Progress 08/01/03 to 07/31/06
Outputs
We mapped & ident. molecular Sub1 locus on chromosome 9S. Sub1 reg. was delineated by recomb. and mol. markers to be ~200 kb. A single gene in this interval was identified as Sub1 gene candidate. We confirmed the presence of 3 ethylene responsive factor-domain containing genes designated Sub1A, 1B & 1C. The corresponding reg. of the publicly available Nipponbare (japonica) genome spans 225 kb & is rearranged, relative to the indica genome. The ERF gene Sub1A is absent from this region in Nipponbare. Sub1A & 1C mRNAs strongly but transiently under submergence and are reduced upon de-submergence in seedling leaves. A test of Sub1 haplotype in 17 indica & 4 japonica varieties identified two Sub1A, 5 1B & 8 1C alleles on the basis of variation in amino acid sequence. Submergence tolerance was correlated with presence of submergence-induced Sub1A-1, & intolerance was assoc.with Sub1A-2 or complete absence of this gene. To validate the role of Sub1A-1, an intolerant japonica
variety Liaogeng was transformed with Sub1A-1 full-length cDNA under the control of the maize ubiquitin1 promoter. Indep. transgenic lines showed a correlation between the presence of the transgene and submergence tolerance. Lines overexpressing Sub1A-1 also showed increased constitutive & inducible alcohol dehydrogenase1 mRNA accumulation. Strong expression of Sub1A-1 confers submergence tolerance to japonica rice. Elucidated mechanisms controlled by the Sub1 haplotype using rice NILs: M202, a submergence intolerant japonica inbred grown in CA & M202(Sub1), a descendent of a mapping population developed by hybridization of a tolerant derivative of the indica line FR13A & M202.In submerging, the tolerant M202(Sub1) displayed restrained leaf & internode elongation, chlorophyll degradation & carbohydrate consumption, but the enzymatic activities of pyruvate decarboxylase & alcohol dehydrogenase were elevated, as compared to the intolerant M202. The transcript levels of genes associated
with carbohydrate consumption, ethanolic fermentation & cell expansion were regulated in the 2 lines. Sub1A & 1C transcript levels were shown to be up-regulated by submergence & ethylene, with the Sub1C allele in M202 also up-regulated by treatment with gibberellic acid (GA). This demonstrated that Sub1 region haplotype determines ethylene-& GA-mediated cellular & develop. responses to submergence thru differential expression of Sub1A & 1C. Submergence tolerance in lowland rice is conferred by a specific allele variant of Sub1A that dampens ethylene production and GA responsiveness causing a growth quiescence in growth correlated with the capacity for re-growth upon de-submergence. The project evaluated the role of Rop signal transduction in response to submergence/oxygen deprivation in the NILs. Rop-GTP levels, H2O2 accumulation & regulation of transcript accumulation of RopGAP (GTPase activating protein) genes in response to submergence were tested. We observed Sub1
genotype-specific increases in H2O2 & transcripts that encode 3 of the 7 RopGAP genes, temporal accumulation of ROP-GTP was not markedly distinct in the 2 lines. Rop signaling is most likely upstream of Sub1 or in a distinct pathway.
Impacts
Flooding inundates approximately 20% of lowland rice paddies throughout the world each year, leading to significant yield losses in Asia and the US. Consequentially, a major goal of rice breeders is to generate cultivars that can withstand 14 d or more of submergence at the seedling stage. In the US, farmers typically plant rice by aerial broadcast of pre-germinated rice seeds into shallow paddies. This pre-germination method requires additional seed use to ensure good stand establishment, however the shallowness of paddies does not adequately discourage weeds. The development of cultivars that can be dry-seeded into flooded fields will significantly reduce the levels of herbicide applied at the pre- and post-emergence stages. This project defined the molecular basis of submergence tolerance by (a) mapping and confirming the Submergence1 locus and (b) elucidating the role of the Sub1 locus in the acclimation response to this stress. The major finding of the completed
project is that the Sub1 locus is comprised of a variable cluster of related genes that encode putative transcription factors. A specific gene in this cluster, Sub1A-1, was shown to be necessary and sufficient to increase submergence tolerance in transgenic rice. This project has yielded the information for production of molecular markers that facilitate the rapid and efficient breeding of submergence tolerant rice lines. This project has greatly increased the understanding of the molecular, physiological and hormonal basis of submergence responses in plants.
Publications
- Fukao, T. and Bailey-Serres, J. (2004) Hypoxia responses in plants: A balancing act. Trends in Plant Sciences. 9(9):449-456.
- Bailey-Serres, J., and Chang, R. (2005) Sensing and signalling in response to oxygen deprivation in plant cells and other organisms. Annals of Botany. 96:507-518.
- Xu, K., Xu, K., Fukao, T., Canalas, P., Maghirang-Rodriguez, R., Heuer, S., Ismail, A., Bailey-Serres, J., Ronald, PC, Mackill, D.J. (2006) Sub1A is an ethylene responsive-factor-like gene that confers submergence tolerance to rice. Nature, 442:705-708.
- Fukao, T., Xu, K, Ronald, P.C. and Bailey-Serres, J. (2006) A variable cluster of ethylene responsive-like factors (ERFs) regulates developmental and metabolic acclimation responses to submergence. Plant Cell. 18(8):2021-2034.
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Progress 01/01/04 to 12/31/04
Outputs
There is genetic variation in submergence tolerance in indica and japonica rice (ORYZA SATIVA). Submergence tolerance is primarily controlled by the SUB1 locus on rice chromosome 9 in the indica rice line FR13A. We mapped SUB1 in a 0.075 cM interval of 150 kb between two markers SSR1A and AFLP209rf where recombination was highly suppressed (2,000 kb/cM). Positional cloning and sequence analysis revealed an open reading frame encoding a putative ethylene responsive factor (ERF) designated GN15 which was rapidly induced in the shoots of the tolerant parent within 1 h of submergence and remained detectable at a moderate and consistent level from 3 h to 11 d. Upon de-submergence, GN15 mRNA gradually decreased, but was still detectable up to 2 of 9 d into the recovery stage. Transcripts were not detectable in the submerged intolerant parent (M-202). GN15 shares 50% amino acid identity with two other ERF genes (GenBank accessions: BAD29170 and BAD29167) in the corresponding
region of chromosome 9 in the japonica cultivar Nipponbare. Over 200 transgenic lines expressing GN15 genomic clone and overexpressing the GN15 cDNA are being evaluated. M-202 with SUB1 introgressed from FR13A (tolerant) and M-202 inbred (intolerant) were the subject of biochemical and molecular analyses of seedlings following oxygen deprivation and two-week old plants following up to 14 days of submergence. Our studies with Arabidopsis (ARABIDOPSIS THALIANA) have implicated the transient activation of a monomeric GTPase, termed ROP, regulates cellular changes in gene expression in response to oxygen deprivation. We hypothesized that ROP signal transduction controls the consumption of carbohydrates and ethanolic fermentation in rice. To test this hypothesis we compared the response physiology and signaling mechanisms in tolerant (SUB1 in M-202) and intolerant (M-202) lines. We found genotypic distinctions in, (1) the reduction in levels of soluble carbohydrate and starch during
submergence; (2) the regulation of the timing and duration of increases in mRNAs that encode proteins involved in carbohydrate consumption and anaerobic metabolism; (3) the regulation of the timing and duration of accumulation of mRNAs that encode components of ROP signaling, including the seven Rop GTPase activating protein (ROPGAP) genes. The biochemical studies are underway to further evaluate the regulation of active ROP (ROP-GTP) in the two lines in response to submergence.
Impacts
Flooding and poor soil drainage can cause severe reductions in yield of rice and other crops. We have made considerable progress in the examination of the hypothesis is that timing and amplitude of Rop signal transduction is critical to survival of flooding. Preliminary evidence indicates that the transformation of a submergence tolerant line with SUB1 from indica rice confers increased tolerance of flooding to young plants. This research should reveal whether manipulation of SUB1 or ROPGAP gene regulation can be used to enhance submergence tolerance in crops.
Publications
- Fukao, T. and Bailey-Serres, J. 2004. Hypoxia responses in plants. A balancing act? Trends in Plant Sciences. 9(9):449-456.
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