Download 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 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 ( 4M)
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
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