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Enhancing aluminium resistance in barley through over-expression of MATE genes


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Zhou, G 2013 , 'Enhancing aluminium resistance in barley through over-expression of MATE genes', PhD thesis, University of Tasmania.

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Acid soils limit crop yields around the world due to nutrient deficiencies and
mineral toxicities. Non-adapted plants grown on acid soils typically have shorter
and thicker root systems because high concentrations of soluble aluminium (Al3+)
inhibit root elongation. This restricts their ability to acquire water and nutrients. An
important mechanism of Al3+ resistance discovered in many plant species relies on
the release of organic anions from roots. The gene controlling this trait are members
of two gene families called the aluminium activated malate transporter (ALMT )
family and multidrug and toxic compound exudation (MATE) family. Members of
the ALMT family encode anion channels which release malate anions from roots
while the MATEs encode co-transporter proteins which facilitate citrate release from
roots. Although barley (Hordeum vulgare) is more sensitive to Al3+ toxicity than
many other cereals including wheat (Triticum aestivum), rye (Secale cereale) and
rice (Oryza sativa) significant genotypic variation in resistance does occur. This
variation is controlled by citrate efflux from the root apices which is encoded by a
MATE gene called HvAACT1. In this study three MATE genes from barley,
Arabidopsis and sorghum (Sorghum bicolor) were transformed into the Al3+-
sensitive barley cultivar ‘Golden Promise’ with a constitutive promoter. These
genes include the major Al3+-resistance genes from barley and sorghum (HvAACT1
and SbMATE respectively) and the Frd3 gene from Arabidopsis thaliana which is
important for iron nutrition. All three are known to encode transport proteins that
facilitate citrate efflux from cells. The resulting transgenic lines were assessed for
transgene expression, citrate efflux from root apices, and Al3+ resistance in
hydroponic solution and acid soil. The control plants included in these experiments
were null segregant lines and the parental barley cultivar. The Al3+-resistant barley
cultivar Dayton was also included as a positive control.
Barley cultivar “Golden Promise” was transformed separately with the MATE genes
using the Agrobacterium method. Several independent T2 or T3 barley lines
homozygous for each transgene were generated as well as null segregant lines. The
transgenic lines released significantly more citrate from their root apices than the
null controls. Plants expressing the HvAACT1 and SbMATE genes required Al3+ in
the external solution to activate citrate efflux while plants expressing Frd3 released citrate in the presence and absence of Al3+. This is consistent with previous studies
showing that HvAACT1 and SbMATE are Al3+-activated proteins. The citrate
efflux from the transgenic lines was similar to, or greater than, the efflux detected
from cv. Dayton.
Transgenic and control seedlings were grown in an aerated hydroponic culture
containing a simple nutrient solution with 0, 1, 2, or 4 μM AlCl3 (pH 4.3). Net root
growth was measured after 4 d. Relative root growth (growth in the Al3+ solution
relative to control solution) was significantly greater in the transgenic lines than the
null controls for most Al3+ treatments and similar results were obtained for the three
MATE genes. The Al3+ resistance of the transgenic lines was similar to the Al3+
resistance of cv. Dayton.
Al3+ resistance of the transgenic and control lines was also assessed in short-term
soil experiments. The acidic ferrosol was either unamended (pH 4.33 with
aluminium being 21% of exchangeable cations) or limed so that pH increased to
5.18 and only 1% of exchangeable cations was aluminium. After 6 d growth the
following measurements were made: length of the longest and second-longest roots,
total root length, total root weight, shoot weight and distribution of root diameters.
In the unamended acid soil root growth of the null lines was inhibited compared to
the limed soil and the roots became thicker. Expression of each of the MATE genes
significantly increased Al3+ resistance with relative length of the longest roots (root
length in acid compared to limed soil) and relative total root length (total root length
in acid compared to limed soil) providing the greatest differences between the
transgenic and null lines. The transgenic lines also maintained a greater percentage
of thinner roots in the acid soil than the null lines.
These results demonstrate that Al3+ resistance in barley can be enhanced by
heterologous expression of the SbMATE and Frd3 genes or by over-expression of
the endogenous HvAACT1 gene. Biotechnology provides important options for
increasing the Al3+ resistance of crop plants which can complement traditional
breeding practices. Both strategies will be important for maintaining and even
increasing food production on acid soils in the future.

Item Type: Thesis - PhD
Authors/Creators:Zhou, G
Keywords: HvAACT1, citrate transporter, aluminium tolerance, transgene
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