Keisuke Nagai

The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water.

Living organisms must acquire new biological functions to adapt to changing and hostile environments. Deepwater rice has evolved and adapted to flooding by acquiring the ability to significantly elongate its internodes, which have hollow structures and function as snorkels to allow gas exchange with the atmosphere, and thus prevent drowning. Many physiological studies have shown that the phytohormones ethylene, gibberellin and abscisic acid are involved in this response, but the gene(s) responsible for this trait has not been identified. Here we show the molecular mechanism of deepwater response through the identification of the genes SNORKEL1 and SNORKEL2, which trigger deepwater response by encoding ethylene response factors involved in ethylene signalling. Under deepwater conditions, ethylene accumulates in the plant and induces expression of these two genes. The products of SNORKEL1 and SNORKEL2 then trigger remarkable internode elongation via gibberellin. We also demonstrate that the introduction of three quantitative trait loci from deepwater rice into non-deepwater rice enabled the latter to become deepwater rice. This discovery will contribute to rice breeding in lowland areas that are frequently flooded during the rainy season.

Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice

Significance Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) in rice encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding. Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1’s allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Stunt or elongate? Two opposite strategies by which rice adapts to floods

Expansion of habitat is important for the perpetuation of species. In particular, plants which are sedentary must evolve specialized functions to adapt itself to new environment. Deepwater rice is cultivated mainly in the lowland areas of South and Southeast Asia that are flooded during the rainy season. The internodes of deepwater rice elongates in response to increasing water level to keep its leaves above the water surface and avoid anoxia. This elongation is stimulated by ethylene-regulated genes, Snorkel1 and Snorkel2. In contrast, when a flash flood occurs at the seedling stage, submergence-tolerant rice, which carries Submergence-1A, remains stunted and survives in water for a few weeks to avoid the energy consumption associated with plant elongation, and restarts its growth using its conserved energy after the water recedes. Interestingly, both Snorkel genes and Submergence-1A encode ethylene-responsive factor-type transcription factor and are connected to gibberellin biosynthesis or signal transduction. However, deepwater and submergence-tolerant rice seem to have opposite flooding response; namely, escape by elongation or remain stunted under water until flood recedes.

Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice

Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice. Deepwater rice obtained the ability for rapid internode elongation to avoid drowning and adapt to flooded condition. How does it regulate internode elongation? Using both physiological and genetic approach, this paper shows that the plant hormone, gibberellin (GA) regulates internode elongation.

Two novel QTLs regulate internode elongation in deepwater rice during the early vegetative stage.

Deepwater rice possesses internode elongation ability to avoid drowning under deepwater conditions. Previous studies identified three QTLs regulating internode elongation ability on chromosomes 1, 3 and 12 using different populations. However, these QTLs only induce internode elongation in response to deepwater conditions from the 7-leaf stage and not during the early leaf stage. In this study, we detected two novel QTLs, qTIL2 and qTIL4 regulating deepwater response at the early leaf stage using an F2 population derived from the cross between NIL1-3-12 carrying the three QTLs regulating deepwater response in T65 (O. sativa ssp. japonica) genetic background and C9285 (O. sativa ssp. indica, deepwater rice). Plants of the BC2F2 population derived from NIL1-3-12/C9285 and the RILs of T65/Bhadua (O. sativa ssp. indica, deepwater rice) possessing these QTLs as well as the three QTLs previously identified also showed internode elongation during the early leaf stage. These results indicate that qTIL2 and qTIL4 regulate early internode elongation and function in coordination with the three major QTLs under deepwater conditions. The results presented here would not only help define the mechanism of deepwater response in rice but also contribute in the breeding of deepwater tolerant rice that is adapted to various water depths.

Loss of function at RAE2, a previously unidentified EPFL, is required for awnlessness in cultivated Asian rice.

Significance This study investigates a previously unidentified cysteine-rich peptide (CRP). CRPs have diverse roles in plant growth and development, such as control of stomata density and guidance of pollen-tube elongation. Despite numerous studies on CRPs in Arabidopsis thaliana, there are still many peptides with unknown function. We identify a previously unidentified rice CRP named Regulator of Awn Elongation 2 (RAE2) and show that it is cleaved specifically in the spikelet to promote awn elongation. We demonstrate that RAE2 was a target of selection during domestication, contributing to loss of awns in Asian but not African rice. The discovery of RAE2 simultaneously deepens our understanding of plant developmental pathways and lends insight into the complex processes underlying cereal domestication. Domestication of crops based on artificial selection has contributed numerous beneficial traits for agriculture. Wild characteristics such as red pericarp and seed shattering were lost in both Asian (Oryza sativa) and African (Oryza glaberrima) cultivated rice species as a result of human selection on common genes. Awnedness, in contrast, is a trait that has been lost in both cultivated species due to selection on different sets of genes. In a previous report, we revealed that at least three loci regulate awn development in rice; however, the molecular mechanism underlying awnlessness remains unknown. Here we isolate and characterize a previously unidentified EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family member named REGULATOR OF AWN ELONGATION 2 (RAE2) and identify one of its requisite processing enzymes, SUBTILISIN-LIKE PROTEASE 1 (SLP1). The RAE2 precursor is specifically cleaved by SLP1 in the rice spikelet, where the mature RAE2 peptide subsequently induces awn elongation. Analysis of RAE2 sequence diversity identified a highly variable GC-rich region harboring multiple independent mutations underlying protein-length variation that disrupt the function of the RAE2 protein and condition the awnless phenotype in Asian rice. Cultivated African rice, on the other hand, retained the functional RAE2 allele despite its awnless phenotype. Our findings illuminate the molecular function of RAE2 in awn development and shed light on the independent domestication histories of Asian and African cultivated rice.

QTL analysis of internode elongation in response to gibberellin in deepwater rice

Gibberellin (GA) is one of the plant hormones which regulates many aspects of plant growth and developmental processes. Rice plants known as deepwater rice can survive during flooding by elongating its internodes to avoid anoxia. Previous studies reported that GA is essential for internode elongation in deepwater rice. However, the interaction between internode elongation and regulator of GA sensitivity is unknown. In this study, we performed a QTL analysis and identified the chromosomal regions that regulate GA responsiveness in deepwater rice. We concluded that deepwater rice could induce internode elongation in response to GA by factors in these regions.

Construction of a versatile SNP array for pyramiding useful genes of rice.

DNA marker-assisted selection (MAS) has become an indispensable component of breeding. Single nucleotide polymorphisms (SNP) are the most frequent polymorphism in the rice genome. However, SNP markers are not readily employed in MAS because of limitations in genotyping platforms. Here the authors report a Golden Gate SNP array that targets specific genes controlling yield-related traits and biotic stress resistance in rice. As a first step, the SNP genotypes were surveyed in 31 parental varieties using the Affymetrix Rice 44K SNP microarray. The haplotype information for 16 target genes was then converted to the Golden Gate platform with 143-plex markers. Haplotypes for the 14 useful allele are unique and can discriminate among all other varieties. The genotyping consistency between the Affymetrix microarray and the Golden Gate array was 92.8%, and the accuracy of the Golden Gate array was confirmed in 3 F2 segregating populations. The concept of the haplotype-based selection by using the constructed SNP array was proofed.

Convergent Loss of Awn in Two Cultivated Rice Species Oryza sativa and Oryza glaberrima Is Caused by Mutations in Different Loci.

A long awn is one of the distinct morphological features of wild rice species. This organ is thought to aid in seed dispersal and prevent predation by animals. Most cultivated varieties of Oryza sativa and Oryza glaberrima, however, have lost the ability to form long awns. The causal genetic factors responsible for the loss of awn in these two rice species remain largely unknown. Here, we evaluated three sets of chromosome segment substitution lines (CSSLs) in a common O. sativa genetic background (cv. Koshihikari) that harbor genomic fragments from Oryza nivara, Oryza rufipogon, and Oryza glaberrima donors. Phenotypic analyses of these libraries revealed the existence of three genes, Regulator of Awn Elongation 1 (RAE1), RAE2, and RAE3, involved in the loss of long awns in cultivated rice. Donor segments at two of these genes, RAE1 and RAE2, induced long awn formation in the CSSLs whereas an O. sativa segment at RAE3 induced long awn formation in O. glaberrima. These results suggest that the two cultivated rice species, O. sativa and O. glaberrima, have taken independent paths to become awnless.

Development of chromosome segment substitution lines harboring Oryza nivara genomic segments in Koshihikari and evaluation of yield-related traits

Chromosome segment substitution lines (CSSLs) are rich genetic resources that can be mined for novel, agriculturally useful loci or that can be used directly as materials for breeding. To date, a number of rice CSSLs have been developed by crossing rice cultivars with its wild relatives as a means to tap into the potential of wild alleles in rice improvement. Oryza nivara is a wild relative of rice that is thought to be a progenitor of O. sativa spp. indica. In the present study, 26 CSSLs that covers the entire genome of O. nivara as contiguous, overlapping segments in the genomic background of a japonica cultivar, O. sativa cv. Koshihikari were developed. Evaluation of the CSSLs for several agriculturally important traits identified candidate chromosome segments that harbors QTLs associated with yield and yield-related traits. The results of the study revealed the potential of O. nivara as a source of novel alleles that can be used to improve the existing japonica cultivar.