Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial β-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (∼38%), and low (∼1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g−1 dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g−1 dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin

Agronomic performance and transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro and control tomato lines under field conditions

Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanumlycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was “HighCaro (HC),” a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to β-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to β-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place

Nicotiana benthamiana as a Production Platform for Artemisinin Precursors

Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-ß-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed

The Fresh water Mussels (Bivalvia, Unionoidea) of the Upper Delaware River Drainage

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The psychology of transition into social institutions: Exploring the relationship between young people’s experience and well-being in higher education

The work presented here is part of a longitudinal study exploring transition in to Higher Education against a background of Widening Participation. 219 first year students from Edge Hill completed measures including coping style, background and well-being, as well as completing narratives. The focus of this presentation examines coping style relationships. It was found that there are two contrasting tendencies. One is that seemingly unhealthy coping styles associate with disengagement (e.g. drug use and denial). Healthy coping styles also tend to cluster together (e.g. active coping and planning ahead). It is suggested that there is a need to take account of the individual in terms of strategy development

The psychology of transition into social institutions: Exploring the relationship between young people’s experiences and well-being in higher education

Policy-makers and researchers, unless they believe in moral decline as an explanatory principle, are forced to make use of psychology in order to explore disaffected youth behaviour. Usually this is an intuitive psychology of cause and effect; so there will be an increasing call for formal psychological investigation in order to understand youth experiences, attitudes, and wellbeing. We seek to extend this approach to understanding the process of integration into higher education institutions, since education is key to current aims to build social inclusivity. In this paper, we present findings on young people's self-esteem, anxiety, and ability to cope in a group for whom disadvantaged backgrounds and first-generation entry to higher education are frequent. Using extended narrative accounts, we combine studies of subjective experience with these formal indicators of well-being and sense of personal empowerment. We outline a longitudinal programme for studying young people as they move into higher education, and offer our results as a contribution to understanding (a) the relationship of social transition to health-related outcomes for individuals, and (b) dynamic interrelationships of youth behaviours and perceptions, and (c) social and individual factors in adjustment to higher education. For example, initial data suggests some evidence of disengagement and poor coping strategie

Additive synthesis with DIASS-M4C on Argonne National Laboratory`s IBM POWERparallel System (SP)

DIASS-M4C, a digital additive instrument was implemented on the Argonne National Laboratory`s IBM POWER parallel System (SP). This paper discusses the need for a massively parallel supercomputer and shows how the code was parallelized. The resulting sounds and the degree of control the user can have justify the effort and the use of such a large computer