Exogenous metabolite application is a potential strategy for expanding the use of direct rice seeding with the aim of reducing seeding costs

Direct-seeded rice (DSR) is increasingly adopted to reduce production costs and labor, but germination and stand establishment are hampered by environmental stresses, particularly cold and submergence during water seeding. Climate variability exacerbates these constraints, leading to lower germination and higher seeding costs. This study addresses how to improve germination and early seedling vigor under cold and submergence to expand feasible use of DSR. The authors screened 66 rice varieties for germination, morphological, and physiological-biochemical performance under normal, cold, submergence, and combined cold plus submergence conditions. They aimed to identify tolerant varieties, uncover molecular and metabolic mechanisms underlying stress responses, and evaluate whether exogenous metabolite application can serve as a cost-effective seed enhancement strategy to reduce seeding costs while maintaining or improving establishment under adverse conditions.

The authors conducted a literature review via Web of Science using the keyword "enhance rice germination rate" and explored relevant MeSH headings such as gene expression regulation in plants, genetically modified plants, and signal transduction. Prior work indicates two major routes to improve germination: (1) crop breeding leveraging genetic diversity, including transgenic and genome editing approaches that accelerate improvement; and (2) seed enhancement technologies, notably exogenous hormone application and seed coating, to promote germination and early vigor. Related studies highlight roles of transcriptional regulation, hormone-mediated signaling, and metabolic adjustments in stress tolerance during germination and early growth.

- Germplasm and growth conditions: Sixty-six rice varieties commonly grown in Southern China were obtained from South China Agricultural University (SCAU). Seeds were cleaned, surface-sterilized (2% NaClO, 15 min), and germinated in boxes with filter paper and quartz sand using deionized water under controlled conditions (12 h light/12 h dark, 60% RH). - Stress treatments: Four environments were applied for 5 days, followed by 5 days recovery at normal conditions: T1S1 (control, 25°C, no submergence), T1S2 (25°C, 5 cm submergence), T2S1 (15°C, no submergence), and T2S2 (15°C, 5 cm submergence). Each treatment had four replicates. - Phenotyping and integrated evaluation: Germination rate of sprouted seeds and morphological and physiological-biochemical indicators were recorded under stress. A comprehensive D value integrating germination, morphology, and physiological-biochemical performance across the 66 varieties was calculated to rank stress tolerance and select contrasting varieties. - Variety selection for omics: Jingliangyou 1468 (RR, resistant) exhibited high D values; Basimadi 385 (SR, sensitive) showed low D values, particularly under cold and submergence. These two were selected for omics analyses. - Transcriptomics: RNA was extracted and sequenced (Illumina NovaSeq 6000). Reads were processed with fastp, aligned to the reference genome using HISAT2, assembled with StringTie, and quantified with RSEM. Differential expression was assessed with DESeq2 (|log2FC| > 1, adjusted p < 0.05). GO enrichment was analyzed using GOATOOLS. qRT-PCR on selected NAC and WRKY genes validated RNA-Seq expression patterns, with relative expression calculated by the 2^−ΔΔCt method. - Metabolomics: Leaf samples (n ≥ 3 biological replicates) were extracted (methanol:water 4:1 with internal standard) and profiled by UHPLC-Q Exactive HF-X MS in positive and negative ion modes. QC samples were run periodically. Data-dependent acquisition covered m/z 70–1050. Differentially expressed metabolites (DEMs) were identified, and pathway enrichment analyses were performed to identify pathways overrepresented among common and unique DEMs in RR and SR. - Seed enhancement (metabolite priming) experiment: Six glycerophospholipid-pathway-related compounds were tested: ethanolamine (Eth), choline (Cho), O-phosphorylethanolamine (PEth), phosphorylcholine (PCho), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Seeds were soaked 24 h at room temperature in solutions at 0, 25, 50, 75, or 100 μmol/L, then subjected to T1S1, T1S2, T2S1, and T2S2 for 5 days, followed by 5 days recovery. Germination and seedling growth indices (germination rate, germination index, dry weights) were measured. Cost analysis compared metabolite application cost to estimated cost savings from improved germination (details in Supplementary Notes 6–7). All experiments used completely randomized designs with three biological replicates unless otherwise specified.

- Variety screening: Selecting rice varieties with superior germination capacity under cold and submergence improved germination rates by 39.43%. - Transcriptomics: Across conditions, 37 DEGs were common to both varieties and enriched for signal transduction and hormone-mediated signaling pathways. NAC and WRKY transcription factor families were prominent; their expression levels were generally higher in the resistant variety (RR) than the sensitive variety (SR), and qRT-PCR validated expression patterns. Combined cold plus submergence elicited transcriptional responses similar to submergence alone. Hormone biosynthesis gene expression suggested roles for ethylene and auxin under different stresses. - Metabolomics: A total of 918 metabolites were identified. Venn analysis revealed 91 RR-common-induced DEMs and 58 SR-common-induced DEMs, with 10 shared DEMs enriched in the glycerophospholipid metabolism pathway. Unique DEMs in each variety were predominantly associated with amino acid and lipid metabolism. - Seed enhancement: Exogenous application of glycerophospholipid-pathway metabolites had stronger effects in SR than RR. Eth, Cho, PEth, and PCho significantly increased SR germination under stress by 8.61% to 20.97% (average 14.13%) compared with control. - Cost-effectiveness: Estimated cost of applying metabolites ($7.59) was lower than the cost savings from improved germination ($29.93), indicating economic viability of metabolite-based seed enhancement for varieties with poor germination. - Additional gene-level observations: Stress-upregulated genes included Os03g0745000 (HSF-related) and Os02g0758000, potentially contributing to stress responses.

The study demonstrates that strategic variety selection combined with targeted seed enhancement can mitigate the negative impacts of cold and submergence typical of water seeding in DSR. Transcriptomic data indicate that stress responses are mediated by hormone-related signaling, with NAC and WRKY transcription factors prominently involved, and that combined cold plus submergence resembles submergence responses, suggesting flooding can attenuate cold damage in early stages. Metabolomic analyses highlight glycerophospholipid metabolism as central to stress adaptation; functional validation via exogenous metabolite priming confirms this pathway’s contribution to improved germination and early growth under stress. The observed germination improvements, especially in sensitive varieties, translate into meaningful cost savings, supporting practical adoption. These findings connect molecular mechanisms with agronomic outcomes, offering a feasible route to expand DSR while controlling seeding costs and maintaining establishment in variable climates.

This work integrates germplasm screening, multi-omics analyses, and functional validation to propose a practical strategy for DSR: select varieties with strong germination under cold and submergence and apply specific glycerophospholipid-related metabolites (Eth, Cho, PEth, PCho) as a seed enhancement treatment. The approach improves germination (up to 39.43% via variety selection; 8.61–20.97% via metabolite priming in sensitive genotypes) and is cost-effective, reducing seeding costs. The study advances understanding of hormone-mediated signaling and glycerophospholipid metabolism in early stress responses and translates these insights into an actionable, economical intervention. Future work should include field trials across environments and seasons, validation of candidate genes through genetic approaches (e.g., knockouts), optimization of metabolite formulations and doses, integration with precision hill-drop drilling for stand uniformity, and assessment of impacts on later growth stages and final yield across diverse rice ecotypes and regions.

Findings are based on controlled indoor experiments and may not fully predict field performance; high germination does not guarantee higher yield. Direct-seeded rice often has a shorter growth period and may show weaker resistance to biotic/abiotic stresses later. High seeding rates in DSR can cause dense stands and disease pressure, though precision hill-drop drilling can mitigate this. Comparisons with transplanted rice indicate transplanted systems can yield higher on average, and outcomes may vary with seeding method and regional climate. The tested varieties are primarily from Southern China, which may limit generalizability; broader multi-location field validations are needed. Some transcriptomic inferences (e.g., roles of specific DEGs) require genetic and biochemical validation.