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Arginine Culture Turns on the Elusive Nitrogen Starvation Signal During Robust Phototrophic Growth in Chlamydomonas

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Arginine Culture Turns on the Elusive Nitrogen Starvation Signal During Robust Phototrophic Growth in Chlamydomonas bioRxiv preprint doi: https://doi.org/10.1101/416594; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title 2 Arginine culture turns on the elusive nitrogen starvation signal during robust 3 phototrophic growth in Chlamydomonas 4 5 Authors 6 Jacob Munz1, Yuan Xiong1, Thamali Kariyawasam1, Nolan Shelley1, Jenny Lee1, 7 Ran Ha Hong1, Jaoon Young Hwan Kim2, Young Joon Sung3, Seung-Bum Seo4, 8 Sang Jun Sim3, EonSeon Jin4, and Jae-Hyeok Lee1 9 10 Address 11 1. Department of Botany, University of British Columbia, Vancouver, British 12 Columbia V6T1Z4, Canada 13 2. Convergence Research Division, National Marine Biodiversity Institute of 14 Korea, Chungcheongnam-do 33662, Republic of Korea. 15 3. Department of Chemical and Biological Engineering, Korea University, Seoul 16 136-713, Republic of Korea 17 4. Department of Life Sciences, Research Institute for Natural Sciences, 18 Hanyang University, Seoul 133-791, Republic of Korea 19 20 E-mail address 21 Jacob Munz: [email protected] 22 Yuan Xiong: [email protected] 23 Thamali Kariyawasam: [email protected] 24 Nolan Shelley: [email protected] 25 Jenny Lee: [email protected] 26 Ran Ha Hong: [email protected] 27 Jaoon Young Hwan Kim: [email protected] 28 Young Joon Sung: [email protected] 29 Seung-Bum Seo: [email protected] 30 Sang Jun Sim: [email protected] 31 EonSeon Jin: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/416594; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 32 33 Corresponding Author: 34 Jae-Hyeok Lee 35 #1326C-6270 University Blvd. 36 1-604-827-5973 37 [email protected] 38 39 Date of submission: September 13th, 2018 40 The number of figures: 8 41 The number of tables: 2 42 Word count: 6409 words 43 Supplementary data: 6 figures and 3 tables 44 bioRxiv preprint doi: https://doi.org/10.1101/416594; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 45 Title 46 Arginine culture turns on the elusive nitrogen starvation signal during robust 47 phototrophic growth in Chlamydomonas 48 49 Running title 50 Arginine decouples N starvation responses from growth arrest 51 52 Highlights 53 Arginine catabolism leads to the activation of nitrogen starvation responses while 54 supporting robust photosynthesis and growth, presenting ways to investigate N 55 starvation signaling mechanisms in photosynthetic organisms. 56 57 Abstract 58 Under nitrogen (N) starvation, photosynthetic organisms search for other N 59 sources while slowing down photosynthesis by downregulating light harvesting 60 and electron transport to balance the carbon/nitrogen ratio and eventually 61 stopping growth due to N limitation. To investigate the elusive N starvation- 62 specific signaling mechanisms, we sought a way to induce N starvation 63 responses without limiting photosynthesis or cell growth. In the chlorophyte 64 Chlamydomonas reinhardtii, gametogenesis is exclusively induced during N 65 starvation except in arginine culture. We showed that the arginine-grown culture 66 turned on N starvation responses including hundreds-fold induction of N 67 starvation-induced genes, reduced chlorophyll content, and increased carbon 68 storage in the form of lipid droplets. Arginine culture supported robust 69 phototrophic growth but not heterotrophic growth, indicating that arginine 70 catabolism contributes CO2 to Rubisco without directly fueling ATP synthesis. 71 Based on in silico analysis, we propose the possible routes of arginine 72 catabolism that may bypass critical steps for monitoring of cellular N status and 73 thereby trigger N starvation responses. Our results describe a study system 74 where the N starvation responses are constantly induced without compromising bioRxiv preprint doi: https://doi.org/10.1101/416594; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 75 photosynthesis or growth, paving ways to discover the mechanisms that sense 76 and respond to cellular N status in eukaryotic phototrophs. 77 78 Keywords: 79 Arginine catabolism, Chlamydomonas, Nitrogen catabolite repression, Nitrogen 80 starvation, Phototrophic growth, Nitrogen signaling 81 82 Abbreviations: 83 ADC = arginine decarboxylase 84 ADI = arginine deiminase 85 CCM = carbon concentrating mechanism 86 DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylure 87 FA = fatty acid 88 FAME = fatty acid methyl ester 89 NCR = nitrogen catabolite repression 90 TAG = triacylglycerol 91 TAP = tris-acetate-phosphate medium 92 bioRxiv preprint doi: https://doi.org/10.1101/416594; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 93 Introduction 94 95 All living organisms require constant nitrogen (N) supply for their growth and 96 survival. Two adaptive strategies are generally considered when N sources are 97 not in sufficient supply: 1) to search for new N sources by inducing high-affinity 98 transporters and/or by changing growth patterns or chemotaxis, known 99 collectively as N scavenging, and 2) to mobilize internal N reserves such as 100 storage proteins and N-rich molecules, known collectively as N salvaging (Ono et 101 al., 1996; Chalker-Scott, 1999; Ding et al., 2005; Diaz et al., 2006). Expression of 102 genes involved in N scavenging and N salvaging are suppressed when preferred 103 N sources are available. This regulation has been described as nitrogen 104 catabolite repression (NCR) and its molecular mechanisms have been studied in 105 detail using Saccharomyces cerevisiae as a model (Cooper, 1982; reviewed in 106 Zhang et al., 2018). 107 108 N homeostasis of photosynthetic organisms is challenging as they acquire N - + 109 primarily from inorganic N sources such as nitrate (NO3 ) and ammonium (NH4 ). 110 Assimilation of inorganic N sources requires a significant amount of carbon 111 skeletons and reducing equivalents thereby becoming a primary sink of 112 photosynthetic products. Photosynthetic organisms must coordinate N 113 assimilation and photosynthesis which has been documented in microalgae. 114 Abrupt N deprivation predicts the accumulation of high-energy electrons that are 115 otherwise used for N assimilation (reviewed in Turpin et al., 1988). Such a redox 116 imbalance can be reversed by diverting excess high-energy electrons to a new 117 sink such as carbon storage molecules including starch and triacylglycerol (TAG) 118 as a protective response (Li et al., 2012; Johnson & Alric, 2013). Conversely, N 119 assimilation is inhibited when high-energy electron donors are in low 120 abundances, such as during the dark phase or when limited exogenous carbon 121 sources are provided in the absence of photosynthesis (Syrett, 1953a; Syrett, 122 1953b; Amory et al., 1991; Huppe & Turpin, 1994). 123 bioRxiv preprint doi: https://doi.org/10.1101/416594; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 124 Triggering N limitation responses requires signaling mechanisms that sense 125 external and internal N availabilities. Using Chlamydomonas reinhardtii as a 126 system, transcriptional/proteomic changes upon N starvation have been intensely 127 studied in the interest of knowing the trigger and routes of TAG accumulation in 128 microalgae (Lee et al., 2012, Wase et al., 2014). N limitation rapidly induces 129 genes including the common N catabolite genes for N scavenging and N 130 salvaging, suggesting that C. reinhardtii actively remobilizes N from purines and 131 amino acids while searching for alternative external N sources (Schmollinger et 132 al., 2014; Park et al., 2015). N limitation also downregulates photosynthesis 133 genes including the majority of antenna and photosystem genes (39 out of 42 in 134 Miller et al., 2010). It remains to be studied whether transcriptional or post- 135 transcriptional level regulations are involved in the rapid changes in gene 136 expression upon N limitation. 137 138 Physiological responses to N limitation in C. reinhardtii include the transition to 139 sexually competent gametes (gametogenesis), degradation of photosynthetic 140 proteins/chlorophylls, ribosome turnover, and the accumulation of carbon storage 141 molecules (Sager & Granick, 1954; Siersma & Chiang, 1971; Martin & 142 Goodenough, 1975; Bulté & Wollman, 1992; Weers & Gulati, 1997). Of which, 143 gametogenesis is a response unique to N starvation as the removal of any single 144 component other than N from the growth medium does not elicit this response. 145 Cultures produce <1% sexually competent gametes when the preferred N + - 146 sources, NH4 or NO3 , are present (Sager & Granick, 1954; Matsuda et al., 147 1992; Pozuelo et al., 2000). 148 + 149 C. reinhardtii can grow on various N sources ranging from simple NH4 and 150 amino acids to complex nucleic acids and their derivatives (Sager & Granick, 151 1953; Cain, 1965).
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