Cytosolic GABA inhibits anion transport by wheat ALMT1.

Anion transport by aluminium-activated malate transporter (ALMT) proteins is negatively regulated by gamma-aminobutyric acid (GABA), which increases in concentration during stress. Here, the interaction between GABA and wheat (Triticum aestivum, Ta) TaALMT1 heterologously-expressed in Xenopus laevis oocytes was investigated. GABA inhibited anion transport by TaALMT1 in membrane patches from the cytosolic, not extracellular membrane face, via a reduction in open probability (NPopen ), not an inhibition of channel current magnitude. TaALMT1 currents in patches frequently exhibited rundown with complete removal of cytosolic factors, but were partially sustained by protein kinase C dependent phosphorylation. When applied to whole oocytes a GABA-analogue-BODIPY conjugate inhibited TaALMT1 anion currents from the cytoplasmic face only, whereas free GABA inhibited from both the inside and outside consistent with GABA traversing the TaALMT1 pore then acting from the inside. We propose GABA does not competitively inhibit ALMT conductance through the same pore but rather leads to an allosteric effect, reducing anion channel opening frequency. Across plants GABA is a conserved regulator of anion transport via ALMTs - a family with numerous physiological roles beyond Al3+ tolerance. Our data suggests that a GABA-ALMT interaction from the cytosolic face has the potential to form part of a novel plant signalling pathway.

Elevated CO2 (free-air CO2 enrichment) increases grain yield of aluminium-resistant but not aluminium-sensitive wheat (Triticum aestivum) grown in an acid soil.

BACKGROUND AND AIMS: Soil acidity currently limits root growth and crop production in many regions, and climate change is leading to uncertainties regarding future food supply. However, it is unknown how elevated CO2 (eCO2) affects the performance of wheat crops in acid soils under field conditions. We investigated the effects of eCO2 on plant growth and yield of three pairs of near-isogenic hexaploid wheat lines differing in alleles of aluminium-resistant genes TaALMT1 (conferring root malate efflux) and TaMATE1B (conferring citrate efflux). METHODS: Plants were grown until maturity in an acid soil under ambient CO2 (aCO2; 400 µmol mol-1) and eCO2 (550 µmol mol-1) in a soil free-air CO2 enrichment facility (SoilFACE). Growth parameters and grain yields were measured. KEY RESULTS: Elevated CO2 increased grain yield of lines carrying TaMATE1B by 22 % and lines carrying only TaALMT1 by 31 %, but did not increase the grain yield of Al3+-sensitive lines. Although eCO2 promoted tiller formation, coarse root length and root biomass of lines carrying TaMATE1B, it did not affect ear number, and it therefore limited yield potential. By contrast, eCO2 decreased or did not change these parameters for lines carrying only TaALMT1, and enhanced biomass allocation to grains thereby resulting in increased grain yield. Despite TaMATE1B being less effective than TaALMT1 at conferring Al3+ resistance based on root growth, the gene promoted grain yield to a similar level to TaALMT1 when the plants were grown in acid soil. Furthermore, TaALMT1 and TaMATE1B were not additive in their effects. CONCLUSIONS: As atmospheric CO2 increases, it is critical that both Al3+-resistance genes (particularly TaALMT1) should be maintained in hexaploid wheat germplasm in order for yield increases from CO2 fertilization to be realized in acid soils.

Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing.

Introgression of a 4D chromosomal fragment into durum wheat confers aluminium tolerance.

BACKGROUND AND AIM: Aluminium (Al(3+)) inhibits root growth of sensitive plant species and is a key factor that limits durum wheat (Triticum turgidum) production on acid soils. The aim of this study was to enhance the Al(3+) tolerance of an elite durum cultivar by introgression of a chromosomal fragment from hexaploid wheat (Triticum aestivum) that possesses an Al(3+) tolerance gene. METHODS: A 4D(4B) substitution line of durum wheat 'Langdon' was backcrossed to 'Jandaroi', a current semi-dwarf Australian durum. In the second backcross, using 'Jandaroi' as the recurrent parent, a seedling was identified where TaALMT1 on chromosome 4D was recombined with the Rht-B1b locus on chromosome 4B to yield an Al(3+)-tolerant seedling with a semi-dwarf habit. This seedling was used in a third backcross to generate homozygous sister lines with contrasting Al(3+) tolerances. The backcrossed lines were characterized and compared with selected cultivars of hexaploid wheat for their Al(3+) and Na(+) tolerances in hydroponic culture as well as in short-term experiments to assess their growth on acid soil. KEY RESULTS: Analysis of sister lines derived from the third backcross showed that the 4D chromosomal fragment substantially enhanced Al(3+) tolerance. The ability to exclude Na(+) from leaves was also enhanced, indicating that the chromosomal fragment possessed the Kna1 salt tolerance locus. Although Al(3+) tolerance of seminal roots was enhanced in acid soil, the development of fine roots was not as robust as found in Al(3+)-tolerant lines of hexaploid wheat. Analysis of plant characteristics in the absence of Al(3+) toxicity showed that the introgressed fragment did not affect total grain yield but reduced the weight of individual grains. CONCLUSIONS: The results show that it is possible to increase substantially the Al(3+) tolerance of an elite durum wheat cultivar by introgression of a 4D chromosomal fragment. Further improvements are possible, such as introducing additional genes to enhance the Al(3+) tolerance of fine roots and by eliminating the locus on the chromosomal fragment responsible for smaller grain weights.

Genome-wide association studies and transcriptome sequencing analysis reveal novel genes associated with Al tolerance in wheat.

Aluminum (Al) toxicity is a major threat to the productivity and quality of wheat on acid soil. Identifying novel Al tolerance genes is crucial for breeders to pyramid different tolerance mechanisms thus leading to greater Al tolerance. We aim to identify novel quantitative trait loci (QTL) and key candidate genes associated with Al tolerance in wheat. Herein, we investigated the genotypic variation in Al tolerance among 334 wheat varieties using an acid soil assay. Genome-wide association study (GWAS) and transcriptome were carried out to identify key genes for Al tolerance. GWAS identified several QTL associated with acid soil tolerance including one major QTL on chromosome 1A, in addition to the QTL on 4D where TaALMT1 is located. The four significant markers around the newly identified QTL explained 27.2% of the phenotypic variation. With the existence of reported markers for TaALMT1, more than 97% of the genotypes showed tolerance to Al. For those genotypes with the existence of the novel QTL on 1A but without TaALMT1, more than 90% of genotypes showed medium or high tolerance to Al, confirming the existence of the Al tolerance gene(s) on chromosome 1A. By combining GWAS and RNA-seq analysis, we identified 11 candidate genes associated with Al tolerance. The results provide new insights into the genetic basis of Al tolerance in wheat. The identified genes can be used for the breeding of Al tolerant accessions.

A sterile hydroponic system for characterising root exudates from specific root types and whole-root systems of large crop plants.

BACKGROUND: Plant roots release a variety of organic compounds into the soil which alter the physical, chemical and biological properties of the rhizosphere. Root exudates are technically challenging to measure in soil because roots are difficult to access and exudates can be bound by minerals or consumed by microorganisms. Exudates are easier to measure with hydroponically-grown plants but, even here, simple compounds such as sugars and organic acids can be rapidly assimilated by microorganisms. Sterile hydroponic systems avoid this shortcoming but it is very difficult to maintain sterility for long periods especially for larger crop species. As a consequence, studies often use small model species such as Arabidopsis to measure exudates or use seedlings of crop plants which only have immature roots systems. RESULTS: We developed a simple hydroponic system for cultivating large crop plants in sterile conditions for more than 30 days. Using this system wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) plants were grown in sterile conditions for 30 days by which time they had reached the six-leaf stage and developed mature root systems with seminal, nodal and lateral roots. To demonstrate the utility of this system we characterized the aluminium-activated exudation of malate from the major types of wheat roots for the first time. We found that all root types measured released malate but the amounts were two-fold greater from the seminal and nodal axile roots compared with the lateral roots. Additionally, we showed that this sterile growth system could be used to collect exudates from intact whole root systems of barley. CONCLUSIONS: We developed a simple hydroponic system that enables cereal plants to be grown in sterile conditions for longer periods than previously recorded. Using this system we measured, for the first time, the aluminium-activated efflux of malate from the major types of wheat roots. We showed the system can also be used for collecting exudates from intact root systems of 30-day-old barley plants. This hydroponic system can be modified for various purposes. Importantly it enables the study of exudates from crop species with mature root systems.