Plant Physiology Preview. Published on May 16, 2018, as DOI:10.1104/pp.17.01780
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Short title: PI3K is a gatekeeper of algal biofuel synthesis
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Article Title: The Ancient Phosphatidylinositol 3-Kinase Signaling System is a
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Master Regulator of Energy and Carbon Metabolism in Algae
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Rishiram Ramanana,b#, Quynh-Giao Tranb,d#, Dae-Hyun Chob, Jae-Eun Jeongc,
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Byung-Hyuk Kimb, Sang-Yoon Shinb,d, Sae-Hae Choib, Kwang-Hyeon Liue, Dae-Soo
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Kimc, Seon-Jin Leef, José L. Crespog, Hee-Gu Leef, Hee-Mock Ohb,d, Hee-Sik
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Kimb,d,1
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a
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University of Kerala, Tejaswini Hills, Periya, Kasaragod, Kerala, India
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b
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Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, Republic of Korea
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c
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Republic of Korea
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d
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Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu,
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Daejeon, Korea
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e
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f
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Daejeon, Republic of Korea
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g
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Cientificas, Universidad de Sevilla, Sevilla, Spain
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1
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Tel. +82-42-860-4326; Fax. +82-42-860-4594.
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#
Department of Environmental Science, School of Earth Science Systems, Central Cell Factory Research Center, Korea Research Institute of Bioscience and
Genome Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon, Department of Environmental Biotechnology, KRIBB School of Biotechnology,
College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea
Biomedical Genomics Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Instituto de Bioquimica Vegetal y Fotosintesis, Consejo Superior de Investigaciones Correspondence: E-mail:
[email protected] These authors equally contributed to this work.
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ORCID IDs: 0000-0002-4641-9603 (R.R.); 0000-0003-3514-1025 (J.L.C.)
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One Sentence Summary:
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Phosphatidylinositol 3-kinase signaling influences biofuel yields in algae by
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regulating membrane lipid hydrolysis, lipogenesis, tricarboxylic acid cycle, and
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mitochondrial ATP synthesis.
34 1 Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Copyright 2018 by the American Society of Plant Biologists
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LIST OF AUTHOR CONTRIBUTIONS
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R.R., and H.-S.K. designed the study. R.R., Q.-G.T., S.-Y.S., B.-H.K., K.-H.L. and S.-
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H.C. conducted experiments; R.R., D.-H.C., J.-E.J., D.-S.K., S.-J.L., J.L.C., K.-H.L.,
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Q.-G.T. and H.-S.K. analyzed the data; R.R., H.-G.L., H.-M.O., J.L.C., and H.-S.K.
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wrote the manuscript with the contribution of all authors.
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FUNDING INFORMATION
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This work was supported by a grant from the host institution through the KRIBB
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Research Initiative Program (www.kribb.re.kr), the Advanced Biomass R&D Center
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(ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future
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Planning (2015M3A6A2065697), and a grant from Marine Biotechnology Program
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funded by Ministry of Oceans and Fisheries (20150184). The authors declare that
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they have no conflicts of interest.
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Abstract
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Algae undergo a complete metabolic transformation under stress by arresting cell
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growth, inducing autophagy, and hyper-accumulating biofuel precursors such as
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triacylglycerols and starch. However, the regulatory mechanisms behind this stress-
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induced transformation are still unclear. Here, we use biochemical, mutational, and
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‘omics’ approaches to demonstrate that phosphatidylinositol 3-kinase (PI3K)
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signaling mediates the homeostasis of energy molecules and influences carbon
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metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown
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(KD) of algal class III PI3K led to significantly decreased cell growth, altered cell
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morphology, and higher lipid and starch contents. Lipid profiling of wild-type and
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PI3K KD lines showed significantly reduced membrane lipid breakdown under
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nitrogen starvation (–N) in the KD. RNA-seq and network analyses showed that
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under –N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis
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by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic
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acid cycle downregulation and via acetyl-CoA synthesis from glycolysis. Remarkably,
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autophagic responses did not have primacy over inositide signaling in algae, unlike
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in mammals and vascular plants. The mutant displayed a fundamental shift in
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intracellular energy flux, analogous to that in tumor cells. The high free fatty acid
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levels and reduced mitochondrial ATP generation led to decreased cell viability.
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These results indicate that the PI3K signal transduction pathway is the metabolic
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gatekeeper restraining biofuel yields, thus maintaining fitness and viability under
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stress in algae. This study demonstrates the existence of homeostasis between
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starch and lipid synthesis controlled by lipid signaling in algae and expands our
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understanding of such processes, with biotechnological and evolutionary
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implications.
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Keywords: Green algae; Chlamydomonas reinhardtii; Signaling network;
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Autophagy; Lipids; Starch; Transcriptomics; Pathway engineering
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INTRODUCTION
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Chlamydomonas reinhardtii, which possesses both animal and plant characteristics,
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has emerged as a model alga for studying lipid metabolism, flagellar assembly,
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photosynthesis, and other cellular processes (Merchant et al., 2007). Nutrient
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starvation in C. reinhardtii and other model algae leads to the synthesis of
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triacylglycerols (TAGs) by inducing membrane lipid hydrolysis, and starch synthesis
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(Hu et al., 2008; Kajikawa et al., 2015; Schmollinger et al., 2014). The membrane
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lipid hydrolysis is mediated by lipases that release Acyl-CoA from membrane lipids
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because these substrates are incorporated into the glycerol backbone of TAGs,
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which form the inner core of LDs. There are several lipases in C. reinhardtii that
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efficiently catalyze this conversion (Li et al., 2012; Yoon et al., 2012).
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In plant vegetative tissues, the breakdown of membrane lipids to TAGs under
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stress conditions is supposed to circumvent the harmful effects of free fatty acid
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(FFA) and reactive oxygen species (ROS) accumulation (Chapman et al., 2012; Hou
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et al., 2016). TAGs in mammalian tissues constantly accumulate and are recycled to
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FFA based on a complex signaling network involving insulin (Nemazanyy et al.,
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2015; Wymann and Schneiter, 2008). On the other hand, it has been proved that
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inositide signaling inhibits Akt, a serine/threonine kinase that in turn regulates
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glucose homeostasis and protein translation thereby affecting insulin signaling
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growth (Chakraborty et al., 2010). In fact, several studies have demonstrated the
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major role of lipid signaling in growth, carbon metabolism, autophagy, apoptosis, and
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disease in mammalian models (Nemazanyy et al., 2015; Shanware et al., 2013;
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Vanhaesebroeck et al., 2012; Wymann and Schneiter, 2008). Although it is widely
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known that algae hydrolyze membrane lipids similar to plants, and homeostasis
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exists between TAG and membrane lipid hydrolysis, a definite mechanism has not
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been elucidated (Trentacoste et al., 2013). Moreover, the roles of algal lipid signaling
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and regulation in stress-induced responses are largely unexplored, although such
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mechanisms are known to occur in mammalian and plant models (Couso et al.,
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2016; Hou et al., 2016; Wymann and Schneiter, 2008). Algae are unique eukaryotes,
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as they produce both starch and LDs as a response to any stress or starvation
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condition, linking plants and animals (Merchant et al., 2007). Therefore, algae are
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excellent model organisms for studying signaling and interactions between FFA,
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TAG, membrane lipids, and starch synthesis (Boss and Im, 2012; Shanware et al.,
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2013; Vanhaesebroeck et al., 2012). 4 Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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Algae are considered to be a renewable feedstock for various bioproducts,
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including biofuels (Wijffels and Barbosa, 2010). Commercialization of algal biofuels
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has several bottlenecks including current fossil fuel prices, high biofuel production
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costs owing to inadequate yields, and harvest inefficiencies. The most significant
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technical challenge in algal biofuel production is the trade-off between growth and
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accumulation of biofuel precursors, leading to modest yield. TAG and starch
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granules are precursors of biodiesel and bioethanol, respectively (Hu et al., 2008;
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Merchant et al., 2012; Sheehan et al., 1998; Wijffels and Barbosa, 2010). Even after
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several decades of research in this area, the molecular mechanism for this
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dichotomy is still unresolved (Sheehan et al., 1998). Therefore, studying the
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signaling mechanism behind growth, starvation, and starvation-related responses is
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critical to the success of algal biofuels.
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Phosphatidylinositol 3-kinase (PI3K) signaling in higher eukaryotes has been
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connected to starvation-induced processes, including autophagy, lipid metabolism,
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and cell survival (Boss and Im, 2012; Chapman et al., 2012; Shanware et al., 2013;
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Vanhaesebroeck et al., 2012). PI3K is also known to have a major role in insulin-
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dependent glucose homeostasis in higher eukaryotes (Nemazanyy et al., 2015).
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Class III PI3K enzyme is the oldest class of this enzyme present in all known
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eukaryotes and is the only class in algae, yeasts, and plants (Vanhaesebroeck et al.,
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2012). Moreover, phosphatidylinositol 3-phosphate (PI3P) is thought to be the first
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known phosphatidylinositol lipid to appear during evolution, having been found in
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both bacteria and archaea (Irvine, 2016). Earlier studies on PI3K in Chlamydomonas
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have shown that the protein is encoded by a single copy gene, Vacuolar protein
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sorting 34 (VPS34) (Molendijk and Irvine, 1998). It has been speculated that PI3P
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may act as a signaling molecule based on its rapid turnover in pulse-chase
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experiments in Chlamydomonas eugematos and that PI3K in algae may act as a
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signaling system (Irvine et al., 1992; Molendijk and Irvine, 1998; Munnik et al., 1994).
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In addition, a recent study on inositide signaling in Chlamydomonas reinhardtii
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implicated the signaling network in nutrient sensing and starvation-triggered
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responses (Couso et al., 2016).
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Here, we show that PI3K regulates the homeostasis of membrane lipids,
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TAGs, starch, FFA, and ATP by influencing other components of the signaling
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network. Hence, the PI3K signaling cascade independently influences the energy
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homeostasis. The PI3K KD lines also display significant differences in phenotype 5 Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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and genotype related to growth and lipid metabolism. PI3K is known to play a major
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role in autophagosome formation in higher eukaryotes (Shanware et al., 2013). The
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selected mutant line reveals autophagy induction under stress, thereby nullifying any
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role of selective autophagic processes in the observed phenotype. Therefore, this
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cascade in algae performs a plethora of functions critical for the survival and fitness
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of the organism, especially in an oligotrophic environment. From a biofuel
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perspective, this study sheds light on the regulatory mechanisms behind the tradeoff
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between growth and biofuel precursor accumulation. Our findings demonstrate that
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signal transduction pathways might provide vital clues to several outstanding
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research bottlenecks in algae and that engineering signaling pathways might be one
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way forward.
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RESULTS
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Inhibition and Knockdown of Class III PI3K Increases Lipid and Starch
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Contents in Algae
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The green algal PI3K protein encoded by the VPS34 gene is highly conserved and is
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more closely associated with homologs from higher plants than other protists and
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mammals, which suggests conservation in green lineage (green algae and land
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plants) (Fig. 1A) (Finet et al., 2010; Mikami, 2014). The C. reinhardtii PI3K is an
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extraordinarily large 125 kDa protein with a novel insert region in the N-terminus of
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the catalytic domain (Molendijk and Irvine, 1998). PI3K is known to be associated
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with starvation-related responses and lipid metabolism in higher eukaryotes (Boss
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and Im, 2012; Chapman et al., 2012; Shanware et al., 2013; Vanhaesebroeck et al.,
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2012). Therefore, we tested the related phenotypic responses of C. reinhardtii after
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treatment with different PI3K inhibitors. Significantly enhanced TAG fluorescence
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was observed in C. reinhardtii cc124 (wild type, WT) upon PI3K inhibition with 3-
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Methyladenine (3MA) and Wortmannin, especially under Nitrogen (N) starvation
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(Supplemental Fig. 1). Further studies with 3MA showed that PI3K inhibition also
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increased lipid content in WT under N-sufficient (+N, 9 days, unless otherwise
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mentioned) and acute starvation (–N, 48 hours, unless otherwise mentioned)
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conditions (Supplemental Fig. 1). Similarly, 3MA treatment in Scenedesmus obliquus
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also resulted in increased TAG, confirming our initial hypothesis that PI3K has a
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critical role in regulating lipid metabolism in algae like in higher eukaryotes
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(Supplemental Fig. 1). Therefore, PI3K knockdown (KD) C. reinhardtii mutants were
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generated to further study the role of PI3K in lipid signaling in algae.
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C. reinhardtii KD lines displayed similar morphological characteristics
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compared to WT and Mock (empty vector) lines. The KD mutants had reduced
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growth particularly in photoautotrophic conditions, in the presence and absence of N
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(Fig. 1B). Most PI3K KD lines showed increased total lipid content especially in –N
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conditions, increased TAG content, and increased cell size compared to both the
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Mock and WT lines. KD lines were also more circular in shape compared to WT and
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Mock lines, and displayed an inability to move in +N conditions (Supplemental Fig. 2;
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Supplemental Dataset 1). Among the three KD lines, T1B3 and T411 lines showed
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altered growth patterns (Fig. 1C) while T411 line showed severely impaired growth
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under both mixotrophic and photoautotrophic conditions in both +N and –N
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conditions. Principal component analysis (PCA) of the above morphological, 7 Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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physiological, and genotypic traits of the mutants showed that all KD lines clustered 8
Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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together whereas WT and Mock lines clustered separately. It must be noted that KD
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lines under +N and –N conditions clustered separately, whereas WT and Mock lines
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clustered together even though the conditions differed. T1B3 and T411 lines
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displayed significantly decreased VPS34 transcript levels (p≤0.05) compared to the
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Mock and WT lines (Supplemental Fig. 2E&F) under +N and –N conditions. Although
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both T1B3 and T411 lines had similar phenotypic, physiological, and genotypic
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characteristics, confirming the knockdown of PI3K, the T411 line was selected for
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further studies due to its phenotypic stability across generations (Fig. 2). These
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studies confirmed that the T411 line consistently showed highly reduced cell growth
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in +N and –N conditions across generations and a milder green hue that was
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reinforced with decreased PI3P generation, confirming reduced PI3K enzyme activity
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(p≤0.01). However, there were no significant changes to protein and chlorophyll
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content in T411 in +N and –N conditions while the starch content increased
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significantly in T411, reaching 38% DCW in –N conditions (Fig. 2F).
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Furthermore, the T411 line showed insignificant changes in TAG levels in +N
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and –N conditions (Fig. 3A). However, higher TAG fluorescence was observed under
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high-light stress condition (Supplemental Fig. 3). Repeated efforts to knock down the
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VPS34 gene in starchless mutants consistently resulted in lethal mutations. This
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result, taken together with reduced cell numbers in KD lines, shows that severe 9 Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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knockdown of PI3K would result in unviable 10 mutants possibly because of issues in Downloaded from on May 22, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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cell division. These results indicate that substantial metabolic changes occur in the
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mutant related to growth and starch and lipid metabolism. Therefore, we performed
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additional lipidomics analyses in WT and T411 cells grown in +N and –N conditions.
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PI3K Regulates Membrane Lipid Hydrolysis in N Starvation
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Algal lipids were profiled using direct infusion nanoelectrospray-tandem mass
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spectrometry in both ionization modes. Twenty-nine lipid species [11 DGTS, 11 TAG,
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4 Lyso-DGTS, and 3 DGDG] were identified in the positive ion mode. Thirteen lipid
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species [5 PG, 4 SQDG, 3 MGDG, and 1 PI] were identified in the negative ion mode
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(Supplemental Table 1 and 2). The statistical analysis of algal lipid profiles showed a
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clear separation among all sample groups (WT+N, WT–N, T411+N, T411–N)
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(p
