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Mechanisms of aluminium toxicity, tolerance and amelioration in wheat


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Wherrett, Timothy 2006 , 'Mechanisms of aluminium toxicity, tolerance and amelioration in wheat', PhD thesis, University of Tasmania.

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Acid soil stress, particularly aluminium (Al) toxicity, is one of the biggest
constraints to food production worldwide, particularly in tropical developing
countries. Despite decades of extensive research both our knowledge of the
mechanisms of Al tolerance and toxicity and consequently our management strategies
are very limited. The aim of this project was to deepen understanding of the
physiology of Al toxicity, tolerance and amelioration, which may later lead to more
sustainable management of acid soils. Because many different genes with diverse
actions confer Al stress tolerance, significant genotypic variation exists between
species and cultivars. This project specifically examines two near isogenic cultivars of
wheat (Triticum aestivum), ET8 (Al-tolerant) and ES8 (Al-sensitive), that differ at a
single genetic locus that confers Al tolerance.
It is generally thought that the mechanism conferring Al tolerance to ET8 is
restricted to Al induced malate efflux from the root apex. Consequently the most
commonly reported difference between ET8 and ES8 genotypes is root elongation in
the presence of Al. Other aspects ofroot morphology are less studied. In this work, a
significant difference in root hair growth was found between Al-exposed ET8 and
ES8 varieties. It is suggested that this differential tolerance was either expressed
directly at the root hairs or remotely from the root tip via hormone production or
retention ofmalate in the cell wall. The higher tolerance ofET8 root hairs was also
reflected in nutrient acquisition; this effect was not attributed to changes in the root
hair surface area between cultivars, implying greater tolerance of nutrient acquisition
in ET8.
Application of external ameliorants had significant effects on root, shoot and root
hair growth. Ca, Mg and silicate all increased root length significantly and there was no interaction observed with cultivar (ET8 or ES8) suggesting that the mechanism of
amelioration is identical in each cultivar and is not associated with enhanced malate
efflux. This was further confirmed by measuring Ca2+ and K+ fluxes at the root apex.
Ca and Mg both significantly increased root hair surface area in the presence and
absence of Al. In comparison, silicate reduced root hair density in ET8 in the presence
of Al and completely abolished root hair development in ES8 with or without Al.
Amelioration by Mg and Ca appears general to the whole root and is probably
associated with reduced membrane binding of Al3
+, whereas amelioration by silicate
seems to be restricted to the apex.
Examining the Al-induced malate tolerance mechanism more closely, the
hypothesis that Al-activated malate and other ionic fluxes would induce changes in
the membrane potential (Vm) and that these responses would differ between ET8 and
ES8 was examined. Within minutes of exposing wheat roots to 50 μM AlCh, a
significant depolarisation was measured in the elongation zone of the ET8 genotype,
but not in ES8. Underlying ionic mechanisms that may be responsible for these
changes in V m were investigated by measuring real-time fluxes of er, H+ and K+ at
the root apices of wheat seedlings. Addition of 50 μM AlCh to the bathing solution
stimulated an increase in K+ efflux and H+ influx in ET8 but not in ES8. The
differences between the genotypes were sustained for 24 h and were observed only at
the elongating zone and not the meristematic zone. After 24 h Al3+ increased er
influx in ET8, but inhibited ES8 influx in a dose dependent manner. These results
provide the first real time kinetics of K+ flux kinetics from intact roots showing K+
efflux to charge balance malate efflux in tolerant, but not sensitive cultivars and that
this is maintained even after 24 hours exposure. It is also suggested that er may play a significant role in both charge balance and maintenance of cell turgor in longer term
tolerance of Al.
Disrupted Ca2+ homeostasis has been often suggested as a primary mechanism of
aluminium toxicity, with reports showing that Al induces a greater increase in
cytosolic Ca2+ in ES8 than ET8. It is unknown whether this cultivar difference is a
result of Al-malate complexing or a direct difference in ET8 versus ES8 Ca2+
signalling in response to Al. In this work, patch-clamp experiments revealed that ET8
and ES8 wheat cultivars differed significantly in slow vacuolar (SV) channel
properties. The SV channel density was about 35% lower in ET8 than ES8 and was
not altered by 24hr aluminium pre-treatment. Al3+ at the vacuolar side reduced the
inward ( cytosol-directed) current through single open SV channels, suggesting Al3+
enters the pore and is permeable. In ET8, but not ES8, stabilisation of the closed states
of SV by Al3+ was observed. SV channel activity was significantly increased in Al
pre-treated vacuoles ofES8 by some unknown low molecular weight compound.
Taken together, this data suggests that the observed differences in SV channel
properties between ET8 and ES8 may be responsible for some the differences
observed in Al disruption of ca.2+ homeostasis and may have significant benefits for
Al tolerance in ET8.
The precise signalling pathway involved in Al induced elevation of cytoplasmic
Ca+ remains unknown, with a large number of second messengers and channels from
endomembranes and the plasma membrane being suggested. In order to determine a
possible signalling pathway underlying Al stress signalling, the capacity of SV
channel to mediate vacuolar Ca release and the effect of a number of second
messengers on vacuolar Ca2+ fluxes were studied in a model system, red beet
vacuoles. It was found that at the measured natural intravacuolar activity ofK+, Na+, Ca2+ and Mg2+ the SV channel becomes insensitive to intravacuolar Ca2+ changes, but
still responds to cytosolic Ca2+ and Mg2+. Ca2+ efflux observed in Ca free solutions
was enhanced by exogenous Ca2
+ and blocked by Zn2
+, suggesting that SV mediates
the majority of the observed Ca2+ efflux. The effect of 26 different combinations of
common second messengers and other molecules implied in Al toxicity or Ca2+
signalling on Ca2+ efflux are reported. In similar experiments using wheat cultivars
ET8 and ES8, significantly larger efflux from vacuoles was observed in ES8
confirming the earlier patch observation of higher ES8 SV density and the possible
involvement of SV density in Ca2+ homeostasis. Higher efflux in ES8 than ET8 was
also observed in all second messenger treatments. The importance of SV channel
density in Al tolerance was conclusively demonstrated by growth experiments of
Arabidopsis mutants over and under expressing the SV channel. Mutants under
expressing the S V channel ( tpc- 1-2) had a better relative root growth in the presence
of Al than wild type (Columbia) and over expressing mutants (TPCl 5-6 and TPCl
10-21 ).
In its entirety this project raises serious questions about the validity of the
assumption that the difference between ET8 and ES8 is simply associated with an Alinduced
plasma membrane channel that mediates malate efflux. Rather it is suggested
that the Altl locus encodes a number of genes associated with a more complex suite
of tolerance mechanisms. Furthermore this thesis provides significant new knowledge
of the physiological mechanisms by which ET8 and ES8 differ in Al tolerance, malate
and K+ release and Ca2+ homeostasis.

Item Type: Thesis - PhD
Authors/Creators:Wherrett, Timothy
Keywords: Wheat, Acid soils, Aluminum
Copyright Holders: The Author
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Copyright 2006 the author

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Available for library use only and copying in accordance with the Copyright Act 1968, as amended. Thesis (PhD)--University of Tasmania, 2006. Includes bibliographical references

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