Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L.

The wide natural variation present in rice is an important source of genes to facilitate stress tolerance breeding. However, identification of candidate genes from RNA-Seq studies is hampered by the lack of high-quality genome assemblies for the most stress tolerant cultivars. A more targeted solution is the reconstruction of transcriptomes to provide templates to map RNA-seq reads. Here, we sequenced transcriptomes of ten rice cultivars of three subspecies on the PacBio Sequel platform. RNA was isolated from different organs of plants grown under control and abiotic stress conditions in different environments. Reconstructed de novo reference transcriptomes resulted in 37,500 to 54,600 plant-specific high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g., for protein completeness (BUSCO). About 40% of all identified transcripts were novel isoforms compared to the Nipponbare reference transcriptome. For the drought/heat tolerant aus cultivar N22, 56 differentially expressed genes in developing seeds were identified at combined heat and drought in the field. The newly generated rice transcriptomes are useful to identify candidate genes for stress tolerance breeding not present in the reference transcriptomes/genomes. In addition, our approach provides a cost-effective alternative to genome sequencing for identification of candidate genes in highly stress tolerant genotypes.

Flood resilience loci SUBMERGENCE 1 and ANAEROBIC GERMINATION 1 interact in seedlings established underwater.

Crops with resilience to multiple climatic stresses are essential for increased yield stability. Here, we evaluate the interaction between two loci associated with flooding survival in rice (Oryza sativa L.). ANAEROBIC GERMINATION 1 (AG1), encoding trehalose 6-phosphate phosphatase 7 (TPP7), promotes mobilization of endosperm reserves to enhance the elongation of a hollow coleoptile in seeds that are seeded directly into shallow paddies. SUBMERGENCE 1 (SUB1), encoding the ethylene-responsive transcription factor SUB1A-1, confers tolerance to complete submergence by dampening carbohydrate catabolism, to enhance recovery upon desubmergence. Interactions between AG1/TPP7 and SUB1/SUB1A-1 were investigated under three flooding scenarios using four near-isogenic lines by surveying growth and survival. Pyramiding of the two loci does not negatively affect anaerobic germination or vegetative-stage submergence tolerance. However, the pyramided AG1 SUB1 genotype displays reduced survival when seeds are planted underwater and maintained under submergence for 16 d. To better understand the roles of TPP7 and SUB1A-1 and their interaction, temporal changes in carbohydrates and shoot transcriptomes were monitored in the four genotypes varying at the two loci at four developmental timeponts, from day 2 after seeding through day 14 of complete submergence. TPP7 enhances early coleoptile elongation, whereas SUB1A-1 promotes precocious photoautotrophy and then restricts underwater elongation. By contrast, pyramiding of the AG1 and SUB1 slows elongation growth, the transition to photoautotrophy, and survival. mRNA-sequencing highlights time-dependent and genotype-specific regulation of mRNAs associated with DNA repair, cell cycle, chromatin modification, plastid biogenesis, carbohydrate catabolism and transport, elongation growth, and other processes. These results suggest that interactions between AG1/TPP7 and SUB1/SUB1A-1 could impact seedling establishment if paddy depth is not effectively managed after direct seeding.

A trehalose-6-phosphate phosphatase enhances anaerobic germination tolerance in rice.

Global socioeconomic developments create strong incentives for farmers to shift from transplanted to direct-seeded rice (DSR) as a means of intensification and economization(1). Rice production must increase to ensure food security(2) and the bulk of this increase will have to be achieved through intensification of cultivation, because expansion of cultivated areas is reaching sustainable limits(3). Anaerobic germination tolerance, which enables uniform germination and seedling establishment under submergence(4), is a key trait for the development of tropical DSR varieties(5,6). Here, we identify a trehalose-6-phosphate phosphatase gene, OsTPP7, as the genetic determinant in qAG-9-2, a major quantitative trait locus (QTL) for anaerobic germination tolerance(7). OsTPP7 is involved in trehalose-6-phosphate (T6P) metabolism, central to an energy sensor that determines anabolism or catabolism depending on local sucrose availability(8,9). OsTPP7 activity may increase sink strength in proliferating heterotrophic tissues by indicating low sugar availability through increased T6P turnover, thus enhancing starch mobilization to drive growth kinetics of the germinating embryo and elongating coleoptile, which consequently enhances anaerobic germination tolerance.