgMADS11, using Bayesian analysis to detect selection signatures. Polymorphism at this gene was also associated with flowering time variation in an association mapping framework. Finally, we found that PgMADS11 allele frequencies were closely associated with annual rainfall. Overall, we determined an efficient way to detect functional polymorphisms associated with climate variation in non-model plants by combining genome scan and association mapping. These results should help monitor the impact of recent climatic changes on plant adaptation.

Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum.

BACKGROUND: Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism. RESULTS: The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins. CONCLUSIONS: CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism.

Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis.

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.

Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple.

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.