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Why and how do plants regulate their pH? Sponsored by the DEST program: China Higher Education Strategic Initiatives © The University of Adelaide Aims of the talk • Show why soil features and plant metabolism require plants to have ability to regulate their pH • Show from pH measurements that plants do regulate pH • Assess roles of 1) internal plant pH buffers, 2) cell metabolism and 3) membrane transport processes in regulating plant pH • Introduce the concept of the combined ‘biophysical’ (membrane transport) & ‘biochemical’ pH-stat • Consider (briefly) complications for shoots, that are remote from the soil • Consider (briefly) changes in rhizosphere pH The plant environment - above and below ground Variable soil environment worldwide; affects plant growth via: • nutrient content, • biological activity, • water content, etc • pH (~3-10?) Photo: Western Australia; SE Smith Why might plants need to regulate their pH? Regulate: use metabolic energy to control pH within limits: ‘homeostasis’ - will involve signals & feedback pH = - log[H+]; so pH 7 = 0.1 M H+ _________________________ 1. External pH-related ‘threats’ to growth: Wide pH range in plant growth media (soil & water): - affects H+ movements across plant cell membrane (plasma membrane) - also affects availability and uptake of many soil nutrients, toxic compounds, etc, that may indirectly ‘threaten’ cell pH Why might plants need to regulate their pH? 2. Internal pH-related ‘threats’ to growth: • Synthesis of H+ : e.g. organic acids (malic, oxalic, citric, etc) and acidic amino acids (mainly glutamic & aspartic); ammonium assimilation • Some consumption of H+ (= production of OH-): nitrate, sulphate and (in aquatic plants) bicarbonate assimilation • Assimilation of NH4+ and NO3- are important examples of ‘pH-threatening’ processes Plant metabolism and pH* Plant metabolism is highly pH-dependent: • Most enzymes operate over a narrow pH range (many around pH 7-8; but not all...) • Many important cell components (enzymes, other proteins, nucleic acids, nucleotides, amino acids, organic acids, sugar phosphates etc) are ionizable their ionic state changes with pH • Free energy changes in key reactions depend on pH (e.g. ATP hydrolysis; NADH oxidation) Effects of some metabolic processes on pH Reaction Effect on pH Glucose respiration: C6H12O6 + 6O2 6CO2 + 6H2O ~neutral Ethanolic fermentationa: C6H12O6 2C2H5OH + 2CO2 ~neutral Lactic fermentationa,b: C6H12O6 2C3H4O3- + 2H+ acidifying a Anaerobic conditions; b Synthesis of any organic acid is acidifying. ATP hydrolysisc: ATP4- ADP3- + H+ + HPO42- acidifying Dehydrogenasec: R-H2 + NAD+ NADH + R + H+ acidifying c Under normal conditions, these are cyclic reactions (dephosphorylationphosphorylation; oxidation-reduction) - no net effect on pH. Do plants regulate their pH? Wide range of pH in cells & tissues: Desmarestia (marine alga): Lemon fruit: Unripe grapes: Rhubarb petioles: Ripe tomato fruit: Whole tomato plant: pH 1 (sulphuric acid) 2.4 (citric) 3.0 (malic, tartaric) 3.2 (malic, oxalic) 4.4 (citric, malic) 4.8-5.5 (various)* * depends on form of Nitrogen nutrition Do plants regulate their pH? Wide range of pH in cells & tissues: Desmarestia (marine alga): Lemon fruit: Unripe grapes: Rhubarb petioles: Ripe tomato fruit: Whole tomato plant: pH 1 (sulphuric acid) 2.4 (citric) 3.0 (malic, tartaric) 3.2 (malic, oxalic) 4.4 (citric, malic) 4.8-5.5 (various) Such values mainly reflect pH of vacuoles (up to 95% of cell volume) acid vacuole cyt Variations in cell pH may be relevant to food quality Measurements of cytoplasmic pH (pHcyt) 8.5 - ‘Unstressed’ conditions pHcyt 8.0 - 7.5 - range of pHcyt 7.0 - 4 6 8 10 pHout Measurements are ‘cytosol’ (many plants): Reid & Smith (2002) Effects of treatments on cytoplasmic pH [Values are changes from control values] Treatment Plant light dark Chara (giant alga) light dark Ca starvation Acetic acid (2 mM) NH4+ (0.2 mM) NH4+ (2 mM) NH4+ (10 mM) Anoxia (low O2) CCCP ( 4M) Abscisic acid (ABA) Fusicoccin (10 M) Riccia (liverwort) Chara maize root hairs Chara rice root hair maize root tips maize root tips Chara Vicia guard cells maize root tips Change in pHcyt -0.25 -0.3 (transient) -0.3 -0.7 +0.15 +0.35 +0.25 (transient) -0.3 -0.6 -0.2 -0.1 References & methods in Reid & Smith (2002); Felle (2001, 2002) Plant cell pH buffers: little ability to regulate pH during plant growth • Cell pH buffers are mixtures of ionizable solutes and counter-ions; e.g. organic acids, phosphates, etc (e.g. K+ + H2PO4- K+ + HPO42- + H+) • Cell solutes have poor buffer capacity at cytoplasmic pH (7-8) • In growing cells buffers alone will maintain this pH range for less than one hour • Buffers are products of earlier pH regulation involving membrane transport of H+ and other ions (K+, Na+, Ca2+ etc) Organic acids: the ‘biochemical pH stat’ Involves phosphoenolpyruvate (PEP) carboxylase; malate dehydrogenase & malic enzyme (Davies 1973, 1986): [sugar ] PEP + CO2 + OH- OAA malate pyruvate +CO2 + OH(PEP carboxylase) (malic enzyme) Strong acid Activity malic enzyme 6 PEP carboxylase 7 pHcyt 8 Organic acids: the ‘biochemical pH-stat’ Involves phosphoenolpyruvate (PEP) carboxylase; malate dehydrogenase & malic enzyme (Davies 1973, 1986): [sugar ] PEP + CO2 + OH- OAA malate pyruvate +CO2 + OH(PEP carboxylase) (Malic enzyme) Activity malic enzyme Decreasing pHcyt increases activity of malic enzyme; decreases activity of PEP carboxylase less OH- consumed 6 PEP carboxylase 7 pHcyt 8 Organic acids: the ‘biochemical pH-stat’ Involves phosphoenolpyruvate (PEP) carboxylase; malate dehydrogenase & malic enzyme (Davies 1973, 1986): [sugar ] PEP + CO2 + OH- OAA malate pyruvate +CO2 + OH(PEP carboxylase) (Malic enzyme) Activity malic enzyme 6 PEP carboxylase 7 pHcyt 8 Increasing pHcyt decreases activity of malic enzyme; increases activity of PEP carboxylase more OH- consumed Biochemical pH-stat: issues • No good evidence that the enzyme activity is regulated by pHcyt in vivo. (Regulation is via protein kinases?) • Does the ‘balance sheet’ for OH- & H+ add up, taking into account NAD & ATP? (Sakano 1998). It does as long as NADH & ATP are ‘recycled’, as in normal growth • The pH-stat relies on prior transport of H+ out of cells, plus C+ in, to ‘set up’ the organic acid/anion mixture (C+ not shown in diagrammatic versions) • BUT: organic acid metabolism is important in pH control! See Reid & Smith (2002) for discussion The ‘biophysical pH-stat’ • Membrane transport processes that result in maintenance of cytoplasmic pH within narrow limits • Includes H+ transport, balanced by transport of other ions • Implies that such transport is influenced by cytoplasmic pH, directly or indirectly • Interacts with biochemical processes, especially organic acid metabolism • Can apply to ‘compartments’ other than cytoplasm (e.g. vacuole) See Reid & Smith (2002) for discussion Removing H+: ‘excess’ cation transport • Uptake of cations (‘C+’, e.g. K+, Na+, Ca2+) balanced by H+ transport via ATP-ase(s) • Electric charge must balance: 2H+ produced with malate2-; exchanged for 2C+ (e.g. 2K+, or 1 Ca2+) • Organic anions + inorganic cations mainly accumulate in vacuole (not shown) • Details of reactions & ion transport processes not shown Sugar (organic acid) RCOOH RCOO- H+ H+ C+ C+ cytoplasm ‘Removing OH-’: ‘excess’ anion transport • • • • • ‘Excess’ anion (A-: e.g. NO3- or Cl-) uptake could be balanced by net efflux of HCO3- or OH- or (probably) net uptake of H+ C+ RCOO(organic acid Electric charge must balance anion) In this example, A is mainly cytoplasm stored in the vacuole (not shown) C+ HCO3- (or) Such ‘luxury’ storage of A- (e.g. CO2 + OH- NO3- ) is not a feature of ‘normal’ growth (or) H+ H+ A- ADetails of reactions & ion transport mechanisms not shown • H+ -ATP-ase activity is ‘hidden’ ‘Removing OH-’: ‘excess’ anion transport This scheme does not apply to NO3- assimilation to organic N C+ RCOO(organic acid anion) cytoplasm C+ HCO3- (or) CO2 + OH- (or) H+ H+ A- A- Nitrogen assimilation: ‘threats’ to cell pH • NH4+ [amino acids, etc] + H+ (acidifying) - H+ effluxed (rhizosphere pH : e.g. to pH 4) - remember the increases in pHcyt (earlier Table) • NO3- + [8H] [amino acids, etc] + OH- (alkalizing) - OH- ‘neutralized’ by organic acid synthesis; leading to accumulation of RCOO-, and/or: neutralized by net H+ influx (rhizosphere pH : e.g. to 6.5) - balance between the 2 ‘strategies’ for NO3- depends on external NO3- concentration, plant type, etc Nitrogen assimilation: ‘threats’ to cell pH • NH4+ [amino acids, etc] + H+ (acidifying) - H+ effluxed (rhizosphere pH : e.g. to pH 4) - remember the increases in pHcyt (earlier Table) • NO3- + [8H] [amino acids, etc] + OH- (alkalizing) - OH- ‘neutralized’ by organic acid synthesis; leading to accumulation of RCOO-, and/or: neutralized by net H+ influx (rhizosphere pH : e.g. to 6.5) • Urea: CO(NH2)2; also symbiotic N2 fixation - still partly acidifying (rhizosphere pH : e.g. to 4.5); due to underlying ‘excess cation uptake’ (all cells contain some RCOO-) Membrane ion transport: general principles Plant cell: ignores the wall; vacuole is usually much larger than shown - 0.2 V cytoplasm H+ H+ C+ C+ vacuole NH4+ NH4+ (2H+ 2H+ A- A-) A- ApHcyt 7-8 Many other transporters not shown Membrane ion transport: general principles • H+ transport generates an electric potential difference (0.1 to -0.2 V) • The PD ‘drives’ uptake of many cations • H+ ‘recycles’, bringing in solutes (e.g. anions; amino acids, sugars; and driving out Na+; not shown) • During transport of sum of ions, charge transfer must balance ATP-ase(s) - 0.2 V cytoplasm H+ H+ C+ C+ vacuole NH4+ NH4+ (2H+ 2H+ A- A-) A- ApHcyt 7-8 Many other transporters not shown Membrane ion transport: general principles • H+ transport generates an electric potential difference (- 0.1 to -0.2 V) • This PD is a tiny charge imbalance • PD ‘drives’ uptake of some cations • H+ ‘recycles’, bringing in solutes (e.g. anions; amino acids, sugars; and driving out Na+ not shown) • During transport of sum of ions, charge transfer must balance • pHcyt must not be perturbed beyond 7-8. The transporters (especially those involving H+) are part of the ‘biophysical pH-stat’ - 0.2 V cytoplasm H+ H+ C+ C+ vacuole NH4+ NH4+ (2H+ 2H+ A- A-) A- ApHcyt 7-8 Many other transporters not shown H+ disposal to vacuoles • Vacuoles often accumulate organic acids/anions: malic, citric, oxalic (up to 15% of photosynthate in shoots of chenopods) • They can dispose of some cytoplasmic H+, and RCOO(produced during NO3assimilation in shoots), and help regulate pHcyt • Limits: pHvac, also osmotic • Transport to & from vacuoles is also part of the ‘biophysical pH-stat’ pHcyt 7-8 0 to ~+0.05V? cytoplasm vacuole H+ H+ pHvac <6 (H+ H+ A- A-) A- A- C+ C+ Many transporters not shown Principles of pH regulation: summary • Cellular pH in plants is regulated by a combined ‘biophysical (transport) and biochemical pH-stat’ • The relative importance of each component varies with plant species, and type of organ and cell • Whether or not the ‘biophysical’ component is important depends on feasibility of H+ disposal externally • H+ disposal to vacuoles is limited in terms of growth • [Molecular biology of transporters (H+-ATPases etc) is rapidly developing research field] Can metabolism be controlled by pH? Change in pHcyt can change enzyme activity via changed protein ionization; and can change H+ transport activity via changed substrate (or product) concentration. Does pHcyt act as a ‘signal’ (‘messenger’), as does Ca? Control by pHcyt is implicated in: • Hormonal action • Gravity sensing • Stomatal opening/closing (interacts with Ca) • Defence mechanisms (interacts with Ca) • Membrane channel activity: pHcyt closes aquaporins (water channels); opens anion channels - increases efflux Signalling mechanisms are unknown See Felle (2001, 2002); Reid & Smith (2002); Tournaire-Roux et al. (2003) Whole-plant issues - 1 Cells within tissues (root or shoot) are surrounded by an ‘apoplast’ - cell wall and limited intercellular spaces • Apoplastic volume is too small to dispose of much H+ from cells • Its pH is usually ~5 and seems to be regulated (within limits); as is xylem pH (also ~5) • Treatments that alter pHcyt can alter apoplastic pH See Felle (2001, 2002); Gallardo & Cànovas (2002) Whole-plant issues - 2 • Above-ground cells rely on the ‘biochemical’ pHstat + transport to the vacuole to regulate pHcyt • However, RCOO- can ‘cycle’ through phloem (pH ~8) down to roots • Roots can use both the biophysical & biochemical components of the pHstat; & select pH ‘nonperturbing’ solutes to be sent to the shoot The plant’s underground environment ... Complicated! The plant affects the pH of its root environment Changes in ‘rhizosphere’ pH: • depend on soil pH-buffering capacity • soil nutrient composition • plant growth rate • presence/absence of root hairs • presence/absence of mycorrhizal fungi • time of day (products of photosynthesis) • may not be uniform along root surface Rhizosphere: pH changes • NH4+ assimilation: pH (acid) - often whole root surface • NO3- assimilation: pH (alkaline) - sometimes patches; may be also be pH patches elsewhere on same root Maize (in soil & agar + indicator) NO3- Marschner & Römheld (1983) NH4+ low NO3- Rhizosphere: pH changes • NH4+ assimilation: pH (acid) - often whole root surface • NO3- assimilation: pH (alkaline) - sometimes patches; may be also be pH patches elsewhere on same root These effects of N may be ‘hidden’ or modified: • Buckwheat (Fagopyrum): pH even when grown in NO3- * • Proteoid roots in low P soil: pH in patches* • Fe deficiency: pH in patches* [* due to extrusion of RCOO- + H+; can be considered as an adaptation, or response to stress] • Lowering Al toxicity (acid soil): RCOO- + C+ release (pH or ) Conclusions - 1 • Most processes (transport, biochemistry) that regulate cell pH are known • Perturbations in cell pH are associated with mineral nutrition, toxicity, plant development, defence etc • Signals that perceive & respond to cell (& apoplast) pH changes are unknown • Role of pH as a ‘messenger’ (‘control by pH’) is unresolved (compare Ca: Felle 2001, 2002) • Integration of pH-regulating processes at whole-plant level is unresolved Conclusions - 2 • Many of the issues that relate to regulation of plant pH and pH effects on plant growth relate to plant nutrition, and: • Many originate below-ground Professor Zhang et al. investigating the rhizosphere: low P ‘soil’, Western Australia. Photo: SE Smith Some key references In order of publication: FA Smith & JA Raven (1979). Intracellular pH and its regulation. Annu. Rev. Plant Physiology 30: 289-311 A Kurkdjian & J Guern (1989). Intracellular pH: measurement and importance in cell activity. Annu. Rev. Plant Physiology 40: 271-303 HH Felle (2002). pH as a signal and regulator of membrane transport. In: Handbook of Plant Growth: pH as the master variable (ed. Z Rengel), Marcel Dekker: New York, 107-130 RJ Reid & FA Smith (2002). The cytoplasmic pH stat. In: Handbook of Plant growth… (ed. Z Rengel), 49-71 HH Felle (2002). pH: signal and messenger in plant cells. Plant Biol. 3: 577591 C Tournaire-Roux et al. ( 2003). Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature 425, 393-397 Recent general reference: Z Rengel (ed.) Handbook of Plant Growth: pH as the master variable. Marcel Dekker: New York, 107-130 Acknowledgments Collaborators in pH research & reviews: • 1970’s: John Raven (Dundee) • 1970’s-1980’s: Alan Walker (Sydney) • 1980’s to date: Rob Reid (Adelaide) • many research assistants & students Yongguan Zhu focused my mind on pH again Research support from the Australian Research Council cspi