The heterotrimeric G protein γ3 subunit, RGG3/GS3, integrates sugar-starvation and hormone-responsive signaling pathways to promote coleoptile elongation during anaerobic germination in rice.

BackgroundAnaerobic germination is a critical trait for rice cultivation, particularly in regions that experience flooding or waterlogging immediately after sowing. Under direct-seeded conditions, where rice is sown directly into the field without prior transplantation, the ability of seeds to germinate in anaerobic (oxygen-deficient) conditions becomes essential for successful crop establishment. This trait is especially relevant in areas prone to waterlogging, were traditional methods of rice cultivation, such as puddled transplanting, may be less viable. Understanding the genetic basis of anaerobic germination can lead to the development of rice varieties that are better adapted to such challenging conditions, thus supporting more sustainable agricultural practices.ResultsIn this study, a nested association mapping (NAM) population consisting of 384 breeding lines was utilized to identify genomic regions associated with anaerobic germination in rice. Through comprehensive analysis, 19 significant marker-trait associations (MTAs) were identified, including 12 associations specifically linked to percent seed germination under anaerobic conditions. These associations were distributed across six different chromosomes: 3, 4, 5, 6, 7, and 9. Notably, a cluster of single nucleotide polymorphisms (SNPs) spanning a 6.9 Mb genomic region on chromosome 3 (from 21,089,181 to 28,017,712 bp) was consistently associated with percent germination at 15 and 21 days after sowing over multiple years. Similarly, a 6.4 Mb genomic segment on chromosome 6 (from 18,028,538 to 24,492,161 bp) was also associated with percent germination at the same time points. Specific SNPs within this region, namely S6_18028538 and S6_24492161, were linked to germination at 15 and 21 days, respectively. In addition to these findings, one MTA was identified for days to 50% flowering on chromosome 1, and six MTAs were identified for grain yield across chromosomes 1, 2, 5, 8, and 10. The breeding lines that exhibited both high and stable yields, along with anaerobic germination traits, have the potential to be particularly valuable in genomics-assisted breeding programs aimed at improving rice varieties for flood-prone areas.ConclusionsThis study provides crucial insights into the genetic basis of anaerobic germination in rice, highlighting specific genomic regions associated with this trait under direct-seeded conditions. The identification of significant MTAs across multiple chromosomes, particularly the consistent associations found on chromosomes 3 and 6, underscores the potential for developing rice varieties with enhanced tolerance to anaerobic conditions. The high-yielding breeding lines identified in this research, which also exhibit strong anaerobic germination traits, represent valuable genetic resources for breeding programs. These findings support the use of direct-seeded rice (DSR) as a sustainable alternative to traditional puddled transplanting, particularly in regions prone to flooding, thereby contributing to the development of more resilient rice cultivation practices.

An important and interesting feature of rice is that it can germinate under anoxic conditions. Though several biochemical adaptive mechanisms play an important role in the anaerobic germination of rice but the role of phytoglobin-nitric oxide cycle and alternative oxidase pathway is not known, therefore in this study we investigated the role of these pathways in anaerobic germination. Under anoxic conditions, deepwater rice germinated much higher and rapidly than aerobic condition and the anaerobic germination and growth were much higher in the presence of nitrite. The addition of nitrite stimulated NR activity and NO production. Important components of phytoglobin-NO cycle such as methaemoglobin reductase activity, expression of Phytoglobin1, NIA1 were elevated under anaerobic conditions in the presence of nitrite. The operation of phytoglobin-NO cycle also enhanced anaerobic ATP generation, LDH, ADH activities and in parallel ethylene levels were also enhanced. Interestingly nitrite suppressed the ROS production and lipid peroxidation. The reduction of ROS was accompanied by enhanced expression of mitochondrial alternative oxidase protein and its capacity. Application of AOX inhibitor SHAM inhibited the anoxic growth mediated by nitrite. In addition, nitrite improved the submergence tolerance of seedlings. Our study revealed that nitrite driven phytoglobin-NO cycle and AOX are crucial players in anaerobic germination and growth of deepwater rice.

Plants face a variety of environmental stresses and have evolved molecular mechanisms to survive these challenges. One of these stresses is low oxygen conditions, which can occur under flooding conditions. Rice (Oryza sativa) is somewhat unique for its ability to tolerate and even germinate under low to no oxygen conditions. In this study, we examined global transcriptomic responses over the course of germination and in response to low oxygen and other abiotic stress in rice and Arabidopsis (Arabidopsis thaliana). Over 150 microarray datasets were analyzed in parallel to determine just how unique the low oxygen response is in rice. Comparison of aerobic germination in rice and Arabidopsis, with anaerobic germination in rice revealed conserved transcriptomic responses that are not only conserved across both species but also occur in the absence of oxygen in rice. Thus, these genes may represent functions necessary for the developmental progression of germination, whether or not oxygen is present in rice. Analysis of genes that responded differently in rice compared to Arabidopsis revealed responses specific to anaerobic germination in rice, including the down-regulation of genes encoding redox functions and up-regulation of receptor kinases. Comparison of a range of hypoxia/anoxia studies within and across Arabidopsis and rice revealed both conserved and species specific changes in gene expression (e.g., Arabidopsis specific up-regulation of WRKYs and rice specific down-regulation of heme), unveiling unique transcriptomic signatures of the low oxygen response. Lastly, a comparison of the low oxygen response with cold, salt, drought and heat stress revealed some similarity with the response to heat stress in Arabidopsis, which was not seen in rice. Comparison of these heat-responsive, abiotic stress marker genes in Arabidopsis with their rice orthologs revealed that while low oxygen may be perceived as an abiotic stress in Arabidopsis, this is not the case in rice.

Anaerobic germination (AG) is an important trait for direct-seeded rice (DSR) to be successful. Rice usually has low germination under anaerobic conditions, which leads to a poor crop stand in DSR when rain occurs after seeding. The ability of rice to germinate under water reduces the risk of poor crop stand. Further, this allows the use of water as a method of weed control. The identification of the genetic factors leading to high anaerobic germination is required to develop improved DSR varieties. In the present study, two BC1F2:3 mapping families involving a common parent with anaerobic germination potential, Kalarata, an indica landrace, and two recurrent parents, NSIC Rc222 and NSIC Rc238, were used. Phenotyping was done under two environmental conditions and genotyping was carried out through the KASP SNP genotyping platform. A total of 185 and 189 individuals genotyped with 170 and 179 polymorphic SNPs were used for QTL analysis for the two populations, Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222, respectively. A total of five QTLs on chromosomes 3, 5, 6, 7, and 8 for survival (SUR) and four QTLs on chromosomes 1, 3 (two locations), and 7 for the trait seedling height (SH) across the populations and over the screening conditions were identified. Except for the QTLs on chromosomes 5 and 8, the parent with AG potential, Kalarata, contributed all the other QTLs. Among the five QTLs for SUR, the second-largest QTL (qSUR6–1) was novel for AG potential in rice, showing a stable expression in terms of genetic background and screening conditions explaining 11.96% to 16.01% of the phenotypic variation. The QTL for SH (qSH1–1) was also novel. Considering different genetic backgrounds and different screening conditions, the QTLs identified for the trait SUR explained phenotypic variation in the range of 57.60% to 73.09% while that for the trait SH ranged from 13.53% to 34.30%.

The success of rice (Oryza sativa L.) germination and survival under submerged conditions is mainly determined by the rapid growth of the coleoptile to reach the water surface. Previous reports have shown the presence of genetic variability within rice accessions in the levels of flooding tolerance during germination or anaerobic germination (AG). Although many studies have focused on the physiological mechanisms of oxygen stress, few studies have explored the breadth of natural variation in AG tolerance-related traits in rice. In this study, we evaluated the coleoptile lengths of a geographically diverse rice panel of 241 accessions, including global accessions along with elite breeding lines and released cultivars from the United States, under the normal and flooded conditions in laboratory and greenhouse environments. A genome-wide association study (GWAS) was performed using a 7K single-nucleotide polymorphism (SNP) array and the phenotypic data of normal coleoptile length, flooded coleoptile length, flooding tolerance index, and survival at 14 d after seeding (DAS). Out of the 30 significant GWAS quantitative trait loci (QTL) regions identified, 14 colocalized with previously identified candidate genes of AG tolerance, whereas 16 were potentially novel. Two rice accessions showing contrasting phenotypic responses to AG stress were selected for the transcriptomics study. The combined approach of GWAS and transcriptomics analysis identified 77 potential candidate genes related to AG tolerance. The findings of our study may assist rice improvement programs in developing rice cultivars with robust tolerance under flooding stress during germination and the early seedling stage.

The leading target domain in lowland areas is improving the tolerance for anaerobic germination and submergence, especially in direct-seeded rice (DSR). The present study identified elite genotypes with tolerance for anaerobic germination and submergence among 25 diverse rice genotypes. This study adopted four experimental conditions (three in the lab and one in the field): anaerobic germination in water, anaerobic germination with soil and water, submergence condition and direct seeded rice in the field. The recorded traits showed significant variation under all four stresses. Chitiraikar performed well in anaerobic germination and DSR experiments. CO55 registered the highest trait value of seedling height and early vegetative vigour in the anaerobic experiment with water. Aanaikomban, Karunguruvai, Karuppukavuni and Chitiraikar outperformed other genotypes in most of the above experimental conditions. Thus, choosing lines based on germination percentage, early seedling vigour, elongation index and seedling survival rate can increase tolerance for anaerobic germination and submergence conditions in the lab and DSR method. Among all the experiments conducted, anaerobic germination with soil and water and the DSR method was found to be reliable for screening a large population

Poor germination and seedling establishment under flooded conditions is a major bottleneck in the direct-seeding system of rice. The objectives of this study were to determine the genetic variability for traits associated with anaerobic germination and identify tolerant genotypes. One hundred rice genotypes were screened. Data were collected on survival percentage, shoot length, root length, length of plant above water, and seedling vigor index. Six clusters, which were generally related to the ability of the genotypes to tolerate anaerobic germination, were detected. The first two principal components accounted for 91.1% of the total variation, with survival percentage, seedling vigor index and shoot length being the main discriminatory traits. Genotypic coefficient of variation ranged from 18.21% for shoot length to 50.89% for seedling vigor index. Shoot length and seedling vigor index recorded high broad-sense heritability estimates (>60%), with accompanying high genetic advance as percent of mean (>20%); the latter ranged from 23.12% for root length to 82.21% for seedling vigor index. The high heritability, along with high genetic advance, particularly for shoot length and seedling vigor index, indicated that selection for these traits should be effective in improving tolerance to anaerobic germination. Five tolerant genotypes (OBOLO, ART68-12-1-1-B-B, ART64-31-1-1-B-B, CRI-1-21-5-12, and CRI-Enapa) with high survival percentage (≥70%) under anaerobic conditions were identified. These genotypes could be used to improve commercial rice varieties for tolerance to anaerobic germination.

Direct seeding of rice is a lightweight and simple cultivation method, which can effectively promote rice production. Anaerobic germination tolerance is one of the main traits of rice adaptability to direct seeding. The mining of related genetic loci, analysis of anaerobic traits and screening of tolerance genes provided valuable genetic resources for improving the anaerobic germination ability of direct seeding rice. This study conducted a dynamic genome-wide association study (GWAS) based on coleoptile-related traits of 591 rice natural populations, and a total of 317 SNP sites were detected. Integrated dynamic widely targeted metabolomics analysis, we found that xanthine, l-alanine and GABA may be key biomarkers that are sensitive and respond strongly to hypoxic stress perception. By WGCNA analysis of targeted metabolomics and transcriptomics, a total of 3 modules were obtained that were significantly correlated with the above three marker metabolites, namely dark green, dark gray and light green modules, respectively, and several key structural genes of OsAlaAT1, OsGAD4, OsAAH and Os09g0424600 that may affect hypoxic germination were screened from the 3 modules. Among them, OsAlaAT1 (Os10g0390500), located in Chr10-12877840, which is within the GWAS location range of CVAN3d, is considered to be a more reliable candidate gene. Overall, in addition to providing new insight into the metabolic regulation of l-alanine, GABA and xanthine during hypoxic germination of rice. This study also provided a reference for the basic theoretical research and breeding application research on the related traits of anaerobic germination in direct-seeding rice.

Anaerobic germination (AG) refers to a seed's natural capacity to germinate despite oxygen deficit by obtaining the necessary energy through anaerobic respiration. Assessing the genetic diversity of rice to create direct-seeded rice varieties with these traits is imperative. A collection of 60 rice genotypes from diverse regions of the North Eastern Hill Region was evaluated for characteristics related to anaerobic germination. Genotypes were submerged at an 8 cm depth for 21 days to screen for AG. Each of the parameters, including seedling vigour index, germination percentage, speed of germination, survival percentage, shoot length, root length, fresh weight, dry weight, and number of leaves, showed a highly significant genotype x treatment (anaerobic and control conditions) interaction, indicating substantial variation in the genotypes with respect to submergence. Principal Component Analysis detected a cumulative variance of 63.3% from first two PCs, of which PC1 accounted for 42.9% and PC2 for 20.4%. Four genotypes were determined to be tolerant (Lakang Baso, Sahbhangi Dhan, CAUS 107, CAUS 123), whereas the remaining were susceptible (BLM, Chakhao Poireiton, Phourel, and Mailung). The tolerant genotypes exhibited an average a-amylase activity of 228.51 mol min-1, which was substantially higher than that of the susceptible genotypes (77.15 mol min-1). A strong positive correlation was observed between a-amylase activity and survival percentage (r = 0.84), along with several other morphological parameters. Traits such as seedling vigour index, survival percentage, germination percentage, shoot length, and a-amylase activity were identified as reliable indicators for screening rice genotypes for anaerobic germination tolerance. The contrasting genotypes characterized in this study represent valuable genetic resources for future breeding programs.

Effect of water absorption patterns on anaerobic germination in rice seed

BackgroundRice is one of the most important food crops in the world, and with the development of direct seeding methods for rice, exposure to anaerobic stress has become a major factor limiting its growth.ResultsIn this experiment, we tested the tolerance to anaerobic germination of rice varieties NIP and HD84, and they were used as parents to construct a DH (doubled-haploid) population. The transcriptomes of NIP (highly tolerant) and HD86 (intolerant), and their progeny HR (highly tolerant) and NHR (intolerant) were sequenced from normal and anaerobic environments. The differentially-expressed genes (DEGs) were subjected to GO (Gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and WGCNA analyses. QTL mapping of the DH population identified tolerance to anaerobic germination-related chromosomal segments. The transcriptome results from 24 samples were combined with the anaerobic stress QTL results for 159 DH population lines to construct a metabolic network to identify key pathways and a gene interaction network to study the key genes. Essential genes were initially subjected to rigorous functional validation, followed by a comprehensive analysis aimed at elucidating their potential utility in domestication and breeding practices, particularly focusing on the exploitation of dominant haplotypes.ConclusionThe results show that pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are the starting signals of energy metabolism for coleoptile length growth, the auxin transporter EXPA is the determining signal for coleoptile length growth. The pivotal genes Os05g0498700 and Os01g0866100 exert a negative regulatory influence on coleoptile length, ultimately enhancing tolerance to anaerobic germination in rice. Analyses of breeding potential underscore the additional value of Os05g0498700-hyp2 and Os01g0866100-hyp2, highlighting their potential utility in further improving rice through breeding programs. The results of our study will provide a theoretical basis for breeding anaerobic-tolerant rice varieties.

Anaerobic Germination in Rice

The rising cost of transplanting rice has made direct seeding an affordable alternative for rice establishment, particularly in Africa. However, direct seeding, while cost-effective, faces crop establishment challenges due to flooding. Uncontrolled water, driven by erratic rains in low-lying areas or uneven fields, limit germination. Rice possesses the unique ability of anaerobic germination, enabling it to sprout and emerge in oxygen-deprived conditions. Understanding rice’s response to anaerobic stress during germination is crucial for resilience breeding. Africa, although relying on direct seeding, has made limited progress in addressing flooding during germination compared to Asia. Anaerobic stress tolerance ensures successful crop emergence even in oxygen-limited environments and can help suppress weeds, a significant challenge in direct-seeded rice cultivation. This study aims to contribute by screening for potential rice donors exhibiting anaerobic stress tolerance. We screened 200 rice genotypes at Sokoine University of Agriculture (SUA) in Morogoro, Tanzania, primarily focusing on landraces with untapped potential. Using an alpha lattice design, we conducted two anaerobic experiments in September and October 2022, adding 7 cm of standing water immediately after dry seeding for flooded and maintaining a 2 cm water level after germination in the control for duration of 21 days. We identified potential donors based on selection index computed from genomic estimated breeding values (GEBVs) using eight variables: germination at 14 DAS, germination at 21 DAS, seedling height at 14 DAS, seedling height at 21 DAS, shoot dry matter at 21 DAS, root dry matter at 21 DAS, culm diameter at 21 DAS, and root length at 21DAS. Ten genotypes emerged as the most promising, exhibiting at least 70% germination in floodwater at 21 DAS and greater selection indices. These genotypes were like: Afaa Mwanza 1/159, Rojomena 271/10, Kubwa Jinga, Wahiwahi, Magongo ya Wayungu, Mpaka wa Bibi, Mwangaza, Tarabinzona, IB126-Bug 2013A, and Kanamalia with respective percentages of 75, 74, 71, 86, 75, 80, 71, 80, 70, and 73. These findings contribute to global efforts to mitigate the impacts of flooding during germination. These donors, will be potential to enrich the gene pool for anaerobic germination, providing valuable resources for breeding for flooding tolerance.

Direct seeding of rice often results in poor crop establishment due to unlevelled fields, unpredicted heavy rains after sowing, and weed and pest invasion. Thus, it is important to develop varieties able to tolerate flooding during germination, also known as anaerobic germination (AG), to address these constraints. A study was conducted to identify QTLs associated with AG tolerance from an IR64/Kharsu 80A F2:3 mapping population using 190 lines phenotyped for seedling survival under the stress. Genotyping was performed using a genomewide 384-plex Indica/Indica SNP set. Four QTLs derived from Kharsu 80A providing increased tolerance to anaerobic germination were identified: three on chromosome 7 (qAG7.1, qAG7.2 and qAG7.3) and one on chromosome 3 (qAG3), with LOD values ranging from 5.7 to 7.7, and phenotypic variance explained (R2) from 8.1% to 12.6%. The QTLs identified in this study can be further investigated to better understand the genetic bases of AG tolerance in rice, and used for marker-assisted selection to develop more robust direct-seeded rice varieties.

Direct seeding of rice as a method of crop establishment is increasingly being adopted by farmers as a means of saving labor and reducing costs. However, the method often results in a poor environment for germination as excessive water levels after seeding can cause poor seedling establishment and a concomitant reduction in yield potential, especially in submergence-prone areas. In this study, we discovered QTLs associated with tolerance of anaerobic germination (AG) in new genetic accessions using genotypic data derived from the Illumina 6K SNP chip. The mapping population developed for QTL analysis comprised 285 F2:3 plants derived from a cross between Tai Nguyen and Anda. In order to evaluate AG tolerance within the mapping population, phenotyping was carried out under anaerobic conditions for 21 days. Three QTLs associated with AG tolerance were identified in the population, qAG1a and qAG1b on chromosome 1 and qAG8 on chromosome 8 using composite interval mapping (CIM). The percentage of variance explained by these QTLs ranged from 5.49 to 14.14%. The lines with three QTLs (qAG1b + qAG1a + qAG8) demonstrated an approximate 50% survival rate under anaerobic conditions, while lines with two QTLs including qAG1b demonstrated survival rates of 36 and 32% after the treatment, respectively. The QTLs detected in this study may be used to improve AG tolerance during germination and may be combined with other QTLs for anaerobic germination to enhance adaptation to direct seeding and to broaden the understanding of the genetic control of tolerance of germination under anaerobic conditions.