Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp

[EN] Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloatsafe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.This work was supported by grants BIO2012-39849-C02-01 and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO) (http://www.idi.mineco.gob.es/portal/site/MICINN) to LAC and a fellowship of the JAE-CSIC program to SF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fresquet-Corrales, S.; Roque Mesa, EM.; Sarrión-Perdigones, A.; Rochina, M.; López-Gresa, MP.; Díaz-Mula, HM.; Belles Albert, JM.... (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLoS ONE. 12(9). https://doi.org/10.1371/journal.pone.0184839Se018483912

Adaptation to life on land: how plants developed UV sunscreens

Terrestrial plant life evolved from an aquatic ancestor 400-6-- million years ago. This significant step from water to land involved a suite of genetic adaptations allowing for colonisation of new environments. One such adaptation was for tolerance of incident UV-B radiation. UV-B radiation is particularly damaging to plant life because of its absorption by molecules such as DNA and the production of reactive oxygen species. Therefore the development of UV-B tolerance by early land plants would have been particularly important. Flavonoid synthesis contributes significantly to UV-B tolerance in higher plants, yet it is not known whether the acquisition of flavonoid production allowed the first land plants to tolerate UV-B and colonise the land environment. Here we examine the model bryophyte Marchantia polymorpha, from one of the oldest extant land plant lineages , to determine the effect of flavonoid production in response to UV-B. Under set environmental conditions and UV-B irradiance M. polymorpha is shown to respond to UV-B with an increased production of flavonoid compounds (predominantly flavones). These preliminary results may lead into an understanding of the importance of flavonoid production in enabling the successful colonisation of land by plant life

Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization

Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name “auronidin,” with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants