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Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae Chlamydomonas Reinhardtii and Coccomyxa Sp

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Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae Chlamydomonas Reinhardtii and Coccomyxa Sp University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications from the Center for Plant Plant Science Innovation, Center for Science Innovation 2012 Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169 Joseph Msanne University of Nebraska-Lincoln, [email protected] Di Xu University of Nebraska-Lincoln, [email protected] Anji Reddy Konda University of Nebraska-Lincoln, [email protected] J. Armando Casas-Mollano University of Nebraska-Lincoln, [email protected] Tala Awada University of Nebraska-Lincoln, [email protected] Msanne, Joseph; Xu, Di; Konda, Anji Reddy; Casas-Mollano, J. Armando; Awada, Tala; Cahoon, Edgar B.; and Cerutti, Heriberto, "Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169" (2012). Faculty Publications from the Center for Plant Science Innovation. 176. http://digitalcommons.unl.edu/plantscifacpub/176 This Article is brought to you for free and open access by the Plant Science Innovation, Center for at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Center for Plant Science Innovation by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. See next page for additional authors Follow this and additional works at: http://digitalcommons.unl.edu/plantscifacpub Part of the Plant Biology Commons, Plant Breeding and Genetics Commons, and the Plant Pathology Commons Authors Joseph Msanne, Di Xu, Anji Reddy Konda, J. Armando Casas-Mollano, Tala Awada, Edgar B. Cahoon, and Heriberto Cerutti This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/plantscifacpub/176 M SANNE ET AL., P HYTOCHEMISTRY 75 (2012) Published in Phytochemistry 75 (2012), pp. 50–59; doi: 10.1016/j.phytochem.2011.12.007 Copyright © 2012 Elsevier. Used by permission. Submitted July 1, 2011; revised October 27, 2011; published online January 5, 2012. Supporting information for this article is available following the references. Metabolic and Gene Expression Changes Triggered by Nitrogen Deprivation in the Photoautotrophically Grown Microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169 Joseph Msanne,1,4 Di Xu,2,* Anji Reddy Konda,3,4 J. Armando Casas-Mollano,2,4 Tala Awada,1 Edgar B. Cahoon,3,4 and Heriberto Cerutti2,4 1. School of Natural Resources, University of Nebraska–Lincoln, Lincoln, Nebraska, USA 2. School of Biological Sciences, University of Nebraska–Lincoln, Lincoln, Nebraska, USA 3. Department of Biochemistry, University of Nebraska–Lincoln, Lincoln, Nebraska, USA 4. Center for Plant Science Innovation, University of Nebraska–Lincoln, Lincoln, Nebraska, USA *Present affiliation: Key Laboratory of Marine Genetics and Breeding of the Ministry of Education, Ocean Univer- sity of China, Qingdao 266003, Shandong, People’s Republic of China. Corresponding authors – Edgar B. Cahoon, telephone 402-472-5611, email [email protected], and Heriberto Cerutti, telephone 402-472-0247, email [email protected] Abstract Microalgae are emerging as suitable feedstocks for renewable biofuel production. Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds, such as lipids and car- bohydrates, and the environmental factors influencing their accumulation is necessary to realize the full potential of these organisms as energy resources. The model green alga Chlamydomonas reinhardtii accumulates significant amounts of triacylglycerols (TAGs) under nitrogen starvation or salt stress 1 M SANNE ET AL., P HYTOCHEMISTRY 75 (2012) in medium containing acetate. However, since cultivation of microalgae for biofuel production may need to rely on sunlight as the main source of energy for biomass synthesis, metabolic and gene expression changes occurring in Chlamydomonas and Coccomyxa subjected to nitrogen deprivation were examined under strictly photoautotrophic conditions. Interestingly, nutrient depletion trig- gered a similar pattern of early synthesis of starch followed by substantial TAG accumulation in both of these fairly divergent green microalgae. A marked decrease in chlorophyll and protein contents was also observed, including reduction in ribosomal polypeptides and some key enzymes for CO2 assimilation like ribulose-1,5-bisphosphate carboxylase/oxygenase. These results suggest that turn- over of nitrogen-rich compounds such as proteins may provide carbon/energy for TAG biosynthesis in the nutrient deprived cells. In Chlamydomonas, several genes coding for diacylglycerol:acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regulated. Understanding the interdependence of these anabolic and catabolic processes and their regulation may allow the engi- neering of algal strains with improved capacity to convert their biomass into useful biofuel precur- sors. Keywords: chlorophytes, starch, triacylglycerols, diacylglycerol:acyl-CoA acyltransferase, 3-Ketoacyl- ACP synthase, biomass, biofuels 1. Introduction Algae are a diverse group of eukaryotic organisms with important roles in marine, fresh- water, and even terrestrial ecosystems. For instance, 30–50% of the planetary net photo- synthetic productivity (the difference between autotrophic gross photosynthesis and respiration) is of marine origin and dependent on phytoplankton biomass (Field et al., 1998; Boyce et al., 2010). Recently, the great potential of algal species as feedstocks for re- newable biofuel production has also gained recognition (Hu et al., 2008; Scott et al., 2010; Wijffels and Barbosa, 2010). Unicellular microalgae are capable of harnessing sunlight and CO2 to produce energy-rich chemical compounds, such as lipids and carbohydrates, which can be converted into fuels (Hu et al., 2008; Rodolfi et al., 2009; Wijffels and Barbosa, 2010). However, the commercial production of algal biofuels is currently hindered by limitations in the biological productivity of strains, culture systems, and harvesting/extraction pro- cesses (Sheehan et al., 1998; Hu et al., 2008; Scott et al., 2010; Wijffels and Barbosa, 2010). As recently proposed, a multidisciplinary approach, including advances in basic biology and metabolic engineering of algal strains as well as in culture systems, bioprocessing, and integrated biorefinery design, may be required to realize the full potential of microalgae as sustainable biofuel sources (Hu et al., 2008; Scott et al., 2010; Wijffels and Barbosa, 2010). Desirable strain characteristics include rapid growth rate, high product content, toler- ance to variable environmental conditions, resistance to predators and viruses and ease of harvest and extraction (Griffiths and Harrison, 2009; Rodolfi et al., 2009; Radakovits et al., 2010). An additional algal feature for biodiesel production is the suitability of the lipid composition, with triacylglycerols (TAGs) being the preferred substrate (Schenk et al., 2008; Radakovits et al., 2010). Algae synthesize, under optimal growth conditions, fatty acids predominantly for esterification into glycerol-based membrane lipids (Guschina and 2 M SANNE ET AL., P HYTOCHEMISTRY 75 (2012) Harwood, 2006; Hu et al., 2008; Khozin-Goldberg and Cohen, 2011). However, factors such as temperature, irradiance, and nutrient availability affect both lipid composition and lipid content in many algal species (Guschina and Harwood, 2006; Hu et al., 2008; Khozin-Gold- berg and Cohen, 2011; Siaut et al., 2011). Upon certain environmental stresses (particularly nutrient shortage), various algae accumulate energy-rich storage compounds such as starch and TAGs (Guschina and Harwood, 2006; Hu et al., 2008; Rodolfi et al., 2009; Wang et al., 2009; Dean et al., 2010; Li et al., 2010; Moellering and Benning, 2010; Work et al., 2010; Fan et al., 2011; Siaut et al., 2011). Fatty acids are the common precursors for the formation of both membrane lipids (required for growth) and TAGs (involved in energy storage and fatty acid homeostasis), but it remains to be elucidated how algal cells coordinate the dis- tribution of these precursors to distinct destinations in response to environmental stimuli (Sheehan et al., 1998; Guschina and Harwood, 2006; Hu et al., 2008; Radakovits et al., 2010). Interestingly, biomass productivity and lipid content appear to be inversely correlated in many algae (Sheehan et al., 1998; Hu et al., 2008; Rodolfi et al., 2009) and nutrient limitation stimulates TAG accumulation but at the expense of growth (Rodolfi et al., 2009; Li et al., 2011). Given the outlined constraints, improving algal strain performance will require a greater understanding of carbon allocation between biosynthetic pathways and of the reg- ulatory mechanisms controlling this distribution, particularly in response to environmen- tal stresses. This need is also emphasized by the limited success in increasing total oil content in higher plants and algae by the direct engineering of single lipid biosynthesis components (Dunahay et al., 1996; Durrett et al., 2008; Li et al., 2010; Radakovits et al., 2010; Work et al., 2010). The unicellular green alga Chlamydomonas reinhardtii has emerged as a model system for
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