Abstract
Contemporary climatic stress seriously affects rice production. Unfortunately, long-term domestication and improvement modified the phytohormones network to achieve the production needs of cultivated rice, thus leading to a decrease in adaptation. Here, we identify a 14-3-3 protein-coding gene OsGF14h in weedy rice that confers anaerobic germination and anaerobic seedling development tolerance. OsGF14h acts as a signal switch to balance ABA signaling and GA biosynthesis by interacting with the transcription factors OsHOX3 and OsVP1, thereby boosting the seeding rate from 13.5% to 60.5% for anaerobic sensitive variety under flooded direct-seeded conditions. Meanwhile, OsGF14h co-inheritance with the Rc (red pericarp gene) promotes divergence between temperate japonica cultivated rice and temperate japonica weedy rice through artificial and natural selection. Our study retrieves a superior allele that has been lost during modern japonica rice improvement and provides a fine-tuning tool to improve flood adaptation for elite rice varieties.
Similar content being viewed by others
Introduction
As current climatic stress limits yield increasingly, more consistent crop production must be achieved1,2. A total inundation area of 2.23 million square kilometers, with 255 to 290 million people directly affected by floods in 913 large flood events worldwide from 2000 to 20183, and agricultural production has also been severely damaged. Direct seeding is a vital cultivation method for rice that is increasingly employed in both rainfed and irrigated fields due to its advantages of reduced labor, energy, water use, production costs, and mechanization4,5,6. Heavy rainfall and floods seriously affect crop production, especially germination and seedling development of direct-seeded rice (DSR), which requires that varieties exhibit uniform and rapid germination to establish seedlings in anaerobic conditions caused by flooding. However, the intensive domestication and improvement have led to a decrease in flood adaptation and poor capacity for anaerobic germination (AG)/anaerobic seedling development (ASD)7 to achieve the production needs of cultivated rice1,8,9. Capturing natural genetic variation from germplasm resources and deciphering the mechanism(s) regulating AG/ASD is an opportunity to improve the status quo.
The SUB1A, which encodes an ethylene-responsive factor-type transcription factor (ERF), confers submergence tolerance to rice by limiting gibberellin (GA) response, thereby inhibiting plant elongation and sustaining seedling vigor for up to two weeks under submerged conditions10,11. By contrast, deepwater rice, with the SNORKEL 1 and SNORKEL 2 genes encode SUB1A-like transcription factors, can invest photo-assimilate into the extension of submerged stem internodes by GA biosynthesis12. In addition, under continuous flooding, the transcript accumulation of SD1, known as the ‘green revolution gene’, induced by ethylene signaling, activates GA synthesis and internode growth of stems, allowing deepwater rice to survive13. OsGA20ox1, a paralog of SD1, also has positive functions on seedling vigor in the initial growth of rice14.
Seed germination requires coordination of embryonic development and environmental conditions, in which the balance of two competing hormone signaling pathways, abscisic acid and gibberellin, plays a decisive role15. Although seedling-stage submergence tolerance and avoidance mechanisms ensure rice survival, the mechanism(s) for ensuring uniform and rapid seed germination under flooded-anaerobic conditions remains explored. A trehalose-6-phosphate phosphatase gene, OsTPP7 can enhance starch mobilization to drive growth kinetics of the germinating embryo and elongating coleoptile5. Another AG gene, OsCBL10, acts as a negative regulator of the CIPK15-dependent low-oxygen signaling pathway to enhance total α-amylase activity16. Screening germplasms with strong AG/ASD tolerance and isolating more functional genes are required for DSR development. They will contribute to the understanding of the molecular mechanism(s) underlying the flood adaptation in rice.
Weedy rice (Oryza sativa f. spontanea) competes aggressively with cultivated rice as an invasive paddy weed. It reduces harvests, which is also reputed to be a ‘hidden gold mine’ in global paddy fields due to the substantial stress resistance and the nutritional quality different from cultivated rice17,18,19,20,21. In addition, the origin and evolution of weedy rice have been extensively studied based on population genomic investigations in recent years18,22,23. Here, we identify a 14-3-3 protein-coding gene OsGF14h in weedy rice, which acts as a signal switch to inhibit ABA signaling through interacting with the transcription factors OsHOX3 and OsVP1 and enhance GA synthesis, thereby improving AG and ASD. OsGF14h can improve flood adaptation for elite rice varieties as a fine-tuning tool. We further reveal the critical role of OsGF14h in rice genetic improvement and ecotype differentiation, whereby providing an empirical example of how the genetic network of phytohormones responds to different selection pressures.
Results
OsGF14h isolated from weedy rice is associated with flood adaptation
After years of investigation, we found that weedy rice spontaneously appeared in paddy fields after cultivated rice was transplanted in Northeast China. Therefore, weedy rice can be regarded as a natural DSR due to its AG tolerance and successful seedling establishment under anaerobic conditions. To isolate essential AG tolerance genes in weedy rice, we first used recombinant inbred lines (RILs) population with 168 lines (generation F8) that derived from AG tolerant weedy rice WR04-6 and AG susceptible cultivated rice Qishanzhan to perform QTL mapping analysis. This population has been effectively used to study the genetic basis of agronomic traits in weedy rice (Supplementary Data 1)22. Based on the inclusive composite interval mapping (ICIM) method with a LOD threshold of 2.5, we detected two QTLs. The main QTL was mapped in the physical range of chromosome 11 from 23,790,182 to 23,859,119 in the japonica reference genome (Os-Nipponbare-Reference-IRGSP-1.0) with a LOD value of 5.65 and phenotypic variance explained (PVE) of 15.85 (Fig. 1a).
We also constructed a genome-wide association study (GWAS) panel including 36 temperate japonica weedy rice (GJ-tmp-weedy), 57 temperate japonica landrace (GJ-tmp-land), and 97 temperate japonica cultivar (GJ-tmp-cul) to detect AG associated QTNs (quantitative trait nucleotides) (Supplementary Data 2). The mean germination rate of the accessions in the GWAS panel was in the order of weedy rice (40.05%) > landrace (31.48%) > cultivated rice (26.08%) after 72 h of anaerobic incubation (Fig. 1c). The GWAS based on the linear mixed model with the first two principal components from principal components analysis (PCA) as covariates (Fig. 1d) showed that the strongest associated signals were also detected on chromosome 11 with the ideal distribution of observed P value based on QQ plot analysis (Fig. 1e); the physical position of QTN peaks occurs at 23,556,546 bp (Fig. 1b) with the −log10 (P) value of 8.279. Considering the mapping results of QTL and GWAS, we speculate that genes in this genomic region contribute to the AG tolerance of weedy rice. Then we plotted the linkage disequilibrium (LD) block around the target QTN peak (Fig. 1f) and preselected causal haplotypes of candidate genes that were annotated by MSU Rice Genome Annotation Project Release 7 within the LD block. (Supplementary Fig. 1a–g). Finally, the candidate genes were narrowed down to seven within a 584 kb genomic region, LOC_Os11g39370, LOC_Os11g39450, LOC_Os11g39500, LOC_Os11g39520, LOC_Os11g39530, LOC_Os11g39780, and LOC_Os11g39540.
LOC_Os11g39540 and LOC_Os11g39370 showed the highest expression level among the candidate genes under anaerobic stress (Supplementary Fig. 1h), whereas LOC_Os11g39370 is mainly expressed in leaves and LOC_Os11g39540 is mainly expressed in seed based on two Spatio-temporal expression databases (Supplementary Fig. 2). Thus LOC_Os11g39540 is considered to be the final candidate gene by considering the gene expression model and annotation (Supplementary Data 3). LOC_Os11g39540 gene encodes a 14-3-3 protein and is named OsGF14h, which is the eighth gene in its family based on gene annotation (http://rice.uga.edu/). Comparing cDNA sequences between an AG tolerant weedy rice (WR04-6) and a super-high-yield but AG-sensitive japonica variety (Shennong9816, SN9816) showed that the coding region of OsGF14h in SN9816 was consistent with the Nipponbare (IRGSP-1.0). SN9816 has a total of six polymorphic sites in the coding region compared with WR04-6: one deletion of four bases at the fourth exon of SN9816 causes frameshift mutations and stops amino acid translation; two non-synonymous substitution SNPs appear before the 4 bp deletion; one non-synonymous substitution SNP appears after the 4 bp deletion; and two synonymous substitution SNPs, which results in an incomplete 14-3-3 protein (Fig. 1g). Therefore, we predicted that this natural variation of OsGF14h may have a genetic effect on AG tolerance. The best linear unbiased prediction (BLUP) value of each individual in the GWAS panel to show the corrected allelic effects with the correlation coefficient with the phenotype is 0.603 (Fig. 1c), and the difference in BLUP value between the two groups classified based on 4 bp deletion is more obvious (Fig. 1h).
OsGF14h promotes AG tolerance and ASD in rice
Based on annotations, OsGF14h may act as a chaperone that participates in protein modification and signal regulation (Supplementary Data 3). In WR04-6, the qPCR analysis indicated that OsGF14h transcripts were detected in all tissues and were highly expressed in the developing seeds and endosperm (Fig. 2a). The OsGF14hWR04-6::GFP fusion protein is localized in both the nucleus and cytoplasm (Fig. 2b).
To further study the role of OsGF14h on AG tolerance and ASD, we introduced the coding sequence (CDS) from OsGF14hWR04-6 into SN9816 (WTSN9816) under the control of the CaMV 35 S promoter, whereby two homozygous overexpression lines OxOsGF14hWR04-6-8 and OxOsGF14hWR04-6-25 were used in the present study. Meanwhile, another two overexpression lines of WTSN9816 with its own CDS overexpressed, OxOsGF14hSN9816-5 and OxOsGF14hSN9816-11, were created. We also used a CRISPR/Cas9 system to specifically disrupt the OsGF14h gene in WR04-6 (WTWR04-6) (Supplementary Fig. 12a), whereby two mutants (gf14h-13 and gf14h-15) with other overexpression lines were used to the comparison of genetic effects in the present study. Compared with WR04-6, the two mutants (gf14h-13 and gf14h-15) showed reduced coleoptile elongation and seedling size under anaerobic conditions, whereas the overexpression lines (OxOsGF14hWR04-6-8 and OxOsGF14hWR04-6-25) showed significantly enhanced AG ability and seedling vigor as compared with WTSN9816 (Fig. 2c–e), however, the other two overexpression lines (OxOsGF14hSN9816-5 and OxOsGF14hSN9816-11) showed no significant difference from of WTSN9816 (Fig. 2d). These results indicated that OsGF14h was the target gene promoting AG tolerance and ASD in rice. Additionally, we found that overexpression of OsGF14h boosted the seeding rate from 13.5 to 60.5% under the flooded direct-seeded condition in outdoor anaerobic facilities (Fig. 2f, g).
OsGF14h interacts with OsHOX3 and OsVP1 to inhibit the ABA signaling
To better understand the mechanism(s) of how OsGF14h improve AG, we performed yeast two-hybrid (Y2H) screening with pGBKT7-OsGF14h as bait. In total, 31 candidate proteins were identified to interact with OsGF14hWR04-6, including an HD-Zip family TF OsHOX3 (LOC_Os01g45570), which was reported as a transcription-repressor related to GA regulation24 and an ABA crucial transcription activator OsVP1 (LOC_Os01g68370)25. These two interactions can be verified by transient expression in the luciferase (LUC) complementation imaging assays, and we also confirmed that both OsGF14hWR04-6 and OsGF14hSN9816 can interact with OsHOX3 and OsVP1 in vivo and in vitro by using Y2H and pull-down assays respectively (Fig. 3a–c). Based on the prediction by AlphaFold226, OsGF14WR04-6 encodes a fully functional 14-3-3 protein that, with nine alpha-helix structures, could form a clamp to bind the N-terminal loop of OsHOX3. For the OsGF14SN9816, the frameshift mutation caused by the 4 bp deletion in the coding region led to a loss of one helix in the C-terminus, which may weaken the strength of its interaction with OsHOX3 (Supplementary Fig. 3a,b). The co-immunoprecipitation (co-IP) and Y2H assays further proved the strength of the two interactions between OsGF14hWR04-6 and OsHOX3 and between OsGF14hWR04-6 and OsVP1 was greater than that of OsGF14hSN9816 in vivo (Fig. 3a, d).
We also regenerated an overexpression construct (35 S: OsVP1-GFP) and transformed it into WTWR04-6, then two overexpression lines (OxVP1-3 and OxVP1-5) that were used together with the existing loss-of-function mutant of OsHOX3 (hox3) to verify their physiological functions in AG. The germination and coleoptile elongation of OxVP1 lines and hox3 were found to be blocked compared with their wild-type WTWR04-6 and WTkitaake respectively (Fig. 3e, f). Previous studies reported that OsVP1 could not bind ABA-responsive elements (ABREs) independently, instead of interacting with TRAB1 (a bZIP-TF that can interact with both OsVP1 and ABREs) to inhibit ABA response25. Significantly, 14-3-3 protein mediates the trans-regulation of the ABA-responsive factor OsEM1 by OsVP127. In the present study, the expression level of OsVP1 in the OsGF14h knockout line (gf14h-15) was significantly greater than that in WTWR04-6 during the initial stage and then significantly reduced at the 24th hour after it was flooded (Fig. 3g). Meanwhile, the TRAB1 expression also appears to be negatively regulated by the OsGF14hWR04-6 (Fig. 4a). The above evidence implies that a stronger interaction between OsGF14hWR04-6 and OsVP1 can inactivate the ABA response.
The favored recognition site of OsHOX3 was shown to be composed of two 5-bp half-sites that overlap at a central position, ‘CAAT(G/C)ATTG’28. Based on the prediction of cis-acting elements for the target genes of the OsHOX3 protein, we found that OsPYL5/RCAR5, an ABA receptor in the ABA signaling unit29, was one of the target genes of OsHOX3. Based on yeast one-hybrid (Y1H) assays, we found that OsHOX3 can specifically bind the HD-ZIP3 element in the OsPYL5 promoter region (Fig. 3h). Then, the ChIP-qPCR analysis revealed three enrichments of OsHOX3 in the OsPYL5 promoter segments at P2, P3, and P4 (Fig. 3i). The dual-luciferase (LUC) assay further showed that co-expression of 35 S: OsHOX3 and OsPYL5pro: LUC significantly reduced the LUC/REN ratio compared to the control (Fig. 3j). The OsPYL5 transcription level was significantly increased in the mutant hox3 compared to its wild-type (WTkitaake) (Fig. 3k). Meanwhile its expression also correspondingly exhibited an increase in knockout lines (gf14h-13 and gf14h-15) and a decrease in overexpression lines (OxOsGF14hWR04-6-8 and OxOsGF14hWR04-6-25) compared to their wild types (Fig. 3k). To further reveal whether OsGF14h is directly involved in OsHOX3 moduling OsPYL5, we performed electrophoretic mobility shift assays (EMSA). The result showed that the binding capacity of OsHOX3 to the OsPYL5 promoter was evidently enhanced when adding OsGF14hWR04-6 protein, indicating that OsGF14hWR04-6 contributes to the binding (Fig. 3l).
To further reveal the response of the ABA signaling pathway under the regulation of OsGF14h, in addition to OsTRAB1, we also detected the expression levels of two bZIP-TF genes that are related to ABA-regulation (OsOREB and OsbZIP72) and two typical ABA-responsive genes (OsEM1 and OsRab16A), between knockout lines (gf14h-13 and gf14h-15) and WTWR04-6, and between overexpression lines (OxOsGF14hWR04-6-8 and OxOsGF14hWR04-6-25) and WTSN9816 under anaerobic condition. The results showed that most of these genes were inducted by flooding at the 24 and 48 h (Supplementary Data 4) and the expressions were suppressed significantly by OsGF14h under anaerobic conditions at the 12 h (Fig. 4a). An assay of exogenous ABA with gradient concentrations demonstrated that the sensitivity of the knockout lines (gf14h-13 and gf14h-15) was higher than WTWR04-6 (Fig. 4b). The relative germination rate (germination rate under the treatment of exogenous 1 μM ABA divided by that of under normal conditions) further verified that the ABA sensitivity of two overexpression lines (OxOsGF14hWR04-6-8 and OxOsGF14hWR04-6-25) was higher compared with WTSN9816. In contrast, the other two overexpression lines (OxOsGF14hSN9816-5 and OxOsGF14hSN9816-11) also showed a consistent level of ABA sensitivity with the WTSN9816 (Fig. 4c). On the other hand, the content of endogenous ABA was not significantly different between knockout lines gf14h-15 and WTWR04-6 and between two kinds of overexpression lines OxOsGF14hSN9816-5 and OxOsGF14hWR04-6-8 (Supplementary Fig. 4). The above experimental evidence reveals an ABA signaling pathway in which OsGF14h acts as a signal switch, and OsGF14h interacts with OsHOX3 and OsVP1 to inhibit ABA response and improving flood adaptation.
OsGF14h enhances GA biosynthesis
RNA-seq for OxOsGF14hWR04-6-8 and OxOsGF14hSN9816-5 under both anaerobic and aerobic conditions were conducted for evaluated genome-wide effects of OsGF14h. The results showed that the number of differential expressed genes (DEGs) (OxOsGF14hWR04-6-8/OxOsGF14hSN9816-5 > 2 and FDR <0.01) between OxOsGF14hWR04-6-8 and OxOsGF14hSN9816-5 was much higher under anaerobic conditions (4183 DEGs) than under aerobic conditions (84 DEGs) (Supplementary Fig. 5a). Genes involved in many metabolic pathways that related to catalytic activity, binding, and transporter activity among these 4183 DEGs were enriched based on GO and KEGG analysis (Supplementary Fig. 5b, c). Genes involved in gibberellin biosynthesis are crucial for rice development, especially OsGA20ox1 play an important role in the initial growth stage14. We then focused on 40 GA biosynthesis genes of the anaerobic group and found that all six DEGs, including OsGA20ox1, among the 11 detectable positively regulated genes were upregulated, two DEGs among the five detectable positively regulated genes were upregulated, and the other nine genes were not differentially expressed (Fig. 4d). On the other hand, among 74 detectable genes of 94 ABA-responsive genes, 13 DEGs were downregulated, four DEGs were upregulated in OxOsGF14hWR04-6-8, and 57 genes were not differentially expressed (Supplementary Fig. 6). The expression of OsGA20ox1 in knockout lines (gf14h-13 and gf14h-15), decreased significantly at 12 h under anaerobic conditions compared with that in WTWR04-6. On the contrary, OsGA20ox1 in overexpression lines (OxOsGF14hWR04-6-8 and OxOsGF14hWR04-6-25) increased significantly compared with WTWR04-6 (Fig. 4e). We thus monitored the dynamic changes of four kinds of endogenous GAs (GA9, GA20, GA24, and GA53) that related to the OsGA20ox1 pathway during the germination of WTWR04-6 and gf14h-15 under anaerobic conditions. Compared with WT seeds, GAs’ contents in gf14h-15 seeds showed a decreasing trend from 0 to 24 h after anaerobic incubation (Fig. 4f). This implies that OsGF14hWR04-6 may promote the germination of weedy rice under anaerobic stress by enhancing GAs synthesis. To further verify the genetic function of OsGA20ox1 for anaerobic germination, we generated CRISPR-Cas9-based knockout lines of OsGA20ox1 (ga20ox1-1 and ga20ox1-3) in WTWR04-6 (Supplementary Fig. 12b) and found that the knockout lines showed significantly reduced coleoptile length compared with WTWR04-6 in anaerobic conditions (Fig. 4g). OsHOX3 has been reported as a positive regulator in GAs biosynthetic pathway in the seedling stage16, which has also been partially validated by qPCR during AG in this study. The results showed that the positive regulation of OsHOX3 on the GA biosynthesis was mainly reflected in the degradation pathway, in addition to OsGA20ox1 and KOS4 in the biosynthesis pathway (Supplementary Fig. 7), OsGF14h can enhance GAs biosynthesis via but not limited to OsHOX3 thereby acts as a role in the balance of ABA and GA during anaerobic germination.
Haplotype distribution and evolution of OsGF14h
To further reveal the haplotype distribution and evolution of OsGF14h in different rice ecotypes, we analyzed the OsGF14h haplotype frequency and network using sequence data of 1,596 accessions of Oryza rufipogon (Or-IIIa) and O. sativa from nine ecotypes with the O.bathii as an outgroup. These accessions mainly included widely planted temperate japonica (GJ-tmp-cul) and indica (XI-cul) cultivars from China30; tropical japonica (GJ-trp) and indica landrace (XI-land) from the 3010 O. sativa accessions (3 K panel)31; a collection of japonica type weedy rice (GJ-tmp-weedy) and indica type weedy rice (XI-weedy)19; and NGS sequencing of Or-IIIa in this study.
Ten haplotypes were identified based on 16 polymorphic sites of the CDS region with two frameshift mutations, six non-synonymous codons, and eight synonymous codons (Supplementary Data 5). Hap1 (OsGF14hSN9816) and Hap2 (OsGF14hWR04-6) were respectively defined as the partial loss-of-function and functional types based on experimental evidence in the present study. We further found that Hap2 had the highest frequency among all sampled wild rice, Or-IIIa (Supplementary Data 6), and was defined in the center of the haplotype network with all ten ecotypes (Fig. 5a); Hap2 was thus predicted as the functional-ancestral type of OsGF14h. Hap3, Hap4, Hap5, Hap6, and Hap8 can encode the full-length 14-3-3 protein and also be detected in Or-IIIa, which can be defined as the functional-ancestral type. The frameshift mutation of Hap9 caused by the 1 bp insertion led to the loss of four helix in the coding protein resulting in a complete loss of binding sites with OsHOX3. Thus, two frameshift types, Hap1 and Hap9, were defined as partial loss of function haplotypes, mainly detected in temperate japonica and indica subspecies separately.
There is only one step (the 4 bp deletion) to convert Hap6 to Hap1. However, according to the haplotype network, converting from any other Hap to Hap1 needs more mutational steps. As shown in Fig. 5b, Hap6 and Hap1 showed the closest divergence time based on a time-tree for the ten haplotypes of OsGF14h according to the mcmctree method in PAML software. Based on the above evidence, we infer that Hap1 was likely directly derived from Hap6 (Or-IIIa or GJ-trp). The emergence of Hap9 may be earlier than Hap1 based on the haplotype network and time-tree. Therefore, we infer that the two partial loss-of-function haplotypes, Hap6 and Hap9, originate independently in the two rice subspecies.
We focus on the evolutionary significance of OsGF14h in the japonica subspecies due to the main functional variation OsGF14hSN9816 occurred mainly in temperate japonica planting areas (Fig. 5c). Our results suggest that OsGF14h exhibited a significant differentiation signal between GJ-tmp-weedy and GJ-tmp-cul based on window-based Fst on chromosome 11 (Fig. 6a), which corresponds to the differentiation of haplotype frequency (Fig. 5c). The haplotype frequencies of OsGF14hSN9816 in three ecotypes of GJ-tmp-cul (GJ-tmp-late = 0.91, GJ-tmp-medium = 0.86, and GJ-tmp-early = 0.80) are higher than that of GJ-tmp-weedy (0.17). However, window-based Tajima’ D values (GJ-tmp-weedy and GJ-tmp-cul) and π ratios (πOr-IIIa / πGJ-tmp-weedy and πOr-IIIa / πGJ-tmp-cul) do not support the selection and domestication occurred in OsGF14h region in both GJ-tmp-weedy and GJ-tmp-cul (Fig. 6a). We further compared GJ-tmp-weedy with the three ecotypes of GJ-tmp-cul using a spectrum of the three parameters on chromosome 11 (Supplementary Figs. 8–10). The OsGF14h also exhibited population differentiation signals between GJ-tmp-weedy and GJ-tmp-late (Fig. 6b) and between GJ-tmp-weedy and GJ-tmp-medium (Fig. 6c) based on window-based Fst analysis. Both of these differentiation signals overlap the selection signals (window-based Tajima’ D) of OsGF14h in GJ-tmp-late and GJ-tmp-medium, respectively (Fig. 6b, c). Meanwhile, a selection sweep of OsGF14h also is detected in GJ-tmp-medium defined by window-based π ratio (πOr-IIIa /πGJ-tmp-medium) (Fig. 6c). Interestingly, the differentiation signal between GJ-tmp-weedy and GJ-tmp-early in OsGF14h genomic region becomes weaker, falling below the 5% significant level (Fig. 6d). Meanwhile, the other two parameters also reject this locus as a selection target (Fig. 6d). It is worth noting that GJ-tmp-early is mainly composed of landraces, whereas GJ-tmp-medium and GJ-tmp-late are modern cultivated varieties in temperate japonica planting areas30. Under this context, the OsGF14h exhibits a strong genetic divergence signal between weedy rice and modern cultivated rice but becomes weakening between weedy rice and landrace, implying that OsGF14hSN9816 plays a vital role in the genetic improvement of modern japonica rice, and the 4 bp deletion could be the major causality.
Discussion
The characterization of natural variation in AG genes and understanding their mechanisms are essential for the genetic improvement of DSR. At present, two genes with natural variation (OsTPP7 and OsCBL10) that act on α-amylase activity have been reported to be related to AG5,16. Nonetheless, the genetic mechanism of AG is still largely unknown. In the present study, we identified an ABA response suppressor gene OsGF14h that further broadens our understanding of factors underlying AG and the phytohormones regulatory network. The signaling pathway of ABA on germination and dormancy has been extensively studied in crops32,33, and the effect of ABA-GA antagonism on seed germination has also been thoroughly researched15,34,35. However, the underlying mechanism of seed germination under anaerobic conditions is not fully understood. On the other hand, 14-3-3 proteins are a fascinating and complex protein family that can interact with lots of proteins in both mammals and plants36,37. However, the consequences of these interactions are little known. Here we reveal insight into the mechanisms and functions for the coding gene of 14-3-3 protein, particularly decoding the two major natural variants of OsGF14h deeply. As shown in Fig. 7, we propose a working model to demonstrate how two natural variations of OsGF14h respond to flooding stress during germination. The 14-3-3 proteins are reported to be involved in both GA biosynthetic pathways and GA signaling38,39. OsHOX3 is reported as a positive regulator in GAs biosynthetic pathway in the seedling stage16, which function is further validated during AG by this study. These results indicate that OsGF14hWR04-6 could balance ABA signaling and GA biosynthesis during anaerobic germination. There may be multiple pathways for the positive regulation of GA by OsGF14h, which requires further in-depth study.
Seed dormancy 4 (Sdr4) and red pericarp gene Rc were reported as two essential seed dormancy genes with positive regulatory effects on ABA that can effectively inhibit pre-harvest sprouting (PHS)40,41,42. A recent study further demonstrates an additive effect of Sdr4 and Rc on seed dormancy43. In the present study, we also verified that Rc can reduce PHS by knocking out the Rc gene in WR04-6 (Supplementary Figs. 11a, b and 12c). The non-dormancy allele, sdr4-n, and white pericarp allele, rc, have been nearly fixed in GJ-tmp-cul by the artificial selection with the genetic improvement of japonica subspecies due to red pericarp and strong dormancy were unfavorable phenotypes (Supplementary Data 7). On the other hand, GJ-tmp-weedy requires AG and ASD that are modulated by OsGF14hWR04-6 to ensure population reproduction by natural selection. However, OsGF14hWR04-6 also increases the PHS risk due to the high expression in the filling and maturity stage due to it inhibiting the seed ABA response (Fig. 2a). In particular weedy rice matures early without environmental limits for the PHS. In the context that sdr4-n is also fixed in GJ-weedy (Supplementary Data 7), it is ingenious that Rc, as a signature gene of weedy rice, may be the perfect one to reduce the risk of OsGF14h-induced PHS because not expressed at germination and seedling stages (Supplementary Fig. 11c), and whereby not counteract the ability of AG and ASD that modulated by OsGF14hWR04-6. Thus, we infer that OsGF14h and Rc are co-inheritance and together contribute to the differentiation between GJ-cul-weedy and GJ-tmp-cul by manipulating the balance between ABA and GA under natural and artificial selection (Fig. 8).
The alleles of SUB1A, SK1, SK2, and SD1, identified from wild Oryza species, are crucial to flooding survival8, which indicates that they arose in ancestral populations in flooded ecosystems1. However, domestication has modified the phytohormones network to achieve the production needs for cultivated rice. The competition between cultivated rice and co-existing weedy rice revealed in the present study provides a practical example of how the genetic network of phytohormones responds to different selection pressures. De-domestication, semi-domestication, and outcrossing are the leading hypotheses for the origin of weedy rice, which have been proposed and debated for decades17,18,19,23,44. Regardless of how weedy rice originated, the consequence is the birth of new diversity. More importantly, exploring adaptive genes from weedy rice germplasm resources can retrieve superior alleles that were lost during domestication and modern breeding selection, contributing to crop genetic improvement.
Methods
Plant materials
A recombinant inbred line (RIL) population was created with 168 lines derived from the cross between weedy rice WR04-6 and AG-sensitive cultivated rice Qishanzhan at Shenyang Agricultural University, China. All RILs and the two parents were selected to detect anaerobic germination (AG) values. A total of 190 accessions of the GWAS panel, including japonica weedy rice (GJ-weedy), japonica landrace (GJ-tmp-land), and japonica cultivated are (GJ-tmp-cul) listed in Supplementary Data 2.
Evaluation of germination rate and coleoptile length
Seeds of each variety were grown in the field, and their seeds were harvested 45 days after heading, air dried, and stored at 50 °C for 7 days to break dormancy. Three independent biological replicates with 30 seeds per replicate were then sterilized with 1.5% (v/v) sodium hypochlorite and subsequently incubated in a 50 ml glass (30 mm × 100 mm) at 30 °C under dark conditions for four days under anaerobic conditions. Seeds were considered germinated when white embryo protrusion was visible. The mean germination rates (%) of each variety were recorded after 72 h of treatment for GWAS. The mean coleoptile lengths of each variety were measured on the fourth day.
QTL mapping
The germination level for each line of the 168 RILs after 72 h of anaerobic treatment was used to determine the QTL phenotype in three independent biological replicates with 30 seeds per replicate. Lines that can germinate and develop coleoptile like the weed rice parent, WR04-6, was defined as 1; lines that cannot germinate like the cultivated rice parent, Qishanzhan, were defined as 0; and lines that can germinate but no obvious coleoptile formation were defined as 0.5 (Supplementary Data 1). For QTL genotyping, we used the previously reported genetic map to calculate QTLs22 based on inclusive composite interval mapping (ICIM) methods in IciMapping software (Version 4.1)45.
DNA sequencing and SNP calling
In the GWAS panel, SNPs of the 190 accessions were obtained by NGS sequencing and re-calling fastq files from published NGS database23,31,46,47. First, paired-end sequence data of 114 accessions, including 36 temperate japonica weedy rice, 65 temperate japonica cultivars, and 13 temperate japonica landraces, were generated by Illumina HiSeq4000 covering an average approximate depth of 23.2x for each sample. Then, these fastq files were integrated with published fastq files of 76 japonica samples for joint-calling of SNPs and Indels (Supplementary Data 2). The raw paired-end reads were first filtered into clean data using fastp software (Version 0.20.1)48 with default settings. Clean reads of each accession were mapped to the rice reference genome Os-Nipponbare-Reference-IRGSP-1.0 pseudomolecules using BWA software (Version 0.7.18)49. Then the variants detection were applied by Samtools software (Version 1.12)50 and GATK (Version 4.2) with the HaplotypeCaller program for joint genotyping51. The SNPs and indels set detected in the above process was filtered according to a threshold; –min-alleles 2 –max-alleles 2 –maf 0.05 –max-missing 0.8 –minDP 3 –minQ 30, to reduce the variants false discovery rate using vcftools ver3.0 software. Finally, generating 1,160,454 SNPs and 150,990 indels were subjected to principal components analysis (PCA) based on Plink software (Version 1.9)52.
GWAS and candidate gene detection
For GWAS, we first performed imputation for the genotype of 190 samples using BEAGLE ver4.053. Then the analysis was performed using EMMAX software (Version emmax-intel-binary-20120210)54 by fitting a linear mixed model with 1,311,444 genetic markers (MAF = 0.05) and the first two PCs from PCA as covariates to control population structure. The threshold for genome-wide significance was determined by Bonferroni correction, and the significance level was determined as 8.12 × 10−7 (0.05 significance level) and 1.62 × 10−7 (0.01 significance level). Visualization of GWAS results (manhattan plot and QQ plot) and linkage disequilibrium block were performed using CMplot package (Version 4.0) in R software (Version x64 4.1.1) and LDBlockShow software (Version 1.36)55. The preselection of causal haplotype among candidate genes annotated by MSU Rice Genome Annotation Project Release 7 was conducted using CandiHap package56 of R software (Version x64 4.1.1) by providing the gff file downloaded from http://rice.uga.edu./downloads.shtml. The haplotype of each candidate gene was defined by all detected SNPs and indels within a region of 2000 bp upstream to 500 bp downstream of annotated genes according to the default setting of the CandiHap package of R x64 4.0.1 software. We then conducted expression analysis for seven candidate genes from the seeds of weedy rice WR04-6 after 72-hour anaerobic treatment by qRT-PCR. BLUP analyses were performed based on a subset of SNPs (n = 54,227) that were randomly selected from the GWAS markers using the Sommer package (Version 4.1.7) in R software (Version x64 4.1.1)57.
Evolutionary analysis of OsGF14h
Haplotype frequency analysis for each rice ecotype in the OsGF14hWR04-6 coding region was calculated using DnaSP software (Version 5)58. A haplotype network was built based on 16 mutation sites of the CDS region for ten ecotypes with the O.bathii as an outgroup based on the ‘Minimum Spanning Network’ approach implemented in PopART software (Version 1.7)59. The geographic map with average annual temperature was plotted using ArcGIS software (Version 10.6). Time-tree is constructed for the ten haplotypes of OsGF14h with the O.bathii as an outgroup according to MCMCTREE implemented in PAML software (Version 4.5). Selection sweeps, 100 kb window-based Tajima’ D and FST were calculated using vcftools software (Version 3.0)60.
Plant transformation
To generate the overexpression constructs, the full-length CDS of OsGF14hWR04-6, OsGF14hSN9816, OsGA20ox1, and OsVP1 from WR04-6 were amplified and then cloned into pCAMBIA1301S with a CaMV 35 S promoter to generate p35S:OsGF14h-GFP, p-35S:OsGA20ox1-GFP, and p-35S:OsVP1-GFP constructs, respectively. To generate the knockout vectors, the specific sgRNA targeting OsGF14h, OsGA20ox1, OsVP1, and Rc were designed online (http://crispr.hzau.edu.cn/CRISPR2/). Agrobacterium tumefaciens strain EHA105 carrying the gene knockout vectors were used to infect callus tissue induced from the weedy rice WR04-6. Hygromycin-containing medium was used to select hygromycin-resistant callus, and then, the callus were transferred to regeneration media to generate green plants61. Plant transformation for OsGA20ox1 and Rc were by Biorun Bioscience Co., Ltd. Wuhan, China. The mutation sites of the gf14h, ga20ox1, and rc mutants are shown in Supplemental Fig. 12. Agrobacterium-mediated transformation was used to generate transgenic rice plants. Primer sequences are listed in Supplementary Data 8.
Real-time PCR
Different tissues and organs of rice were excised from different plants and immediately put into liquid nitrogen for RNA extraction. Total RNA was prepared using the RNAiso Plus Reagent (Takara Bio Inc, Dalian, China) according to the manufacturer’s instructions. cDNA was obtained with PrimeScriptTM RT Master Mix (Takara Bio Inc, Dalian, China). For qRT-PCR, the cDNA was mixed with SYBR Premix Ex Taq II (TAKARA, Tokyo, Japan) and analyzed using an Applied Biosystems QuantStudio 3 system (Thermo Fisher Scientific, USA).
Subcellular localization
Rice protoplasts were extracted from a 7-day-old leaf sheath of Nipponbare, which was cultured in the dark. The fusion construct pCAMBIA1301S-35S: OsGF14h-GFP was co-transformed into rice protoplasts with 35S::NLS::CFP, used as a nuclear marker. The constructs were introduced into protoplasts through polyethylene glycol (PEG) mediated transformation62,63. After incubation in the dark for 24–48 h, the subcellular distribution of GFP fluorescence was determined using confocal microscopy (Nikon C2-ER, Japan). Primer sequences are listed in Supplementary Data 8.
Germination test by exogenous application of ABA
A total of 35 seeds of WT and knockout line (gf14h) were anaerobic-incubated at 30 °C with a gradient concentration of ABA solution as treatment and with pure water as control, respectively. The concentration of ABA solution was set to 0, 0.1, 1, 5, 10, and 50 µM, respectively. The germinated rate was recorded at 72 h of incubation.
Determination of endogenous GA concentration
To measure the endogenous content of GA, the seeds were prepared at 0, 6, 12, and 24 HAI (hours after imbibition) under anaerobic stress. Liquid nitrogen frozen germinated seeds (50 mg fresh weight) were ground into powder and extracted with the traction method (methanol/water/formic acid = 15:4:1, V/V/V). The extracts were vortexed and centrifuged at 4694 × g under 4°C for 10 min. The supernatants were dried by evaporation under the flow of nitrogen gas at room temperature, then dissolved in 200 μl of methanol. The sample extracts were analyzed using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system; MS, Applied Biosystems 6500 Triple), and the data were analyzed by Metware Biotechnology Co., Ltd. Wuhan, China. Three replicates of each assay were performed.
Yeast two-hybrid assay
The Matchmaker Gold Yeast Two-Hybrid system (Clontech) was used to perform the yeast two-hybrid (Y2H) screen. Various tissues (including 30-day seedlings and roots, as well as the stems, leaves, young panicles, and seeds at the reproductive stage) were mixed to construct a two-hybrid library. For the Y2H screening, the CDS of OsGF14hWR04-6 was cloned into vector pGBKT7 between the NdeI and EcoRI sites and functioned as bait. The yeast strain Y2H Gold (Clontech, Cat. No. 630489) was used for transformation. To verify the interaction between (OsGF14hWR04-6, OsGF14hSN9816) and (OsHOX3, OsVP1) and between OsGF14h and OsVP1 in yeast, the CDS of OsHOX3 and OsVP1 were cloned into the pGADT7 vector using the NdeI and EcoRI sites; then, the pGBKT7-OsGF14h/pGADT7-OsHOX3 and pGBKT7-OsGF14h/pGADT7-VP1 pairs were separately transformed into yeast strain Y2H Gold and grown on SD/-Leu/-Trp medium. The positive transformants were further detected on SD/-Leu-Trp-His-Ade medium as described in the Yeast Protocols Handbook (Clotech, PT3024-1). The interaction between pGADT7-T7 and pGBKT7-53, and between pGADT7-T7 and pGBKT7-Lam were regarded as a positive and negative control, respectively. Primers used for construction are listed in Supplementary Data 8.
Co-immunoprecipitation assay
For the co-immunoprecipitation (co-IP) assay, the full-length CDS of OsGF14hWR04-6, OsGF14hSN9816, OsHOX3, and OsVP1 were cloned into pBWA(V)HS to construct the vectors of pBWA(V)HS-OsGF14h-GFP, pBWA(V)HS-3×FLAG-HOX3, and pBWA(V)HS-3×FLAG-VP1, respectively. The vectors were transformed into Agrobacterium strain GV3101, then injected into Nicotiana benthamiana leaves. After 48 h, total protein was extracted according to the manufacturer’s instructions, then incubated with GFP-Trap coupled to agarose beads (TransGen, DP501-01) for 2 h, then washed three times with buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 2 mM DTT, 0.1% NP-40). 5×SDS loading buffer was added to the immunoprecipitated proteins and denatured at 95 °C for 10 min and resolved on 10% acrylamide gels. Individual bands were detected using Supersignal West Pico Chemiluminescent Substrate (Thermo) and the ChemDocTM Touch Imaging system (Bio-Rad). GFP protein was used as a negative control in each set of experiments. The dilution for anti-FLAG (MBL, M185-3L) and anti-GFP (TransGen, HT801-02) antibodies was 1:5000. Primer sequences are listed in Supplementary Data 8.
Pull-down assay
The CDS of OsHOX3 or OsVP1 was cloned into pGEX4T-1 to construct GST-OsHOX3 or GST-OsVP1 fusion protein, and the CDS of OsGF14hWR04-6 or OsGF14hSN9816 was inserted into pET28a to construct and His-OsGF14hWR04-6 or His-OsGF14hSN9816, which were expressed in Escherichia coli Rosetta (DE3). The recombinant proteins were induced by 0.5 mM isopropyl-d-thiogalactopyranoside (IPTG) and purified using Ni NTA beads or GST beads (ProbeGene, Xuzhou, China). Then equal quantities of GST-OsHOX3/GST-OsVP1/GST and His-OsGF14hWR04-6/His-OsGF14hSN9816 recombinant proteins were incubated in 1 mL of GST pull-down buffer at 4 °C overnight, and then 50 μL of GST beads were added into the mixture and incubated at 4 °C for 4 h. The beads were washed extensively and boiled. Then the pulled-down proteins were further analyzed by immunoblotting using anti-GST (ABclonal, AE001, 1:5000 dilution) and anti-His (Proteintech Group, 66005-1-Ig, 1:5000 dilution). Primer sequences are listed in Supplementary Data 8.
Luciferase complementation imaging assay
The luciferase (LUC) complementation imaging assays for the interaction between OsGF14hWR04-6 and OsHOX3, between OsGF14hWR04-6 and OsVP1 were performed in N. benthamiana leaves. The full-length CDS of OsGF14hWR04-6 and OsHOX3 and OsVP1 were fused with the pCAMBIA1300-nLuc (pNL) and pCAMBIA1300-cLuc (pCL) plasmids, respectively. The modified plasmids and empty vectors were transformed into Agrobacterium strain GV3101. Those cells were resuspended with infiltration buffer (10 mM MgCl2, 100 mM acetosyringone, 10 mM MES, pH 5.6, 1.0 of OD600 for each), then equal volumes of Agrobacterium suspensions carrying the indicated constructs were co-injected into N. benthamiana and cultured at 23 °C for 48 h64. The infiltrated leaves were sprayed with 1 mM luciferin in darkness for 5 min and LUC activity was analyzed for 48 h after infiltration using chemiluminescence imaging (Tannon 5200). Each plasmid combination was performed in three independent transformations. Primer sequences are listed in Supplementary Data 8.
Yeast one-hybrid assay
Yeast one-hybrid (Y1H) assays were performed using the MatchmakerTM Gold Yeast One-Hybrid System (Clontech). First, the full-length CDS of OsHOX3 was fused with the activation domain of GAL4 protein in the pGADT7 vector as prey. For the construction of bait plasmid, 3×DNA wild and mutated fragment from −1970 to −1948 harboring HD-ZIP3 motif from OsPYL5 promoter was inserted into the pAbAi vector to construct the bait. After linearizing, the pAbAi bait plasmid was transformed into the Y1H Gold yeast strain, and the minimal inhibitory concentration of aureobasidin A (AbA) of the bait strain was screened. The prey and empty pGADT7 vector were transformed into the recombinant bait-reporter strain. The interaction between pGADT7-p53 and pAbAi-p53 was regarded as a positive control. The yeast cells were grown on SD/-Leu culture media with or without AbA for 3–5 days at 30 °C. Primer sequences are shown in Supplementary Data 8.
Dual-luciferase assay
The full-length CDS of OsHOX3 were separately cloned into the pGreenII 62-SK vector as an effector. The OsPYL5 promoter was inserted into the pGreenII0800-LUC vector to generate the reporter. The effector and reporter constructs were separately transferred into A. tumefaciens strain GV3101 with the helper plasmid pSoup-p19. The strains were co-transformed into 4-week-old N. benthamiana leaves and grown for 2 days at 25 °C. The leaves were collected for the determination of firefly luciferase (LUC) and Renilla luciferase (REN) activities using a Dual-Luciferase® reporter assay system (Promega, Madison, WI, USA) with a microplate reader (Infinite M200 Pro; Tecan, Männedorf, Switzerland). The empty pGreenII 62-SK vector was used as a negative control. The REN activity under the control of the CaMV 35 S promoter was used as an internal control, and the LUC activity was normalized to the REN activity. Three independent biological replicates were performed. Primer sequences are shown in Supplementary Data 8.
Electrophoretic mobility shift assay
The CDS of OsHOX3 and OsGF14hWR04-6 were expressed in Escherichia coli Rosetta (DE3) and purified using Ni NTA or GST resin (ProbeGene, Xuzhou, China). For the probes, a 55 bp fragment containing the HD-ZIP3 element as well as a mutated fragment was synthesized and labeled with biotin at the 3′ end, and the unlabeled fragment was used as a competitor. Protein–DNA binding reactions were performed using a Chemiluminescent EMSA Kit GS009 (Beyotime, Shanghai, China) according to the manufacturer’s protocol. A 6% native polyacrylamide gel was used for electrophoresis. After that, the complexes were transferred onto a nylon membrane and cross-linked with a UV-light cross-linker. The chemiluminescence was detected using an Azure Biosystems C500 Imager (USA). The probe sequences are shown in Supplementary Data 8.
Chromatin immunoprecipitation-qPCR assay
Chromatin immunoprecipitation (ChIP) was performed with a 3×FLAG-tagged OsHOX3 expressed in callus. About 2 g callus from the 3×FLAG-OsHOX3-overexpressing line was collected and cross-linked with 1% formaldehyde for 15 min. Then the samples were sonicated for isolation and lysis of nuclei. Antibodies against FLAG (D6W5B, Cell Signaling Technology, 1:50 dilution) were used for immunoprecipitation, and Protein-A + G-agarose beads were used to pull down the protein–DNA complex. After collection, wash, and elution of the immune complex, the precipitated DNA was reversely cross-linked and used for qPCR analysis65. Primers were designed in the promoter regions of OsPYL5 and listed in Supplementary Data 8. Three biological replicates were measured independently.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
The genetic map for QTL mapping has been released previously22. The raw sequencing of RILs are available on NCBI BioProject under the accession number PRJNA513407. The raw sequencing of the GWAS and Or-IIIa datasets have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA) under accession number CRA005027. The raw sequencing of transcriptome analysis used in this study have been deposited in GSA database under accession number CRA006828. In order to identify the haplotypes of OsGF14h and Sdr4 for GJ-weedy and XI-weedy, we downloaded short-read sequence data from GenBank under the BioProject accession number PRJNA606132, PRJNA344937, and PRJNA295802. The OsGF14h and Sdr4 sequences of widely planted GJ-tmp and XI-cul cultivars from China were downloaded from MBKbase Database [http://www.mbkbase.org/rice]30. OsGF14h and Sdr4 sequence data of XI-land and GJ-trp were downloaded from RiceVarMap v2.0 Database [http://ricevarmap.ncpgr.cn/]. Source data are provided with this paper.
References
Bailey-Serres, J., Parker, J. E., Ainsworth, E. A., Oldroyd, G. E. D. & Schroeder, J. I. Genetic strategies for improving crop yields. Nature 575, 109–118 (2019).
IPCC. Summary for policymakers. In: Stocker, T. F. et al. (eds). Climate Change 2013: the Physical Science Basis. Contribution of Working Group to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. 1535 (Cambridge University Press, Cambridge New York, 2013).
Tellman, B. et al. Satellite imaging reveals increased proportion of population exposed to floods. Nature 596, 80–86 (2021).
Yu, S. M., Lee, H. T., Lo, S. F. & Ho, T. D. How does rice cope with too little oxygen during its early life? N. Phytol. 229, 36–41 (2021).
Kretzschmar, T. et al. A trehalose-6-phosphate phosphatase enhances anaerobic germination tolerance in rice. Nat. Plants 1, 15124 (2015).
Godfray, H. C. et al. Food security: the challenge of feeding 9 billion people. Science 327, 812–818 (2010).
Miro, B. & Ismail, A. M. Tolerance of anaerobic conditions caused by flooding during germination and early growth in rice (Oryza sativa L.). Front. Plant Sci. 4, 269 (2013).
Stein, J. C. et al. Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza. Nat. Genet. 50, 285–296 (2018).
Voesenek, L. & Bailey-Serres, J. Flood adaptive traits and processes: an overview. N. Phytol. 206, 57–73 (2015).
Xu, K. et al. Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature 442, 705–708 (2006).
Fukao, T. & Bailey-Serres, J. Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice. Proc. Natl Acad. Sci. USA 105, 16814–16819 (2008).
Hattori, Y. et al. The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature 460, 1026–1030 (2009).
Kuroha, T. et al. Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding. Science 361, 181–186 (2018).
Yano, K. et al. Efficacy of microarray profiling data combined with QTL mapping for the identification of a QTL gene controlling the initial growth rate in rice. Plant Cell Physiol. 53, 729–739 (2012).
Waadt, R. et al. Plant hormone regulation of abiotic stress responses. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/s41580-022-00479-6 (2022).
Ye, N. H. et al. Natural variation in the promoter of rice calcineurin B-like protein10 (OsCBL10) affects flooding tolerance during seed germination among rice subspecies. Plant J. 94, 612–625 (2018).
Wu, D. et al. Weedy rice, a hidden gold mine in the paddy field. Mol. Plant 15, 566–568 (2022).
Li, L. F., Li, Y. L., Jia, Y., Caicedo, A. L. & Olsen, K. M. Signatures of adaptation in the weedy rice genome. Nat. Genet. 49, 811–814 (2017).
Qiu, J. et al. Diverse genetic mechanisms underlie worldwide convergent rice feralization. Genome Biol. 21, 70 (2020).
Wang, W. et al. Weedy rice as a novel gene resource: a genome-wide association study of anthocyanin biosynthesis and an evaluation of nutritional quality. Front. Plant Sci. 11, 878 (2020).
Zhang, G. et al. Comparative multi-omics analysis of hypoxic germination tolerance in weedy rice embryos and coleoptiles. Genomics 113, 3337–3348 (2021).
Sun, J. et al. Population genomic analysis and de novo assembly reveal the origin of weedy rice as an evolutionary game. Mol. Plant 12, 632–647 (2019).
Qiu, J. et al. Genomic variation associated with local adaptation of weedy rice during de-domestication. Nat. Commun. 8, 15323 (2017).
Chen, W. et al. Small Grain and Dwarf 2, encoding an HD-Zip II family transcription factor, regulates plant development by modulating gibberellin biosynthesis in rice. Plant Sci. 288, 110208 (2019).
Hobo, T., Kowyama, Y. & Hattori, T. A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription. Proc. Natl Acad. Sci. USA 96, 15348–15353 (1999).
Bryant, P., Pozzati, G. & Elofsson, A. Author correction: improved prediction of protein-protein interactions using AlphaFold2. Nat. Commun. 13, 1694 (2022).
Schultz, T. F., Medina, J., Hill, A. & Quatrano, R. S. 14-3-3 proteins are part of an abscisic acid-VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1. Plant Cell 10, 837–847 (1998).
Meijer, A. H., de Kam, R. J., d’Erfurth, I., Shen, W. & Hoge, J. H. HD-Zip proteins of families I and II from rice: interactions and functional properties. Mol. Gen. Genet. 263, 12–21 (2000).
Kim, H. et al. A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathway in seed germination and early seedling growth. J. Exp. Bot. 63, 1013–1024 (2012).
Li, X. et al. Analysis of genetic architecture and favorable allele usage of agronomic traits in a large collection of Chinese rice accessions. Sci. China Life Sci. 63, 1688–1702 (2020).
Wang, W. et al. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature 557, 43–49 (2018).
Gubler, F., Millar, A. A. & Jacobsen, J. V. Dormancy release, ABA and pre-harvest sprouting. Curr. Opin. Plant Biol. 8, 183–187 (2005).
Song, S. et al. OsMFT2 is involved in the regulation of ABA signaling-mediated seed germination through interacting with OsbZIP23/66/72 in rice. Plant J. 103, 532–546 (2020).
Liu, F., Zhang, H., Ding, L., Soppe, W. J. J. & Xiang, Y. REVERSAL OF RDO5 1, a homolog of rice seed dormancy4, interacts with bHLH57 and controls ABA biosynthesis and seed dormancy in Arabidopsis. Plant Cell 32, 1933–1948 (2020).
Abley, K. et al. An ABA-GA bistable switch can account for natural variation in the variability of Arabidopsis seed germination time. eLife https://doi.org/10.7554/eLife.59485 (2021).
Muslin, A. J. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 (1996).
de Boer, A. H., van Kleeff, P. J. & Gao, J. Plant 14-3-3 proteins as spiders in a web of phosphorylation. Protoplasma 250, 425–440 (2013).
Fukazawa, J. et al. Repression of shoot growth, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 12, 901–915 (2000).
Schoonheim, P. J., Costa Pereira, D. D. & De Boer, A. H. Dual role for 14-3-3 proteins and ABF transcription factors in gibberellic acid and abscisic acid signalling in barley (Hordeum vulgare) aleurone cells. Plant Cell Environ. 32, 439–447 (2009).
Sugimoto, K. et al. Molecular cloning of Sdr4, a regulator involved in seed dormancy and domestication of rice. Proc. Natl Acad. Sci. USA 107, 5792–5797 (2010).
Gu, X. Y. et al. Association between seed dormancy and pericarp color is controlled by a pleiotropic gene that regulates abscisic acid and flavonoid synthesis in weedy red rice. Genetics 189, 1515–1524 (2011).
Wang, J. et al. Transcription factors Rc and OsVP1 coordinately regulate preharvest sprouting tolerance in red pericarp rice. J. Agric Food Chem. 68, 14748–14757 (2020).
Zhao, B. et al. Sdr4 dominates pre-harvest sprouting and facilitates adaptation to local climatic condition in Asia cultivated rice. J. Integr. Plant Biol. https://doi.org/10.1111/jipb.13266 (2022).
Song, B. K., Chuah, T. S., Tam, S. M. & Olsen, K. M. Malaysian weedy rice shows its true stripes: wild Oryza and elite rice cultivars shape agricultural weed evolution in Southeast Asia. Mol. Ecol. 23, 5003–5017 (2014).
Meng, L., Li, H., Zhang, L. & Wang, J. QTL IciMapping: integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. Crop J. 3, 269–283 (2015).
Yano, K. et al. Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice. Nat. Genet. 48, 927–934 (2016).
Yano, K. et al. GWAS with principal component analysis identifies a gene comprehensively controlling rice architecture. Proc. Natl Acad. Sci. USA 116, 21262–21267 (2019).
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
McKenna, A. et al. The genome analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007).
Browning, S. R. & Browning, B. L. Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering. Am. J. Hum. Genet. 81, 1084–1097 (2007).
Kang, H. M. et al. Variance component model to account for sample structure in genome-wide association studies. Nat. Genet. 42, 348–354 (2010).
Dong, S. S. et al. LDBlockShow: a fast and convenient tool for visualizing linkage disequilibrium and haplotype blocks based on variant call format files. Brief Bioinform. https://doi.org/10.1093/bib/bbaa227 (2021).
Li, X., Shi, Z., Gao, J., Wang, X. & Gao, K. CandiHap: a haplotype analysis toolkit for natural variation study. Preprint at Research Square https://doi.org/10.21203/rs.3.rs-1741665/v1 (2022).
Covarrubiaspazaran, G. Software update: moving the R package sommer to multivariate mixed models for genome-assisted prediction. (2018).
Librado, P. & Rozas, J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452 (2009).
Leigh, J. W. & Bryant, D. POPART: full-feature software for haplotype network construction. Methods Ecol. Evol. 6, 1110–1116 (2015).
Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).
Cui, Y., Zhu, M., Xu, Z. & Xu, Q. Assessment of the effect of ten heading time genes on reproductive transition and yield components in rice using a CRISPR/Cas9 system. Theor. Appl. Genet. 132, 1887–1896 (2019).
Chen, S. et al. A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol. Plant Pathol. 7, 417–427 (2006).
Zhao, F. et al. Visualizing the essential role of complete virion assembly machinery in efficient hepatitis C virus cell-to-cell transmission by a viral infection-activated split-intein-mediated reporter system. J. Virol. https://doi.org/10.1128/JI.0V1720-16 (2017).
Zhou, Z., Bi, G. & Zhou, J. M. Luciferase complementation assay for protein-protein interactions in plants. Curr. Protoc. Plant Biol. 3, 42–50 (2018).
Saleh, A., Alvarez-Venegas, R. & Avramova, Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat. Protoc. 3, 1018–1025 (2008).
Acknowledgements
OsHOX3 loss-of-function mutant hox3 was provided by Prof. Jianmin Wan from the Chinese Academy of Agricultural Sciences. The leaves of Oryza rufipogon (Or-IIIa) used for NGS sequencing were obtained from National Nursery of Wild Rice Germplasm (Guangzhou), Guangdong Academy of Agricultural Sciences; Rice research institute, Guangxi academy of agricultural sciences; Rice research institute, Fujian Academy of Agricultural Sciences. We thank Prof. Yuanhu Xuan from Shenyang Agricultural University, China, for suggestions on the molecular biology Experiment. We thank Dr. Baolei Jia from Chung-Ang University, South Korea, for help in the prediction of protein structure. We thank Prof. Chengzhi Liang from Chinese Academy of Science, for help in the genotyping. This work was supported by the National Natural Science Foundation of China (Grant Agreement No. U1708231, W.C. and J.S.), the National Key R&D Program of China (Grant Agreement No. 2017YFD0100501, J.S. and Z.X), and the Support Program for Young Scientific and Technological Innovation Talents of Shenyang, China (Grant Agreement No. RC210408, J.S.).
Author information
Authors and Affiliations
Contributions
J.S. designed this study. J.S., G.Z., Y.C.H., X.Z., and R.G. performed AG phenotyping and QTL mapping, analysed the data. J.S. and X.Y. performed the GWAS and molecular evolutionary analysis. G.Z., R.G., and H.L. performed genetic transformation experiments and phytohormones content determination. G.Z., Z.C., X.K., Y.Q.H., and Z.L. conducted biochemistry experiments. J.S., G.Z., Z.C., X.K., Q.X., and L.T. wrote the manuscript with contributions from all author. W.C., D.M., Z.X., and J.S. supervised the project.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Vicent Arbona, Shuhua Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Sun, J., Zhang, G., Cui, Z. et al. Regain flood adaptation in rice through a 14-3-3 protein OsGF14h. Nat Commun 13, 5664 (2022). https://doi.org/10.1038/s41467-022-33320-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-022-33320-x
This article is cited by
-
A mediator of OsbZIP46 deactivation and degradation negatively regulates seed dormancy in rice
Nature Communications (2024)
-
UDP-glucosyltransferase OsUGT75A promotes submergence tolerance during rice seed germination
Nature Communications (2023)
-
Identification and substitution mapping of QTLs for anaerobic germination in rice
Euphytica (2023)
-
Progress in the study of functional genes related to direct seeding of rice
Molecular Breeding (2023)
-
Two bHLH transcription factors affect sprouting by regulating the level of ABA
Nature Genetics (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.