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Evaluating the contribution of tonoplast and plasma membrane transporters in salinity tissue tolerance in barley

Adem, GD 2016 , 'Evaluating the contribution of tonoplast and plasma membrane transporters in salinity tissue tolerance in barley', PhD thesis, University of Tasmania.

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Salinity is one of the major abiotic stresses affecting the world food supply. Salt affected soil
has area coverage of 950 million ha which accounts for 10% of the land surface of the globe.
Fifty percent of the irrigated land (230 million ha) is affected by salinity costing the world
food production $US27 billion per annum. In Australia alone, 67% of the agriculture land is
affected by transient salinity costing the Australian economy $AUD 1330 million per annum.
In comparison to other strategies, the use of salt tolerant plants is cost effective and
sustainable way of controlling salinity.
While numerous attempts have been made to develop salt tolerant varieties over the last
few decades, most of the efforts were focused on tackling individual components or genes
contributing to overall salt tolerance. As a result, the progress in the field was much slower
than expected and we still lack truly salt tolerant varieties in the farmers‟ field. Salinity
tolerance is a multi-faceted physiological trait, and all the beneficial effects of improving a
function of one gene/mechanism may be lost or overturned by plethora of other contributing
factors. This calls for a need of “pyramiding” several key traits in one ideotype. Before this
can be implemented into the practice, the essentiality and a relative contribution of various
traits should be quantified, for each particular species. The aim of this PhD study was to
identifying the major contributing mechanisms to salt tolerance in barley. The major focus
was on the following aspects: (1) essentiality of transcriptional vs post translational factors in
mediating plant adaptive responses to salinity; (2) quantifying the relative contribution of
osmo- and tissue-tolerance mechanisms in barley; (3) identifying components involved in Na+
sequestration in vacuole; and (4) revealing the role of H+-ATPase in vacuolar ion
sequestration and overall salinity stress tolerance.
In the first part of this PhD study, the relative contribution of ionic, osmotic and
oxidative stresses to the overall salinity tolerance in barley was studied, both at the whole plant and cellular level. In addition, the gene expression profile of key genes in ionic and
oxidative homeostasis (NHX, RBOH, SOD, AHA and GORK) as a way of comparing the
contribution of transcriptional and post translational factors for salinity tolerance was also
investigated. The major findings can be summarized to two main points. These are: (i) tissue
tolerance is the dominant component in which root K+ retention and lower sensitivity to stress
induced hydroxyl radical production are the main ones; (ii) responses at the post-translational
level is more important than at the transcriptional level for understanding the mechanisms for
salt tolerance. Overall, for better tissue tolerance, sodium sequestration, K+ retention and
resistance to oxidative stress are important salt tolerance mechanisms. It is concluded that
every crop improvement programs for salinity stress tolerance should take into consideration
all this components.
In the second part of this PhD study, we showed that unlike a number of reports for saltsensitive
“salt excluder” species (such as Arabidopsis or rice), expressing AtNHX1 in barley
had no beneficial effect on plant performance under saline conditions. AtNHX1 Arabidopsis
tonoplast Na+/H+ exchanger was expressed in barley (Hordeum vulgare L., cv. Golden
Promise) and the plants grown under saline growth condition. The transgenic plants were
compared with null segregant for biomass, water content, gas exchange, and Na+ and K+
content of the leaf. The transgenic barley plants expressing AtNHX1 has not shown significant
difference from the null segregant for any of the trait at least under our experimental
conditions. The lack of phenotype in barley which adapts “salt including” strategy was
explained by one or more of the following: (i) low level of activity of vacuolar H+-PPiase and
vacuolar H+-ATPase causing poor proton gradient; (ii) the lack of controlling passive leak of
sodium via Na+ permeable slow activating and fast activating channels in the vacuole; (iii)
insufficient ATP pool to assist the H+ pumping activity; (iv) the AtNHX1 protein may not be
folded properly, inactive or mis-targeted.
In the last part of this PhD study, improvement in salinity stress tolerance was obtained in
barley crops by expressing V-ATPase subunit C gene. Most of the reports until now have not
shown improvement at the grain yield level and also have not explained the physiological
mechanisms behind. Also, no previous attempt to express the V-ATPase subunit C was done
in barley. Accordingly, we expressed AtVHA-C gene in barley and grown under 300mM NaCl.
The barley plants expressing the gene were compared with wild type plants for dry biomass,
leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ content. The
transgenic barley plant expressing AtVHA-C have shown smaller reduction in biomass and
grain yield compared to wild type plants. The beneficial effect in the AtVHA-C expressing
plant was due to better maintenance of stomatal conductance resulted from the accumulation
of Na+ and K+ in the leaf that lead to osmotic adjustment and less reliance on the de novo
synthesis of organic osmolytes.
To recap, salinity tolerance is a multigenic physiological trait involving various
mechanisms that may differ between species. For barley, the dominant mechanism appears to
be the tissue tolerance. This includes better K+ retention in the root; reduced tissue sensitivity
to oxidative stress; and more efficient sodium sequestration in the leaf. We have shown that
for a better sodium sequestration all the components such as proton pumping for generating
proton gradient in the tonoplast membrane, control of the back leak of sodium through Na+
permeable SV and FV channels, and ensuring properly folded, active and correctly targeted
NHX protein to the tonoplast all should be considered and modified as one set, to achieve a
positive outcome and improve crop salinity tolerance. Finally, we have also shown the
importance of vacuolar H+-ATPase towards salinity tolerance by the contribution of vacuolar
H+-ATPase for the accumulation of Na+ and K+ in the leaf where they contribute towards
osmotic adjustment, and hence, conservation of energy otherwise spent for organic osmolyte

Item Type: Thesis - PhD
Authors/Creators:Adem, GD
Keywords: Salinity tissue tolerance; Tonoplast Na+/H+ antiporter; Vacuolar H+-ATPase; K+ retention; Vacuolar sequestration
Copyright Information:

Copyright 2015 the author

Additional Information:

Chapter 2 has been published as: Adem GD., Bose J., Zhou M., Shabla S., (2015), Targeting vacuolar sodiumsequestration in plant breeding for salinity tolerance. In H. Wani ,A. Hossain (eds.) Managing salinity tolerance in plants : molecular and genomic perspectives (pp. 35-50), Taylor & Francis.

Chapter 3 appears to be the equivalent of a post-print of an article published as: Adem GD., Roy S., Zhou M., Bowman J., Shabala S., (2014), Evaluating contribution of ionic, osmotic and oxidative stress components towards salinity tolerance in barley, BMC plant biology ,14(1), 113, under a Creative Commons Attribution License (

Chapter 4 appears to be the equivalent of a post-print of an article published as: Adem GD., Roy S., Plett D., Zhou M., Bowman J., Shabala S. (2015), Expressing AtNHX1 in barley (Hordeum vulgare L.) does not improve plant performance under saline conditions, Plant growth regulation, 77(3), 289-297. The final publication is available at Springer via

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