Mechanisms of aluminium toxicity, tolerance and amelioration in wheat

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 ˜í¬¿M 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 ˜í¬¿M 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.