Functional characterization of wheat ALMT1 transporter and its involvement in extreme pH stress tolerance

Abstract

The optimum soil pH for most cultivated plants ranges from pH 6 to 8. This range provides optimal nutrient availability and minimal effects of toxic ions. Soils with pH below 5.5 (acid) and above 8 (alkaline) pose challenges for plant growth and development due to ion toxicities and lack of nutrient availability or nutrient imbalances. Roots of some species such as Triticum aestivum (wheat) exude organic anions such as malate under acidic conditions, providing tolerance against free Al3+ which is highly toxic to roots. In wheat the transporter responsible for this exudation is the Aluminium Activated Malate Transporter (TaALMT1). This thesis examines the role of the TaALMT1 transporter in extreme pH stress tolerance. Plant ALMTs are anion channels named after the first characterized member from wheat roots (TaALMT1). However, most ALMTs are not activated by Al3+, but all those so far investigated are regulated by gamma-aminobutyric acid (GABA). Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA-binding site in mammalian GABAA receptors. In wheat root apices a negative correlation between activation of TaALMT1 and endogenous GABA concentrations ([GABA]i) was previously identified. This is explored here further in both wheat root apices and in heterologous expression systems using inhibitors that are reported to change [GABA]i: amino-oxyacetate (AOA) – a glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) inhibitor, and vigabatrin – a GABA transaminase (GABA-T) inhibitor. It is demonstrated that activation of TaALMT1 reduces [GABA]i because TaALMT1 facilitates GABA efflux. Though TaALMT1 is activated by Al3+ the released GABA does not complex Al3+. TaALMT1 also facilitates GABA transport into cells, demonstrated by a yeast complementation assay and via 14CGABA uptake into TaALMT1-expressing Xenopus laevis oocytes; found to be a general feature of all ALMTs examined. Mutation of the GABA ‘motif’ (TaALMT1F213C) prevented both GABA influx and efflux in yeast and Xenopus laevis oocytes, and resulted in no correlation between malate efflux and [GABA]i. It is concluded that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other’s transport, suggestive of a role for ALMTs in communicating metabolic status. One of the potential roles for GABA is as a pH regulator. Being a zwitterion its exudation into acidic or alkaline solutions will tend to bring pH towards neutrality. Previous field studies have suggested that TaALMT1 in wheat may also confer tolerance to alkaline soil. Soil alkalinity reduces yield and is a major problem worldwide, but very little is known about the physiological mechanisms that allow some plants to tolerate alkaline conditions. Along with its role in Al3+ tolerance at low pH, TaALMT1 is also activated by external anions at alkaline pH. Therefore it was hypothesized that TaALMT1 provides alkaline soil tolerance by exuding malate and GABA facilitating acidification of the rhizosphere. To test this hypothesis, a series of experiments were carried out using wheat NILs; ET8 (Al+3 tolerant, high expression of TaALMT1) and ES8 (Al+3 sensitive, low expression of TaALMT1) and Xenopus laevis oocytes expressing TaALMT1. Under alkaline conditions, root biomass was significantly higher in the ET8 plants compared to ES8 plants and was inhibited by the application of GABA. Shoot gas exchange also differed between NILs but continuous GABA application to roots interfered with shoot gas exchange. In alkaline conditions, a higher concentration of both malate and GABA was found in root exudates from root apices and whole seedling roots with high TaALMT1 expression which appears to decrease the rhizosphere pH more so in ET8 compared with ES8. Xenopus laevis oocytes expressing TaALMT1 also acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accord with a high rate of GABA synthesis in ET8. It is concluded that TaALMT1 plays a role in alkaline soil tolerance by exuding malate and GABA, possibly coupled to proton efflux, facilitating rhizosphere acidification. To further explore the role of TaALMT1 in alkaline soil tolerance, transgenic Golden Promise barley plants expressing TaALMT1 (TaALMT1-GP) were treated with pH 6 and pH 9 nutrient solutions over 5 weeks of growth. There was no significant effect of TaALMT1 expression on shoot and root growth relative to GP wildtype in alkaline conditions. However, root fresh mass was more sensitive to pH for TaALMT1-GP with a significantly larger root fresh mass at pH 9 compared with pH 6. GABA application significantly reduced both root and shoot growth independently of TaALMT1 expression. Malate and GABA efflux was higher in TaALMT1-GP plants than for GP plants at pH 9, however, the opposite was the case at pH 6. GABA application affected malate efflux with different effects between TaALMT-GP and GP. Malate efflux from root apices over 1 h was not significantly different between TaALMT1-GP and GP and both genotypes increased malate efflux at high pH. However, GABA efflux was significantly higher in TaALMT1-GP than GP at pH 9 in buffered solution. It is concluded that the expression of TaALMT1 may be interfering with the endogenous systems that allow barely to tolerate alkaline soils and that future experiments will require the use of null segregants as the appropriate controls rather than wildtype (GP) background. Preliminary experiments were also undertaken to test the effects of externally applied sodium aluminate, calcium, GABA and muscimol, and selected hormones using wheat (ET8 and ES8 NILs) and Barley (TaALMT1-GP and GP, aluminate only) at alkaline pH. Sodium aluminate treatment significantly increased malate and GABA efflux above the already elevated level at pH 9 in plants with high expression of TaALMT1, suggestive of TaALMT1 involvement. Root growth was also higher in response to sodium aluminate in both ET8 and TaALMT1-GP compared with ES8 and GP respectively. Elevated external calcium significantly increased Al3+-activated malate efflux in ET8 compared with ES8 with an optimum at 3 mM CaCl2. In response to external jasmonic acid (JA) and brassinosteroid (BR) at pH 4.5, ET8 showed a higher Al3+activated malate efflux compared to ES8. However, in contrast to malate, GABA efflux was significantly reduced by BR and JA compared with the Al3+ treatment alone. Root growth was significantly reduced in response to JA plus Al3+ compared with Al3+ alone. However, BR plus Al3+ significantly enhanced root growth compared to Al3+ treatment alone. Overall, it is concluded that: 1) TaALMT1 is not only regulated by GABA but also mediates its transport, which is a general feature of ALMTs. 2) TaALMT1 plays a role in alkaline soil tolerance by exuding malate and GABA and facilitating rhizosphere acidification. 3) External application of high concentrations of GABA (10 mM) to roots results in inhibited root growth and alters leaf gas exchange possibly by interactions with other ALMTs. In addition, the following preliminary conclusions are subject to carrying out experiments with TaALMT1 expressed in heterologous systems: 1) TaALMT1 might be calcium sensitive. 2) TaALMT1 may play a role in aluminium tolerance in alkaline conditions. 3) There may be complicated hormonal control over TaALMT1 activity and selectivity.