BackgroundAs the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.Methology/Principal FindingsIn Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.ConclusionsThe sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

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As global demand for livestock products (such as meat, milk and eggs) is expected to double by 2050, necessary increases to future production must be reconciled with negative environmental impacts that livestock cause. This paper describes the LivestockPlus concept and demonstrates how the sowing of improved forages can lead to the sustainable intensification of mixed crop-forage-livestock-tree systems in the tropics by producing multiple social, economic and environmental benefits. Sustainable intensification not only improves the productivity of tropical forage-based systems but also reduces the ecological footprint of livestock production and generates a diversity of ecosystem services (ES) such as improved soil quality and reduced erosion, sedimentation and greenhouse gas (GHG) emissions. Integrating improved grass and legume forages into mixed production systems (crop-livestock, tree-livestock, crop-tree-livestock) can restore degraded lands and enhance system resilience to drought and waterlogging associated with climate change. When properly managed tropical forages accumulate large amounts of carbon in soil, fix atmospheric nitrogen (legumes), inhibit nitrification in soil and reduce nitrous oxide emissions (grasses), and reduce GHG emissions per unit livestock product.The LivestockPlus concept is defined as the sustainable intensification of forage-based systems, which is based on 3 interrelated intensification processes: genetic intensification -the development and use of superior grass and legume www.tropicalgrasslands.info cultivars for increased livestock productivity; ecological intensification -the development and application of improved farm and natural resource management practices; and socio-economic intensification -the improvement of local and national institutions and policies, which enable refinements of technologies and support their enduring use. Increases in livestock productivity will require coordinated efforts to develop supportive government, non-government organization and private sector policies that foster investments and fair market compensation for both the products and ES provided. Effective research-for-development efforts that promote agricultural and environmental benefits of foragebased systems can contribute towards implemention of LivestockPlus across a variety of geographic, political and socio-economic contexts. ResumenDe la misma manera que la demanda global de productos pecuarios (carne, leche, huevos) se duplicará para 2050, se espera que las producciones futuras tengan en cuenta los efectos ambientales negativos ocasionados por este sector. En este documento se describe el concepto LivestockPlus y se demuestra cómo en el trópico los forrajes mejorados pueden llevar a la intensificación sostenible de sistemas de producción mixta que integran forrajes/ganadería y cultivos y/o árboles, produciendo múltiples beneficios sociales, económicos y ambientales. La intensificación sostenible no sólo incrementa la productividad de los sistemas tropicales basados en forra...

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