Ionic Basis of Plant Perception of Salt and Osmotic Stress

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Ionic relations, potassium homeostasis, and
salinity tolerance in cereals implications for
breeding
Sergey Shabala School of Agricultural Science,
University of Tasmania
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The Problem
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Salinity as a worlds quiet crisis

• Salinity affects 7 of the world's land area,
  which amounts to 930 million ha
• Approximately 33 of irrigated land worldwide is
  affected by salinity.
• About 10,000,000 of 15,000,000 hectares of
  irrigated land in Pakistan are becoming saline
• A third of Australia's agricultural area is at
  risk
• By 2020 somewhere between 10 and 25 of
  previously arable land could be out of production

Salinity will cost Australia 1 billion a year by
2100
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Breeding for salt tolerance some principles and
dogmas
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Traditional approach to breeding

• Breeding for better osmotic adjustment
• Breeding for Na exclusion

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Breeding for better osmotic adjustment

• Osmolyte accumulation has long been emphasised as
  a selection criterion in traditional crop
  breeding programs (Morgan 1983 Blum et al. 1983
  Ludlow and Muchow 1990)
• Overexpressing genes responsible for biosynthesis
  of so-called compatible solutes

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Advantages

• Often controlled by only one gene (easy to
  manipulate)
• Overall, some 13 species have been transformed
  with nearly 40 genes between 1993 and 2003
  (Flowers 2004) most of these genes were related
  to biosynthesis of compatible solutes

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Problems

• Lack of direct evidence for the conventional role
  in osmotic adjustment
• Controversial results e.g. less proline
  accumulation in rss mutant higher proline GB
  content in sensitive cultivars
• Concentrations are far too low (lt 1 mM)
• High cost of osmolyte production
• No practical outcomes for farmers

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Breeding for Na exclusion

• Na is toxic for cell metabolism
• Traditional view glycophytes have to exclude Na
  to survive under saline conditions (up to 98
  Munns 2005)
• The latter is done either by excluding Na from
  uptake, or by removing it from the cytosol
  (Na/H antiporter)

Zhang et al (2004) Plant Phys 135 615-621
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Problems

• Exclusion of Na from root uptake does not
  solve the problem of the osmotic component of
  salt stress
• ? Necessity of plants having increased level of
  compatible solutes to be used for cell osmotic
  adjustment
• ? High energetic cost of this process
  (45-50 mol ATP/mol of compatible solute)
• ? Plant growth retarded (no
  energy to invest!)
• Negative correlation between Na accumulation
  and salt tolerance doesnt hold in many cases
  (halophytes Chenopodium family Arabidopsis
  sos1 tomato maize bread wheat)

Back to square one??
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Targeting K homeostasis an alternative approach
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Salinity and K homeostasis

• K is central to cell metabolism
• Poor plant growth under salinity is a result of
  Na replacing K in key metabolic reactions

• It is not Na per se, but K/Na ratio that is
  critical to plant salt tolerance
• Na can be used as a cheap osmoticum in the
  vacuole (compartmentation!)

Rather than restricting Na uptake, lets retain
K in the cell!
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Microelectrode Ion Flux Measurements (the MIFE
technique)

• Non-invasive measurements of up to 3 ions at the
  same time
• Nearly 20 various ions measured
• High temporal (5 sec) and spatial (a few µm)
  resolution
• Long-term measurements for hours if not days
• In planta method

Net flux is calculated from diffusion equations
based on the measured electrochemical gradient
for a particular ion between two positions
J c u (d?/dx)
? ?0 RT ln ?c z F Vb
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Now to physiology.
Zhonghua Chen
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Gairdner
Barley genotypes contrasting in salt tolerance
0 mM NaCl 160 mM NaCl 320 mM NaCl
320 mM NaCl
ZUG293
ZUG293 Gairdner
0 mM NaCl 160 mM NaCl 320 mM NaCl
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NaCl-induced K efflux correlates with salt
tolerance
80 NaCl
increase in salt tolerance
Fluxes were measured from roots of 3-d old
seedlings
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Correlation with growth

• Strong correlation between K flux measured from
  3-d old seedling and growth responses from 2-3
  months old plants grown in glasshouse

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and with plant biomass
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and with plant photosynthetic activity
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and with K content
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K efflux feature is a heritable trait
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The story so far

• Plants ability to retain K correlates with salt
  tolerance in barley
• Very strong correlation between net K leak
  measured from 3-d old roots of barley seedling
  and whole-plant physiological responses in
  glasshouse experiments
• Evidence for inheritance
• K flux measurement as an efficient screening
  tool ?

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Validation
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Large-scale screening

• A glasshouse trial with 70 barley genotypes (560
  pots 5600 seedlings)
• Replicated twice (in 2004 and 2005)

MIFE K flux measurements from 3-d old seedlings
(70 genotypes x 8-10 replicates)
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Ranking barley genotypes
Ranking according to grain yield
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Ranking according to grain yield (continued)
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Ranking barley genotypes
... and shoot biomass
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... and shoot biomass (continued)
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Strong correlation between K flux and plant
physiological responses
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Take-away message

• Measuring K efflux is a sensitive, reliable and
  efficient way of screening plants for salt
  tolerance
• The method is not destructive (prospective plants
  can be grown and used in breeding programs)
• No need for space (seedlings are grown in Petri
  dishes)
• Time efficient (hours vs months)

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Mechanisms which transporters and genes?
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K transport systems in plants

• 75 genes from 7 different families are known for
  K transport
• Total number of cation transporters has 880
  members from 46 unique families (5 of the entire
  Arabidopsis genome)

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Arabidopsis K transporters
KCO channels
Trk/HKT transporters
Shaker-type channels
KUP/HAK/KT transporters
From Maser et al (2001)
K/H antiporters
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GORK as a downstream target
Multiple mechanisms are well combined in order to
withstand saline conditions (1) better control
of membrane voltage so retaining a more negative
membrane potential (2) intrinsically higher H
pump activity (3) better ability of root cells
to pump Na from the cytosol to the external
medium (4) higher sensitivity to supplemental
Ca2

• NaCl-induced K efflux is mediated by GORK
• AKT channel is involved in root osmotic adjustment

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What about other species?
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Lucerne growth data
160 mM NaCl for 5 weeks
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Lucerne K flux kinetics

• Similar to barley, salt tolerance in lucerne can
  be determined by measuring the magnitude of
  NaCl-induced K efflux from roots of 5-6 d old
  seedlings
• Higher salt tolerance is related to roots ability
  to maintain more negative MP

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Wheat growth data

• Two durum and two bread wheat cultivars
  contrasting in their salt tolerance
• - Kharchia (bread, tolerant)
• - Baart 46 (bread, sensitive)
• - Wollaroi (durum, tolerant)
• - Tamaroi (durum, sensitive)

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Wheat K flux kinetics

• Similar to barley, wheat salt tolerance (measured
  as grain yield under saline conditions)
  correlated negatively with the magnitude of
  NaCl-induced K efflux measured from 5-d old
  seedlings
• However, the overall NaCl-induced K efflux from
  wheat roots was about 5 to 10 times lower
  compared with barley

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General Summary

• Our results are consistent with the idea of the
  cytosolic K/Na ratio being a key determinant of
  plant salinity tolerance
• This K/Na ratio is controlled by multiple
  pathways, most likely specific for each species
• Regardless of mechanisms, salt tolerant genotypes
  have the better ability to retain K in their
  tissues
• The above feature is inheritable

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Practical conclusions

• Measuring net K flux is an efficient and highly
  reliable screening tool for salt tolerance at
  least, for barley!
• Plant breeding for salt tolerance can benefit
  from targeting K homeostasis and, specifically,
  functional expression and control of GORK genes

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Acknowledgements

• Members of my group
• Zhonghua Chen
• Dr Tracey Cuin
• Dr Lana Shabala
• Stuart Betts
• My colleagues at Univ. Tasmania
• Dr Meixue Zhu
• Dr Neville Mendham
• Dr Ian Newman
• My International collaborators
• Prof Mark Tester (Univ. Adelaide)
• Prof Mickey Palmgren (Uni. Copenhagen, Denmark)
• Prof Igor Pottosin (Univ. Colima, Mexico)
• Prof Guoping Zhang (Zhejiang Univ., China)
• Funding bodies (ARC and GRDC)