Mechanisms of oxidative stress tolerance in rice and their application in the molecular breeding of genotypes adapted to stress environments
Final Report Abstract
This project aimed at identifying genes and mechanisms involved in oxidative stress tolerance in rice, focusing on three distinct subprojects, each representing the topic of a PhD thesis: (i) Characterization of a quantitative trait locus (QTL) for ozone tolerance (Subproject 1), (ii) ascorbate biosynthesis in rice and its involvement in tolerance to zinc (Zn) deficiency (Subproject 2), and (iii) identifying genes and mechanisms involved in tolerance to iron (Fe) toxicity (Subproject 3). Additional cross-project studies aimed at linking these subprojects and identifying common genes or transcriptional patterns involved in multiple stress tolerance. In Subproject 1, a gene hypothetically underlying the previously identified ozone tolerance QTL OzT9 was characterized in-depth. Although this gene had been annotated as an ‘ascorbate oxidase family gene’, heterologous expression and complementation assays did not confirm enzymatic ascorbate oxidase activity. On the other hand, the protein was localized in the apoplast as shown by a GFP- fusion assay, and its involvement in ozone tolerance was confirmed using rice mutant lines. The gene was thus re-annotated as OZONE RESPONSIVE APOPLASTIC PROTEIN1 (OsORAP1). Sequence polymorphisms in the regulatory part of the gene (promoter and 5’UTR) were highly correlated with mRNA expression and ozone tolerance in rice, further corroborating the hypothesis the OsORAP1 contributes to the effect of the ozone tolerance QTL OzT9. As a more applied aspect of this subproject, QTL pyramiding lines were developed that contained both OzT9 and a second QTL OzT8 in a sensitive background variety. These breeding lines were significantly more tolerant than their parents in terms of biomass, yield components, and rice grain quality when exposed to high ozone concentrations in season-long ozone fumigation trials. In Subproject 2, ascorbate biosynthesis in rice was characterized using gene knockout mutants for several orthologues of genes previously characterized in Arabidopsis thaliana. Three of these genes, i.e. two isoforms of GDP-D-mannose-3’,5’-epimerase (OsGME), and one GDP-L-galactose phosphorylase (OsGGP) were confirmed to be involved in ascorbate biosynthesis in rice. Lack of ascorbate in rice knockout lines affected photosynthetic efficiency, growth and phenology, and negatively affected tolerance to ozone stress and Zn deficiency, but not Fe toxicity. In additional experiments, the role of the ascorbate metabolism in Zn deficiency was investigated in-depth using contrasting genotypes differing in Zn efficiency. It was concluded that high expression of ascorbate biosynthesis genes and high ascorbate and ascorbate precursor levels were correlated with tolerance, and that lack of ascorbate led to a redox imbalance preceding the formation of visible stress symptoms. In Subproject 3, two different bi-parental mapping populations were screened for Fe toxicity tolerance. The resulting QTL were localized on the physical rice genome map and co-localization with previously reported QTL was analyzed to identify consensus regions. Subsequently, contrasting lines were subjected to physiological investigations to identify mechanisms of Fe exclusion, as well and Fe tissue (shoot) tolerance. The ability to diffuse oxygen into the rhizosphere was proposed as a Fe exclusion mechanism, and was facilitated by morphological traits such as large numbers of lateral fine roots. Shoot tolerance was examined in a series of transcriptomic and biochemical experiments, which suggested that low ascorbate turnover was associated with Fe tolerance. This was explained with a pro-oxidant activity of ascorbate in the presence of high Fe levels, which stimulated the production of reactive oxygen species via the ‘Fenton reaction’. In addition to these specific subprojects, a series of genome-wide association studies was conducted to identify loci associated with tolerance to diverse stress conditions such as ozone, Fe toxicity, boron toxicity and manganese toxicity. Also, a transcriptome meta-analysis was conducted to identity common expression patterns of genes involved in redox homeostasis in multiple abiotic stress conditions affecting rice. Together, these results have advanced our understanding of oxidative stress response in rice and identified target genes and traits for the breeding of adapted varieties.
Publications
- (2013) A critical comparison of two high-throughput ascorbate analyses methods for plant samples. Plant Physiology and Biochemistry 70, 418-423
Ueda Y, Wu L, Frei M
(See online at https://doi.org/10.1016/j.plaphy.2013.06.006) - (2014) Ascorbate metabolism in rice genotypes differing in zinc efficiency. Planta 239, 367-379
Höller S, Hajirezaei M, von Wirén N, Frei M
(See online at https://doi.org/10.1007/s00425-013-1978-x) - (2014) Genetic and physiological analysis of tolerance to acute iron toxicity in rice. Rice, 7(1):8
Wu LB, Shhadi MY, Gregorio G, Matthus E, Becker M, Frei M
(See online at https://doi.org/10.1186/s12284-014-0008-3) - (2014) Pyramiding of ozone tolerance QTL OzT8 and OzT9 confers enhanced tolerance to season-long ozone exposure in rice. Environmental and Experimental Botany 104, 26-33
Wang Y, Yang L, Höller M, Zaisheng S, Pariasca-Tanaka J, Wissuwa M, Frei M
(See online at https://doi.org/10.1016/j.envexpbot.2014.03.005) - (2014) Zinc deficiency differentially affects redox homeostasis of rice genotypes contrasting in ascorbate level. Journal of Plant Physiology 171, 1748-1756
Höller S, Meyer A, Frei M
(See online at https://doi.org/10.1016/j.jplph.2014.08.012) - (2015) A novel gene OZONE RESPONSIVE APOPLASTIC PROTEIN1 enhances cell death in ozone stress in rice (Oryza sativa L.). Plant Physiology 169, 873-889
Ueda Y, Siddique S, Frei M
(See online at https://doi.org/10.1104/pp.15.00956) - (2015) Ascorbate biosynthesis and its involvement in abiotic stress tolerance and plant development in rice (Oryza sativa L.). Plant Molecular Biology 88(6), 545-560
Höller S, Ueda Y, Wu LB, Wang Y, Hajirezaei MR, Ghaffari MR, von Wirén N, Frei M
(See online at https://doi.org/10.1007/s11103-015-0341-y) - (2015) Genetic dissection of ozone tolerance in rice (Oryza sativa L.) by a genome-wide association study. Journal of Experimental Botany 66, 293-306
Ueda Y, Frimpong F, Qi Y, Matthus E, Wu LB, Höller S, Kraska T, Frei M
(See online at https://doi.org/10.1093/jxb/eru419) - (2015) Loci, genes and mechanisms associated with tolerance to ferrous iron toxicity in rice (Oryza sativa L.). Theoretical and Applied Genetics 128, 2085-2098
Matthus E, Wu LB, Ueda Y, Höller S, Becker M, Frei M
(See online at https://doi.org/10.1007/s00122-015-2569-y) - (2016) Microarray meta-analysis focused on the response of genes involved in redox homeostasis to diverse abiotic stresses in rice. Frontiers in Plant Science 6:1260
De Abreu Neto J, Frei M
(See online at https://doi.org/10.3389/fpls.2015.01260) - (2016) Physiological and genotype specific factors associated with grain quality changes in rice exposed to high ozone. Environmental Pollution 210, 397-408
Jing LQ, Dombinov V, Shen SB, Wu YZ, Yang LX, Wang YX, Frei M
(See online at https://doi.org/10.1016/j.envpol.2016.01.023) - (2016) Shoot tolerance mechanisms to iron toxicity in rice (Oryza sativa L.). Plant Cell & Environment
Wu LB, Ueda Y, Lai SK, Frei M
(See online at https://doi.org/10.1111/pce.12733)
