Nitric Oxide Is Important for Sensing and Survival Under Hypoxia in Arabidopsis
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bioRxiv preprint doi: https://doi.org/10.1101/462218; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Nitric oxide is important for sensing and survival under 2 hypoxia in Arabidopsis 3 4 Aakanksha Wany1†, Alok Kumar Gupta1†a, Yariv Brotman^2, Sonika Pandey^1, Abhay 5 Pratap Vishwakarma1, Aprajita Kumari1, Pooja Singh1, Pradeep K Pathak1, Abir U. 6 Igamberdiev3, Kapuganti Jagadis Gupta1* 7 8 1National Institute of Plant Genome Research, Aruna Asaf Ali Marg, 110067, New 9 Delhi 10 2 Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, 11 Israel 12 3Department of Biology, Memorial University of Newfoundland, St. John’s, NL, A1B 13 3X9, Canada 14 15 Correspondence 16 *Kapuganti Jagadis Gupta 17 National Institute of Plant Genome Research 18 Aruna Asaf Ali Marg 19 110067 20 New Delhi 21 Email: [email protected] 22 Phone +91-11-26735111 23 Email addresses 24 Aakanksha Wany [email protected] 25 Alok Kumar Gupta [email protected] 26 Yariv Brotman [email protected] 27 Sonika Pandey [email protected] 28 Abhay Pratap Vishwakarma [email protected] 29 Aprajita Kumari [email protected] 30 Pooja Singh [email protected] 31 Pradeep Kumar Pathak [email protected] 32 Abir U. Igamberdiev [email protected] 1 | P a g e bioRxiv preprint doi: https://doi.org/10.1101/462218; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 33 Kapuganti Jagadis Gupta [email protected] 34 Number of words 7053 35 Number of figures: 8 36 Supplementary files: 2 37 38 † Equal first authors 39 ^ Equal second authors 40 @ Current address: Food microbiology and nutrition lab, Amity Institute of 41 Biotechnology, Amity University Uttar Pradesh, Noida, UP 201313 42 43 44 Key words: antioxidant, haemoglobins, hypoxia, metabolism, nitric oxide, nitrate 45 reductase 46 47 Running title: NO role in gene induction under hypoxia 48 49 Highlight: Hypoxia-induced nitric oxide plays a central role in activation of genes and 50 metabolites involved in fermentation, TCA cycle, nitrogen and antioxidant 51 metabolism 52 53 54 55 56 57 58 59 60 61 62 63 2 | P a g e bioRxiv preprint doi: https://doi.org/10.1101/462218; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 Abstract 65 Nitric oxide (NO) is a free radical molecule that plays an important role in hypoxic 66 stress. We studied the impact of hypoxia-induced NO production on the expression 67 of genes and production of metabolites involved in carbon, oxygen sensing, nitrogen, 68 and antioxidant metabolism using wild type (WT), non-symbiotic haemoglobin- 69 overexpressing (Hb+) and nitrate reductase double mutant (nia1,2) of Arabidopsis. 70 Futher application of NO scavenger cPTIO was used to confirm NO role .We found 71 that imposing hypoxia leads to the increaseof NO and reactive oxygen species 72 (ROS) levels in WT, while the reduced levels of NO and higher levels of ROS were 73 observed in roots of Hb+ and nia1,2 mutant. Expression of the genes encoding 74 oxygen sensing and the enzymes involved in fermentative pathways, their activities 75 and metabolite levels were highly induced in WT suggesting that NO plays a role in 76 the induction of fermentation. Several genes and metabolites involved in the TCA 77 cycle were also induced in WT in comparison to Hb+ and nia mutant line suggesting 78 that NO can accelerate TCA cycle to regenerate reducing equivalents under hypoxia. 79 Interestingly, we found that the genes and metabolites involved in the ascorbate- 80 glutathione cycle were modulated by NO under hypoxia.The alternative oxidasegene 81 (AOX1A) was induced under hypoxia in WTdue to increased levels of NO rather than 82 ROS. Futher, we found that NO improves plant survival. Overall these findings 83 suggest that NO is a major player in plant survival under hypoxia by modulating gene 84 expression and metabolite levels of carbon, nitrogen, oxygen sensing and 85 antioxidant metabolism. 86 87 88 89 90 91 92 93 3 | P a g e bioRxiv preprint doi: https://doi.org/10.1101/462218; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 94 Introduction 95 Oxygen is critical for life as it acts as a terminal electron acceptor in mitochondrial 96 electron transport chain, where ATP generation takes place for cellular metabolism, 97 moreover, this process leads to regeneration of NAD+ from NADH which can be 98 useful for accelerating fermentation under hypoxia. The importance of oxygen is 99 documented in various cellular pathways such as haem, sterol, amino acid and lipid 100 biosynthesis (Geigenberger, 2003). Unlike animals, plants do not possess 101 sophisticated mechanisms for delivery of oxygen hence, they experience hypoxia 102 during their development due to very low internal oxygen concentrations (Gupta et 103 al., 2009; 2014; Rolletschek et al., 2002). Transient and severe flooding, 104 waterlogging or increased oxygen-consuming microbial activity in soils leads to 105 hypoxia and eventually anoxia (Ricard et al., 1994). During hypoxia, cytochrome c 106 oxidase (COX) activity is ceased that leads to reduced levels of ATP. Under hypoxia, 107 plants produce during oxidation of one hexose only less than one tenth of ATP 108 produced under normoxia but the flux of fermentation can exceed the aerobic 109 metabolic rate and this acceleration leads to production of energy. In addition to 110 energy production, fermentation leads to production of ethanol and lactate (Drew, 111 1997), and may result in production of harmful levels of reactive oxygen species 112 (ROS) even at low oxygen levels due to the increased redox state (Vergara et al., 113 2012). Severe reduction in root biomass and the allocation of root metabolites to the 114 shoot was also documented (Huang et al. 1994). 115 Plants have several strategies to cope with hypoxic stress. These include various 116 physiological, biochemical and anatomical changes (Bailey-Serres et al., 2012). 117 Among the anatomical adaptations, internode elongation (Hattori et al., 2009), 118 petiole elongation in Arabidopsis (Mustroph et al., 2010; Colmer & Voesenek, 2009), 119 adventitious root formation (Mergemann and Sauter, 2000); lysigenous aerenchyma 120 formation (Abiko et al., 2012; Wany et al., 2017), leaf gas films clinging to the surface 121 of leaves of many semi-aquatic species (Winkel et al., 2011) were reported. Several 122 metabolic changes were also reported in response to oxygen deprivation 123 (Geigenberger, 2003). Fermentation is combined with some partial activity of the 124 TCA cycle which can help in coping hypoxia (Fox et al., 1994). Low oxygen alters 125 gene expression, energy consumption, cellular metabolism and growth. In particular, 126 cells of aerobic organisms have evolved adaptive responses to compensate for the 4 | P a g e bioRxiv preprint doi: https://doi.org/10.1101/462218; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 127 energy loss caused by oxygen deprivation (Gupta et al., 2009, 2014; Rolletschek et 128 al., 2002; Zabalza et al., 2009). Examples of such metabolic adaptations to hypoxia 129 include the down-regulation of storage metabolism and the shift from invertase to 130 sucrose synthase pathways of sucrose utilization (Geigenberger, 2003). 131 Under hypoxic stress, it has been shown that plants produce high amount of nitric 132 oxide (NO), a free radical which plays a role in wide range of processes that include 133 seed germination, stomatal function, root development, cell wall elongation, 134 senescence and programed cell death (Mur et al., 2013). In plants, NO can be 135 generated via multiple pathways, those include oxidative and reductive reactions. In 136 the oxidative pathways, NO is produced via oxidation of arginine or hydroxylamine, 137 while the reductive pathways operate via reduction of nitrate and nitrite. The 138 oxidative pathways include NO synthase-like activity (Corpas et al., 2009), the 139 polyamine-mediated pathway (Tun et al., 2006), and the hydroxylamine-mediated 140 pathway (Rumer et al., 2009), these pathways are active under normoxic conditions 141 (Igamberdiev and Hill, 2004; Gupta et al., 2011). NO production from oxidative 142 pathways still needs more characterization. In contrast, the reductive pathways such 143 as the nitrate reductase (NR) pathway (Sakihama et al., 2002; Planchet et al., 2005), 144 the mitochondrial electron transport pathway (Planchet et al., 2005; Gupta et al., 145 2011), and the plasma membrane nitrite–NO pathway (Stohr et al., 2001) operate in 146 response to the decrease in oxygen availability. 147 Under hypoxic conditions, nitrite reduction to ammonia is inhibited, therefore the 148 accumulated nitrite can act as a substrate for NO production. It was shown that 149 nitrite is a limiting factor for NO production (Planchet et al., 2005). Some amount of 150 nitrite is also transported into mitochondria where the complexes III and IV of the 151 electron transport chain produce NO (Gupta et al., 2005) which is then scavenged by 152 the cytosolic non-symbiotic haemoglobins (nHb1) via operation of haemoglobin-nitric 153 oxide cycle. So far the function of hypoxically generated NO is attributed to oxidation 154 of NADH (Igamberdiev et al., 2006) and generation of limited amount of ATP in roots 155 and nodules (Stoimenova et al., 2007; Horchani et al., 2011).