Why and how do plants regulate their pH?

Transcript Why and how do plants regulate their pH?

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

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 NH 4 + and NO 3 of ‘pH-threatening’ processes are important examples

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: C 6 H 12 O 6 + 6O 2  6CO 2 + 6H 2 O Ethanolic fermentation

: C 6 H 12 O 6  2C 2 H 5 OH + 2CO 2 Lactic fermentation

a,b

: C 6 H 12 O 6

acidifying

 2C 3 H 4 O 3 + 2H +

~ ~ neutral neutral a

Anaerobic conditions;

Synthesis of any organic acid is acidifying .

ATP hydrolysis

: ATP 4  ADP 3 + H + + HPO 4 2 Dehydrogenase

: R-H 2 + NAD +  NADH + R + H +

acidifying acidifying c

Under normal conditions, these are cyclic reactions (dephosphorylation phosphorylation; oxidation-reduction) - no net effect on pH.

These reactions take place in the cytoplasm

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

plants regulate their pH?

Wide range of pH in cells & tissues:

Desmarestia

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 (pH cyt ) 8.5 pH

cyt

8.0 -

‘Unstressed’ conditions

7.5 7.0 -

range of pHcyt

4 6 8 10 pH

out

Measurements are ‘cytosol’ (many plants): Reid & Smith (2002)

Effects of treatments on cytoplasmic pH [Values are changes from control values]

Treatment

light  dark light  dark

Plant

Chara (giant alga) Riccia (liverwort)

Ca starvation

Chara

Acetic acid (2 mM) NH 4 + (0.2 mM) NH 4 + (2 mM) NH 4 + (10 mM) Anoxia (low O 2 ) CCCP ( 4  M) Abscisic acid (ABA) Fusicoccin (10  M) 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

-0.3

(transient)

-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 + + H 2 PO 4  K + + HPO 4 2 + 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 + , Ca 2+ etc)

Organic acids: the ‘biochemical pH stat’ Involves phosphoenolpyruvate (PEP) carboxylase; malate dehydrogenase & malic enzyme (Davies 1973, 1986 ): [sugar  ] PEP + CO 2 + OH  OAA  malate  pyruvate +CO 2 + OH (PEP carboxylase) Strong acid (malic enzyme) Activity malic enzyme PEP carboxylase 6 7 8 pH

cyt

Organic acids: the ‘biochemical pH-stat’ Involves phosphoenolpyruvate (PEP) carboxylase; malate dehydrogenase & malic enzyme (Davies 1973, 1986) : [sugar  ] PEP + CO 2 + OH  OAA  malate  pyruvate +CO 2 + OH (PEP carboxylase) (Malic enzyme) Activity malic enzyme Decreasing pH

cyt

increases activity of malic enzyme; decreases activity of PEP carboxylase less OH consumed pH

cyt

PEP carboxylase 6 7 8

cyt

Increasing pH

cyt

decreases activity of malic enzyme; increases activity of PEP carboxylase more OH consumed

Biochemical pH-stat: issues • No good evidence that the enzyme activity

regulated by pH

cyt 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 + plus C + out of cells, 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 + , Ca 2+ ) balanced by H + transport via ATP-ase(s) • Electric charge must balance: 2H + produced with malate 2 ; exchanged for 2C + (e.g. 2K + , or 1 Ca 2+ ) • Organic anions + inorganic cations mainly accumulate in vacuole (not shown) • Details of reactions & ion transport processes not shown

Sugar



(organic acid) RCOOH



RCOO H + C +

cytoplasm

 

H + C +

‘Removing OH ’: ‘excess’ anion transport • ‘Excess’ anion (A : e.g. NO 3 or Cl ) uptake could be balanced by net efflux of HCO 3 -

OH (probably) net uptake of H +

• Electric charge must balance • In this example, A is mainly stored in the vacuole (not shown) • Such ‘luxury’ storage of A NO 3 (e.g. ) is not a feature of ‘normal’ growth • Details of reactions & ion transport mechanisms not shown

C + RCOO (organic acid

cytoplasm

anion)



C + HCO 3 -

(or)



CO 2

(or)

A -

 

+ OH H +

 

A H +

• H + -ATP ase activity is ‘hidden’

‘Removing OH ’: ‘excess’ anion transport This scheme does not apply to NO 3 to organic N assimilation

C + RCOO (organic acid

cytoplasm

anion)



C + HCO 3 -

(or)



CO 2

(or)

A -

 

+ OH H +

 

H + A -

Nitrogen assimilation: ‘threats’ to cell pH • NH 4 + - H +  [amino acids, etc] + H + effluxed (rhizosphere pH  : e.g. to pH 4) (acidifying) - remember the

increases

in pHcyt (earlier Table) • NO 3 + [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 NO 3 external NO 3 concentration, plant type, etc depends on

Nitrogen assimilation: ‘threats’ to cell pH • NH 4 + - H +  [amino acids, etc] + H + effluxed (rhizosphere pH  : e.g. to pH 4) (acidifying) • - remember the

increases

Urea: CO(NH partly acidifying

RCOO )

2 ) 2 ; also symbiotic N 2 fixation - still

(rhizosphere pH  : e.g. to 4.5); due to underlying ‘excess cation uptake’ (all cells contain

some

Membrane ion transport: general principles Plant cell: ignores the wall; vacuole is usually much larger than shown

cytoplasm - 0.2 V

H +



C +



H + C + NH 4 +



NH 4 +

vacuole

(2H + A -

 

A 2H +

) pH

cyt

7-8

A -



A -

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)

cytoplasm - 0.2 V

H +



C +



H + C + NH 4 +



NH 4 +

vacuole

(2H + A -

 

A 2H +

) pH

cyt

7-8

A -



A -

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’

cytoplasm - 0.2 V

H +



C +



H + C + NH 4 +



NH 4 +

vacuole

(2H + A -

 

A 2H +

) pH

cyt

7-8

A -



A -

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 NO 3 assimilation in shoots), and help regulate pH

cyt

• • Limits: pH

vac

, also osmotic

Transport to & from vacuoles is also part of the ‘biophysical pH-stat’

cyt

7-8

0 to ~+0.05V? cytoplasm vacuole

vac

H +



H + (H + A H + 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

pH?

Change in pH

cyt

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:  pH

cyt

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 pH

cyt

can alter apoplastic pH See Felle (2001, 2002); Gallardo & Cànovas (2002)

Whole-plant issues - 2 • Above-ground cells rely on the ‘biochemical’ pH stat + transport to the vacuole to regulate pH

cyt

• However, RCOO can ‘cycle’ through phloem (pH ~8) down to roots • Roots can use both the biophysical & biochemical components of the pH stat; & select pH ‘non perturbing’ 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 • •

NH 4 + assimilation:

pH 

(acid)

- often whole root surface

NO 3 assimilation:

may be also be pH  pH 

(alkaline)

- sometimes patches; patches elsewhere on same root

Maize (in soil & agar + indicator)

NO 3 NH 4 + low NO 3 Marschner & Römheld (1983)

Rhizosphere: pH changes • • •

NH 4 + assimilation:

pH 

(acid)

- often whole root surface

NO 3 assimilation:

may be also be pH  pH 

(alkaline)

- sometimes patches; patches elsewhere on same root • • •

These effects of N may be ‘hidden’ or modified: Buckwheat

(

Fagopyrum

): pH  even when grown in NO 3 -

Proteoid roots

in low P soil: pH  in patches * *

Fe deficiency:

pH  in patches * [ * 

due to extrusion of RCOO + H + adaptation, or response to stress] ; can be considered as an

Lowering Al toxicity (acid soil):

(pH  or  ) RCOO + C + release

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

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: 577 591 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

Why and how do plants regulate their pH?

Transcript Why and how do plants regulate their pH?

Why and how do plants regulate their pH?

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