The transcripted response of barley (Hordeum vulgare L.) to boron toxicity.

The occurrence of Boron (B) toxicity in Australian soils is recognised as a limiting factor for cereal productivity. A number of loci conferring tolerance to B toxicity have been identified in barley and chromosomally mapped. However, a lack of knowledge relating to the physiological and molecular events that occur under B toxicity and the molecular basis for B stress tolerance has been a bottleneck in harnessing available genetic diversity in barley and wheat. The recent advances in functional genomics provided an opportunity to examine B stress in barley in more detail. The aim of this project was to analyse genes differentially expressed under B stress in tolerant and intolerant barley to identify candidate genes involved in B toxicity tolerance. Two experimental approaches, Suppression Subtractive Hybridization (SSH) and microarray were adopted. Firstly, SSH was performed to examine gene expression in roots of selected tolerant and intolerant doubled haploid lines from a Clipper (B intolerant) X Sahara 3771 (B tolerant) mapping population, grown under moderate B stress. The SSH experiment aimed to investigate the early transcriptional response of B tolerant barley lines to B stress in order to identify the basis for B toxicity tolerance in roots. Differential screening of the subtracted library generated from B treated plants identified a total of 111 non-redundant clones up-regulated in bulked tolerant lines. On the other hand 94 clones were differentially expressed under non-treated conditions. Among the clones identified from subtracted library generated from B treated plants, metabolism was the largest functional category, representing 21% of the clones. The largest functional category in the subtracted library generated from non treated plants was cellular transport, representing 19% of the clones. Based on sequence similarity, about 170 transcripts identified in this experiment were assigned to chromosomal segments (bins) on the three homoeologous genomes of bread wheat. In total, 36 clones from the subtracted library generated from B treated plants were analysed as candidates. Nine were genetically mapped within the region of B tolerance QTL on three chromosomes (2H, 4H and 6H). The genes mapped to 4H and 6H QTL have the highest association with these loci in the Clipper X Sahara 3771 doubled haploid mapping population. A 4H B tolerance QTL candidate gene was identified as a B transporter gene with similarity to the Arabidopsis BOR1 gene. Genes identified to be differentially expressed in the tolerant lines from SSH suggest activation of a diverse defence response in the roots of barley plants under B stress. Data from SSH experiment indicate that cell wall-plasma membrane cytoskeleton continuum constitute the first action site against B toxicity and the influence of toxic B on K+ uptake could be the key initiating factor. In the second approach, the Affymetrix 22K Barley1 GeneChip(TM) was used to investigate B stress adaptation processes in barley. Gene expression was profiled in leaves of Sahara 3771 and Clipper plants grown under various B concentrations. The results show that the two genotypes respond differently to B toxicity. The B intolerance of Clipper is expressed through the induction of a high number of probe sets (2310) even at a low B concentration of 100 µM. In contrast, Sahara 3771 responded to a high B concentration (2000 µM) through the induction of only a few hundred (266) probe sets. In Sahara 3771 no change in the expression level of any probe sets was observed at 100 µM B. Altogether 286 probe sets showed differential expression in Sahara 3771 under three levels of B treatment (500, 1000 and 2000 µM). About 30% of these were down-regulated and about 70% were up-regulated in Sahara 3771 in response to B treatment. Most of the probe sets (59%) up-regulated in Sahara 3771 did not respond to B treatment in Clipper. These genes are either salt stress responsive or related to plant defense and thus could play a key role in protecting barley plants from the toxic effects of B. Two differentially expressed probe sets annotated as B transporters were identified between Sahara 3771 and Clipper under control condition. These two B transporter probe sets did not respond to B treatment but showed opposing expression patterns in the two varieties. One of these probe sets (Contig21126_at) is similar to the B transporter gene isolated from the SSH experiment that maps to the 4H tolerance locus. The map location and expression of this B transporter gene suggest that it could be the borate anion efflux transporter predicted by the proposed efflux model of B tolerance in Sahara 3771 barley. The other B transporter gene (Contig14139_at) showed over expression in Clipper under control condition and could be contributing to high B accumulation in Clipper which needs further investigation. Data from both experiments have indicated that B toxicity triggers oxidative stress and that jasmonate-based signaling plays a key role in B toxicity tolerance. SSH data indicate that Sahara 3771 which evolved in the harsh environment of Africa is more efficient in osmoregulation and ROS scavenging than Clipper. This trait is likely to give Sahara 3771 an edge over Clipper in tolerating toxic the effect of B. In addition to the efflux mechanism, which becomes less efficient with increasing B supply, Sahara 3771 appears to apply a number of other mechanisms for alleviating or withstanding toxic B induced stress to sustain growth. Some of these mechanisms are already known to be used by plants to cope with a number of stresses.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 200