Introduction to plant nutrition Energizing the membrane H + -ATPases and H + -PPases Potassium

1. Plant Nutrition 1: Membrane energetics and transport, potassium nutrition and sodium toxicity • Introduction to plant nutrition • Energizing the membrane • H+-ATPases and H+-PPases • Potassium • Uptake, transport and homeostasis • Sodium • Toxicity, transport and tolerance

2. Plant nutrition: Introduction Plants are ~70 to >90% water by weight CO2, photo-synthesis N Nitrogen 42% Carbon P Phosphorus K Potassium Ca Calcium 7% Hydrogen Mg Magnesium 7% Other, from soil 44% Oxygen S Sulfur Si Silicon 93% of plant dry mass is composed of C, O and H Cl Chlorine Other These elements are obtained mainly from soil, are often referred to as mineral nutrients, and are the subject of the topic Plant Nutrition H2O water

3. Nutrient uptake, assimilation and utilization involve many processes Nutrient acquisition efficiency Nutrient usage efficiency Root exudates Root system architecture Assimilation and remobilization efficiency NH3 Intercellular transport efficiency N N Transporters and pumps Regulatory and homeostatic networks Symbioses R-X X Rhizosphere microbiota P P

4. Nutrients are concentrated in the plant relative to the environment Energy is expended to assimilate nutrients against a steep concentration gradient The driving force of the nutrient’s chemical gradient is outwards [H2PO4-]o [HPO42-]o < 1 μM [NO3-]o <100 μM – >1 mM [NH4+]o <100 μM – >1 mM Soil abundance (ranges or typical values) [K+]o 0.1 – 1 mM Cell [K+]i 50 - 100 mM [H2PO4-]i [HPO42-]i 5 - 10 mM [NO3-]i 10 mM [NH4+]i ~1 mM

5. The electrochemical gradient is important for ion transport The electrochemical gradient defines the energetic demands for transport, and integrates the electrical and concentration gradients The cell’s electrical gradient drives anions OUT and cations IN [H2PO4-]o [HPO42-]o < 1 μM [NO3-]o <100 μM – >1 mM [NH4+]o <100 μM – >1 mM [K+]o 0.1 – 1 mM [K+]i 50 - 100 mM [H2PO4-]i [HPO42-]i 5 - 10 mM [NO3-]i 10 mM [NH4+]i ~1 mM ~ -150 mV Em =

6. Transport can be down or against an electrochemical gradient Down an electrochemical gradient (Diffusion or facilitated diffusion) Against an electrochemical gradient (Active transport) Symport Antiport OUT IN ATP ADP + Pi Through membrane Through channel Through carrier Secondary active transport: Indirectly coupled to ATP hydrolysis Primary active transport: Directly coupled to ATP hydrolysis

7. Solutes cross membranes through different types of transporters • Carriers / • Coupled Transporters • are membrane proteins • can be active or inactive • can move more than one solute at a time • the driver (usually H+ in plants) moves down its electrochemical gradient, which provides the energy for the co-transported solute’s transport ATP ADP • Channels: • are protein-formed holes in the membrane • can be open or closed • move one type of solute at a time • do not provide an energy source for the movement; solutes can only move down their electrochemical gradient Channels are often drawn as two adjacent ovals (or a cross-section of a doughnut) Pumps are often drawn like lollipops, with a large cytoplasmic catalytic domain H+ H+ • Pumps: • move solutes against a chemical or charge gradient • couple transport to hydrolysis of ATP or pyrophosphate X X X Coupled transporters are often drawn as circles with arrows indicating the direction of flow for each ion X X

8. Energizing the membrane: Plant H+-ATPases and VH+-PPases The plasma-membrane H+-ATPase uses energy from ATP to pump protons out of the cell The vacuolar-type proton pumps transport protons into the lumen of endomembrane compartments (e.g., vacuole) The VH+-ATPase is a multimeric protein complex The H+-PPase uses energy stored in pyrophosphate Sze, H., Li, X. and Palmgren, M.G. (1999). Energization of plant cell membranes by H+-pumping ATPases: Regulation and biosynthesis. Plant Cell. 11: 677-689.

9. The PM H+-ATPase is a “master enzyme” and “powerhouse” By pumping protons out of the cell, PM H+-ATPases produce electric and pH gradients pH ~ 5 - 6 + + + + + pH ~ 7.5 - - - - - ATP ~ -150 mV ADP H+ The electrochemical gradient produced by the PM H+-ATPase drives other transport processes Antiporters Uniporters Channels Symporters Anions Cations H+ H+ See Palmgren, M.G. (2001). Plant plasma membrane H+-ATPases: Powerhouses for nutrient uptake. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 817-845; Figure adapted from Michelet, B. and Boutry, M. (1995). The plasma membrane H+-ATPase. Plant Physiol. 108: 1-6.

10. Vacuolar pumps pump protons into the vacuole and endocompartments pH 5 - 6 H+ pH 7.5 H+ H+ ADP + Pi ATP PPi 2 x Pi • Protons are pumped into the vacuole by: • Vacuolar H+-ATPases (VH+-ATPases) and • Vacuolar pyrophosphatases (H+-PPases) H+ H+ Em = ~ -30 mV pH 3 - 6 Sze, H., Li, X. and Palmgren, M.G. (1999). Energization of plant cell membranes by H+-pumping ATPases: Regulation and biosynthesis. Plant Cell. 11: 677-689. Isayenkov, S., Isner, J.C. and Maathuis, F.J.M. (2010). Vacuolar ion channels: Roles in plant nutrition and signalling. FEBS letters. 584: 1982-1988.

11. K+ and Na+ - “The twins”. So alike yet so different NaCl toxicity Potassium deficiency • Sodium (Na) and potassium (K): • Same column of the periodic table • Both have a single electron in the outer shell so form monovalent cations • Both are very abundant elements And yet, potassium is an essential nutrient, and sodium frequently is toxic K FAO

12. Potassium uptake, transport and homeostasis Potassium is an essential macronutrient Enhances fertility Maintains turgor and reduces wilting Promotes stress tolerance Regulates stomatal conductance, photosynthesis and transpiration Regulates enzyme activities Symptomsof potassium deficiency Strengthens cell walls Maintains ionic homeostasis Stimulates photosynthate translocation [K+] in soil = ~0.1 – 1 mM [K+] in plant cell cytoplasm = ~100 mM See Wang, M., Zheng, Q., Shen, Q. and Guo, S. (2013). The critical role of potassium in plant stress response. Intl. J. Mol. Sci. 14: 7370-7390; Sin Chee Tham /Photo; Purdue extension; Onsemeliot.

13. Potassium is an essential plant nutrient K+ is a counter ion for negatively charged molecules including DNA and proteins K+ moves in and out of the vacuole through specific transporters As the major cation in the vacuole, K+ contributes to cell expansion and movement, including that of guard cells K+ uptake involves high and low affinity transporters K+ is a cofactor for some enzymes Reprinted from Maathuis, F.J.M. (2009). Physiological functions of mineral macronutrients. Curr. Opin. Plant Biol. 12: 250-258 with permission from Elsevier.

14. Potassium homeostasis: Responses to low K+ availability Low K Membrane hyperpolarization Hormonal changes (auxin, ethylene) Enhanced root growth and gravitropic responses Calcium signaling Direct effects Transcriptional induction of HAK5 K+ channel Indirect effects More efficient uptake through K+ channels K+ uptake Adapted from Chérel, I., Lefoulon, C., Boeglin, M. and Sentenac, H. (2013). Molecular mechanisms involved in plant adaptation to low K+ availability. J. Exp. Bot. 65: 833-848.

15. K+ mobilization is critical for K+ homeostasis As K+ becomes limiting, it becomes preferentially allocated to the cytosol Cytosol Vac. Prioritized K+ can be remobilized from less essential tissues into prioritized tissues such as growing and photosynthetic tissues Non-Prioritized Adapted from Amtmann, A., and Leigh, R. (2010). Ion homeostasis. In Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic Foundation, A. Pareek, S.K. Sopory, H.J. Bohnert and Govindjee (eds) (Dordrecht, The Netherlands: Springer), pp. 245 – 262.

16. Sodium toxicity, transport and tolerance You can’t take salt out of soil easily; once it is there it stays there To demonstrate his (fake) madness, Odysseus plowed salt into his field Colum, P. (1918). The Adventures of Odysseus and the Tale of Troy. Project Gutenberg; USDA, USDA; FAO

17. How can we address the problems caused by soil salinization? Avoid adding to the problem by better management of fragile soil systems Learn about salt tolerance from naturally salt-tolerant species (halophytes) Areas of concern Salicornia europaea Identify halophytes that can be used as food or energy crops Arthrocnemum macrostachyum Identify responses to salt stress in salt-sensitive species (glycophytes) Chenopodiumquinoa Thinopyrumponticum Introduce salinity-tolerance traits into crop plants through breeding and engineering Munns, R., James, R.A., Xu, B., Athman, A., Conn, S.J., Jordans, C., Byrt, C.S., Hare, R.A., Tyerman, S.D., Tester, M., Plett, D. and Gilliham, M. (2012). Wheat grain yield on saline soils is improved by an ancestral Na+ transporter gene. Nat Biotech. 30: 360-364. CSIRO; The State of Victoria; Maurice Chédel; Marco Schmidt Geng, Y., Wu, R., Wee, C.W., Xie, F., Wei, X., Chan, P.M.Y., Tham, C., Duan, L. and Dinneny, J.R. (2013). A spatio-temporal understanding of growth regulation during the salt stress response in Arabidopsis. Plant Cell. 25: 2132-2154. Study salt-tolerant relatives of crop plants

18. Plant species have a broad range of salinity tolerances Saltbush (Atriplex amnicola) is a halophyte that can tolerate very salty soil Q. Can we identify and exploit the mechanistic basis of increased salinity tolerance? A. YES! Arabidopsis and rice are quite sensitive Reprinted by permission of Annual Reviews from Munns, R. and Tester, M. (2008). Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59: 651-681.

19. Mechanisms of sodium toxicity and tolerance SALINITY STRESS Ionic stress: K+ deficiency / excess Na+ influx Osmotic stress Oxidative stress Inhibition of: water uptake, growth, photosynthesis Inhibition of: enzyme activity, protein synthesis, photosynthesis Leaf senescence Detoxification strategies Osmotic adjustment: Accumulation of solutes Ion homeostasis: Na+ extrusion, Na+ exclusion, Na+ compartmentation Adapted from Horie, T., Karahara, I. and Katsuhara, M. (2012). Salinity tolerance mechanisms in glycophytes: An overview with the central focus on rice plants. Rice. 5:11; see also Munns, R. and Tester, M. (2008). Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59: 651-681and Shabala, S. and Pottosin, I. (2014). Regulation of potassium transport in plants under hostile conditions: implications for abiotic and biotic stress tolerance. Physiol. Plant. 151: 257-279.

20. General sodium tolerance strategy: Keep sodium out of cytosol & shoot OUT Na+ IN Na+ 1. Keep Na+ from entering plant / cells “OUT” K+ 2. Pump out any Na+ that leaks in Na+ 5. Accumulate K+ to maintain a high ratio of K+ to Na+ 3. Compartmentation of Na+ in vacuole 4. Extrude Na+ via salt glands Compatible solutes 6. Synthesize compatible solutes for osmotic balance Na+ 7. Prevent Na+ from moving into the shoot and leaves

21. Breeding and engineering for salt tolerance Salt tolerance can be attributed to three non-exclusive mechanisms Salinity tolerance can be enhanced by breeding or engineering Reprinted from Roy, S.J., Negrão, S. and Tester, M. (2014). Salt resistant crop plants. Curr. Opin. Biotech. 26: 115-124.

22. Wheat yield on saline soils improved by an ancestral Na+ transporter gene A pair of genes derived from a relative of wheat confers enhanced salinity tolerance Because these species are closely related, the genes can be introduced into cultivated wheat without using GM methods Durum wheat carrying salt-tolerance genes Tetraploid pasta wheat Hexaploid bread wheat Huang, S., Spielmeyer, W., Lagudah, E.S. and Munns, R. (2008). Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance. J. Exp. Bot. 59: 927-937 by permission of Oxford University Press; Credit: Dr Richard James, CSIRO

23. Interaction between K+ nutrition and Na+ toxicity Cytosol Vac. K+ / Na+ ratio Plants must coordinate the actions of K+ and Na+ transporters to maintain a high ratio of K+ to Na+ in prioritized tissues Prioritized Non-Prioritized K+ / Na+ ratio Adapted from Amtmann, A., and Leigh, R. (2010). Ion homeostasis. In Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic Foundation, A. Pareek, S.K. Sopory, H.J. Bohnert and Govindjee (eds) (Dordrecht, The Netherlands: Springer), pp. 245 – 262.

24. Summary and ongoing research • Nutrient uptake is extremely energetically demanding • Proton motive force generated by proton pumps is essential for nutrient uptake • Dozens of membrane transporters are involved in uptake, allocation and homeostasis of mineral nutrients • Most plants require a high cytosolic ratio of K+ to Na+ • Plants require large amounts of potassium for optimal growth PO43- PO43- PO43- K+ K+ • Sodium toxicity is a real and growing problem • The mechanisms of sodium tolerance are being identified and exploited for plant breeding NO3- NO3-