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Springer US New York

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10.1186/s12284-024-00734-8
Research
OsRAV1 Regulates Seed Vigor and Salt Tolerance During Germination in Rice
Gao Yingbo 12
Zhao Xinyi 12
Liu Xin 12
Liu Chang 3
Zhang Kunming 12
Zhang Xiaoxiang 4
Zhou Juan 12
Dong Guichun 12
Wang Youping 2
Huang Jianye 12
Yang Zefeng zfyang@yzu.edu.cn

12
Zhou Yong zhouyong@yzu.edu.cn

12
Yao Youli yaoyl@yzu.edu.cn

12
1 https://ror.org/03tqb8s11 grid.268415.c Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009 China
2 https://ror.org/03tqb8s11 grid.268415.c Key Laboratory of Plant Functional Genomics of the Ministry of Education/ Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, College of Agriculture, Yangzhou University, Yangzhou, 225009 China
3 https://ror.org/03tqb8s11 grid.268415.c Guangling College, Yangzhou University, Yangzhou, 225000 China
4 Jiangsu Lixiahe District Institute of Agricultural Sciences, Yangzhou, 225007 China
2 9 2024
2 9 2024
2024
17 5614 7 2024
22 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Seed vigor is a complex trait encompassing seed germination, seedling emergence, growth, seed longevity, and stress tolerance, all are crucial for direct seeding in rice. Here, we report that the AP2/ERF transcription factor OsRAV1 (RELATED TO ABI3 AND VP1) positively regulates seed germination, vigor, and salt tolerance. Additionally, OsRAV1 was differently expressed in embryo and endosperm, with the OsRAV1 localized in the nucleus. Transcriptomic analysis revealed that OsRAV1 modulates seed vigor through plant hormone signal transduction and phenylpropanoid biosynthesis during germination. Haplotype analysis showed that rice varieties carrying Hap3 displayed enhanced salt tolerance during seed germination. These findings suggest that OsRAV1 is a potential target in breeding rice varieties with high seed vigor suitable for direct seeding cultivation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12284-024-00734-8.

Keywords

OsRAV1
Seed vigor
Salt tolerance
Postgraduate Research & Practice Innovation Program of Jiangsu ProvinceXKYCX18_083 Yangzhou Modern Agriculture ProjectYZ2022043 http://dx.doi.org/10.13039/501100013058 Jiangsu Provincial Key Research and Development Program BE2022335 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 31571608 Natural Science Foundation of JiangsuBK20201219 issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcBackground

Rice (Oryza sativa) serves as a staple food for billions of people worldwide, highlighting the critical importance of producing and preserving rice seeds for food security. Seed vigor, encompassing traits like seed germination, seedling emergence and establishment, growth, seed longevity, and stress tolerance, is essential for successful direct seeding in rice (Zhao et al. 2021; Li et al. 2023). High-vigor seeds consistently exhibit rapid and uniform germination, robust seedling establishment, and resilience under stress conditions in agricultural production (He et al. 2019, 2020a; Zhao et al. 2021). However, seed vigor declines over time during storage, posing challenges to food production and genetic integrity (Yin et al. 2017).

Seed longevity, or seed aging, refers to a seed’s ability to remain viable in a dormant, dry state during prolonged storage. Factors such as high temperature and humidity during storage can diminish seed vitality, leading to reduced germination rates, abnormal seedling growth, and decreased tolerance to stress. Methods to study seed longevity include natural aging, where seeds deteriorate under ambient storage conditions, and artificially accelerated aging, involving controlled exposure to high temperature (around 42℃) and humidity (approximately 80%) over a specified duration (Zhou et al. 2024; Hay et al. 2018). Variability in seed responses among rice varieties underscores the need for refined methods in artificial aging studies; for instance, varieties like Kasalath can remain a 80% germination rate even after 18 days of artificial aging (Lee et al. 2019; Wang et al. 2022).

In field conditions, seed germination faces challenges from abiotic stresses such as drought, low temperature, and salinity, which particularly hinder rice seedling establishment (Zhao et al. 2021). Among these stresses, salt stress poses persistent obstacles, significantly impairing both seed germination and seedling’s growth (He et al. 2019). Thus, enhancing seed germination and seedling establishment under adverse conditions is a priority in rice breeding. Understanding the physiological and molecular mechanisms underlying abiotic stresses in seed vigor is crucial for safeguarding genetic resources, ensuring food security, and promoting consistent seedling establishment in simplified cultivation systems.

Recent researches have elucidated relationships between seed germination, longevity, and salt tolerance. For instance, precise regulation of gene expression levels, such as OsLOX2, ZmDREB2A, and ZmPIMT2, can balance regulation germination rate and seed longevity (Huang et al. 2014; Han et al. 2020; Zhang et al. 2023c). Moreover, overexpression of OsGLYI3 and ZmGOLS2 enhances both germination rate and salt tolerance during germination (Gu et al. 2016; Liu et al. 2022), whereas overexpression of OsLOX10 and ZmAGA1 may improve salt tolerance but compromise seed longevity (Zhang et al. 2021; Wang et al. 2023). Strategic use of allelic variations in gene promoters or coding regions, such as OsNAC3 and OsPK5, can modulate transcription levels or protein activity to optimize seed germination, longevity, and salt tolerance (Aung et al. 2023; Huang et al. 2024; Yamasani et al. 2023; Yang et al. 2022; G. Zhang et al. 2023).

APETALA2/ethylene response factors (AP2/ERFs) comprise a large transcription factors super-family in plants, characterized by one or two AP2/ERF domains crucial for DNA binding (Nakano et al. 2006; Sharoni et al. 2011). RNA-seq analyses highlight the involvement of AP2-domain-containing regulators in seed germination in rice (He et al. 2020b). Within this regulatory network, OsRAV1 interacts notably with OsbZIP23, known for its positively regulation of seed vigor (Wang et al. 2022). Previous studies suggest that OsRAV1 may play a role in salt tolerance regulation based on its induction in salt-tolerant varieties (Liu et al. 2024), implicating OsRAV1 in governing seed vigor from germination through early seedling stages.

In this study, we explore the regulator role of OsRAV1 in seed vigor. Overexpression of OsRAV1 significantly enhances seed vigor, germination, and salt tolerance, whereas disruption of OsRAV1 reduces seed vigor. RNA-Seq data indicate that OsRAV1 may modulate seed vigor through plant hormone signal transduction and phenylpropanoid biosynthesis pathways. Moreover, an elite haplotype of OsRAV1 found in the Geng accession version shows promise for seed germination improvement under salt stress, suggesting OsRAV1 as a potential target for genetic improvement of seed germination, vigor, and salt tolerance in rice, particularly for direct seeding applications in the future.

Results

OsRAV1 Expression Pattern in Different Rice Tissues and Subcellular Localization

To analyze the function of OsRAV1 in the growth and development of rice plant, we examined its expression profile in various tissues at the booting stage using RT-qPCR. The results demonstrate constitutive expression of OsRAV1 across different tissues, with higher levels in mature leaves and sheaths, and lower expression in newly formed organs. Expression was lower in young panicles but increased in later stages of panicle development (Fig. 1A). To validate these patterns, we constructed a vector with the GUS gene driven by the OsRAV1 promoter and used it for rice transformation. Importantly, GUS activity was the highest in panicle tissues and roots during the booting stage, indicating a crucial role of OsRAV1 in rice panicle developmental (Additional file 1: Fig. S1A. GUS activity was notably strong in roots and sheaths at 14d after germination (Additional file 1: Fig. S1B). Subcellular localization studies using an OsRAV1-green fluorescent protein (GFP) fusion construct under the CaMV 35 S promoter showed that OsRAV1 localized in the nucleus, confirmed by confocal microscopy in both Nicotiana benthamiana leaf epidermal cells and rice protoplasts (Fig. 1B).

Fig. 1 Expression patterns of OsRAV1 and its subcellular localization in rice. A Expression patterns of OsRAV1 in various developmental stages in rice by RT-qPCR. B Subcellular localization of OsRAV1 with GFP in tobacco leaves and rice protoplasts. Scale bars = 50 μm (tobacco leaves) or 10 μm (rice protoplast). C Expression of OsRAV1 in response to salt (NaCl). Rice seedlings (14 days old) were treated with 120 mM NaCl. RNA from shoots (leaf) were used for RT-qPCR, with OsActin as an internal control. The error bars indicate the SE based on three independent replicates

Next, we investigated OsRAV1 expression in response to various abiotic stress and hormone treatment. Our findings indicate that OsRAV1 expression is notably induced by salt (NaCl), ABA, and osmotic stress (PEG) (Fig. 1C; Additional file 1: Fig. S1C and D), suggesting its responsiveness to a broad range of abiotic stressors.

OsRAV1 Improves Germination Rate and Seed Vigor

To further explore OsRAV1 function, we generated overexpression (OE) lines by inserting the OsRAV1 coding region under the 35 S promoter. Increased OsRAV1 in the OE lines were confirmed by RT-qPCR (Additional file 1: Fig. S2A). Additionally, OsRAV1 knockout mutants (osrav1-1 and osrav1-2) were generated using CRISPR/Cas9 technology, resulting in premature termination of OsRAV1 and disruption of the AP2 and B3 domains (Additional file 1: Fig. S2B and C).

The OsRAV1 expression is upregulated and linked to gene networks related to seed vigor in Kasalath (Wang et al. 2022). Our germination assay showed that under control conditions, OE lines and Nipponbare (NIP) had similar germination rates, whereas osrav1 mutants showed lower germination rate than NIP (Fig. 2A and B). Notably, only OE lines maintained seed vigor after one year of indoor storage (S-1Y), with germination rates reaching 28–48% within 120 h (Fig. 2A and C). As storage time increased, seeds aged and lost germination ability. OsRAV1 appears to play a crucial role in enhancing seed vigor. Seed accelerated aging, a method for rapid seed vigor assessment, was employed in aging seeds of NIP, OE lines, and osrav1 mutants at 42℃ and 80% relative humidity for 11、14、17, and 20 days (Fig. 2D-G). With increasing aging time, initial germination rates of OE lines were higher than those of NIP, while osrav1 mutants almost completely lost their germination ability at 17 and 20 days.

Fig. 2 OsRAV1 positively regulates seed vigor under aging conditions. A Images of germination. B Newly harvested seeds. C The seeds after stored in room conditions for one year (S 1Y). C-G The seeds aged under 42 ℃ and 80% RH for various period as indicated

Given the lengthy process of traditional aging method (high temperature and humidity), we developed an improved method using seeds soaked in saturated NaCl solution and stored in darkness at 42℃ (Fig. 3). For germination, the seeds were firstly rinsed in tap water, then imbibed in distilled water for 72 h at 4℃ and followed by placing on moistened filter paper at 28℃ under alternating dark and light (12 h/12 h) conditions. Germination rates decreased rapidly with aging time, with a majority of seeds losing germination ability by 9 days (Fig. 3H). Germination rates (36 h, 48 h, and 120 h) and other indices of OE lines were significantly higher than those of NIP at aging times of 5 and 7 days (Fig. 3I-L), confirming that OsRAV1 positively regulates seed vigor.

Fig. 3 OsRAV1 positively regulates seed vigor. A-H The germination rate after aging treatment. The seeds aged in saturated NaCl solution at 42℃ for various length as indicated. I-K The germination rates at 24, 36, and 48 h. L Germination index. Different small letters indicate significant differences at p ≤ 0.05 within the comparison group

OsRAV1 Expression Patterns in Seeds at Various Germination Stages

To further comprehend OsRAV1’s physiological role, we examined its expression patterns during seed germination and aging stages using RT-qPCR and GUS activity assays (Fig. 4). OsRAV1 transcript were prominently detected the imbibition phase (1–3 days), decreasing significantly in early germination stages (Fig. 4B). Further analysis revealed significant OsRAV1 expression in both embryo and endosperm during imbibition, with higher expression in the embryo (Fig. 4C). To exclude low-temperature effects on OsRAV1 expression patterns, we examined transcript levels during germination without prior imbibition. OsRAV1 transcripts were highly expressed during early germination (3 and 6 h), with higher levels in the embryo, though the gap narrowed over time (Fig. 4D). During aging treatments, OsRAV1 expression in the endosperm increased significantly, surpassing levels observed in the embryo. This pattern resembled that found in dry seeds (Fig. 4E), suggesting distinct roles for OsRAV1 during seed germination and storage conditions.

Fig. 4 OsRAV1 expression and GUS activity were observed in germination stages. A Histochemical GUS staining reporting OsRAV1 activity during seed germination. I-1d, I-2d, and I-3d refer to seeds imbibed in distilled water at 4℃ for 1, 2, and 3 days, respectively. G-1d, G-2d, and G-3d indicate seeds germination in distilled water at 28℃ for 1, 2, and 3 days after imbibed, respectively. BOsRAV1 expression in whole seeds. COsRAV1 expression in embryo and endosperm during imbibition (4℃). DOsRAV1 expression in embryo and endosperm during germination (28℃), without prior imbibition (4℃). EOsRAV1 expression in embryo and endosperm under aging

OsRAV1 Improve Seed Vigor and Direct Seeding under Salinity Stress

Salt stress severely limits rice production globally, particularly under direct seeding conditions. Under control conditions, OE lines exhibited significantly higher germination rates than NIP at 24 h, but this trend reversed at other time points (Fig. 5A and B). As NaCl concentration increased, NIP germination rates gradually decreased compared to the OE lines, particularly evident at 200mM NaCl (Fig. 5C, D, F, and G). Conversely, osrav1 mutants showed greater sensitivity to increasing NaCl concentration compared to NIP, with significantly reduced germination rates (Fig. 5).

Fig. 5 OsRAV1 enhances initial germination rate under salt stress. A Images of seed germination. B-D The germination rate under mock (control treatment), 120, and 200 mM NaCl. E-G The initial germination rates (24, 36, and 48 h) under 0, 120, and 200 mM NaCl. Different small letters indicate significant differences at p ≤ 0.05 within the comparison group

To assess OsRAV1’s utility in direct seeding, we compared seedling establishment and growth between NIP, OE lines, and osrav1 mutants directly sown in soil (Fig. 6). Under normal conditions, no significant differences were observed in seedling percentage between NIP and OE lines at 7 days after sowing (Fig. 6B). However, OE lines exhibited significantly longer roots and greater root dry weights than NIP. In contrast, seedling percentage, shoot length, shoot dry weight, root length, and root dry weight were significantly reduced in osrav1 mutants compared to NIP (Fig. 6). Under 0.5% NaCl stress, OE lines showed significantly higher seedling establishment and growth than NIP. osrav1 mutants exhibited heightened sensitivity to increasing NaCl concentrations, ultimately losing the ability to germinate.

Fig. 6 Seedling growth of direct seeding in soil. A Seedling establishment and seedling growth under normal conditions or salinity stress at 7 days after seeding. B-F The seedling percentage, shoot lengths, root lengths, shoot dry weight, and root dry weight of OE, osrav1, and NIP. Different small letters indicate significant differences at p ≤ 0.05 within the comparison group

Association of OsRAV1 Haplotype with Seed Germination under Salinity Stress

To investigate whether OsRAV1 allelic variation affects seed germination under salinity stress, the 1 kb upstream region of OsRAV1 were analyzed using SNP and saline-alkali tolerance germination data from 603 accessions (McCouch et al. 2016; Zhang et al., 2023) (Fig. 7A; Additional file 3: Table. S2). Analysis of SNP correlation with germination identified four haplotypes (n > 15 accession) of OsRAV1. Hap3 showed the highest mean relative germination rate (RGR), suggesting it as the favorable haplotype for saline-alkali tolerance, predominantly found in Geng accessions (Fig. 7B and C).

Fig. 7 Haplotype of OsRAV1 associated with germination under salt stress. A Haplotypes in the 1 kb promoter region upstream of OsRAV1 CDS. B Distribution of relative germination rate (RGR) for the four OsRAV1 haplotypes. C Frequency of four OsRAV1 haplotypes in accessions. Different small letters indicate significant differences at p ≤ 0.05 within the comparison group

RNA-seq and Differential Expression Analysis

Given the significant difference in germination rates between osrav1 mutants and OE lines, and the sustained higher germination rates in OE lines, we performed RNA-seq to elucidate the molecular pathways underlying OsRAV1-regulated seed germination. Post-imbibition seeds of NIP, OE lines, and osrav1 mutants at initial germination stages (0 and 24 h) were sequenced (RNA-seq), with 30 cDNA libraries constructed representing three biological replicates for each of NIP, OE1, OE2, osrav1-1, and osrav1-2. Principal component analysis (PCA) revealed close overlap among biological replicates of OE lines and osrav1 mutants, respectively (Fig. 8A). DESeq (differential gene expression analysis) identified DEGs based on |log2FoldChange| ≥ 1 and P value ≤ 0.05 criteria. Venn diagram analysis revealed 306 genes (0H2) differentially expressed in osrav1_0H and OE_0H, while osrav1_24H and OE_24H shared 528 genes (24H2). Notably, 4124 DEGs (24H1) were exclusively present in osrav1_24H (Fig. 8B), indicating profound transcriptome changes at 24 h of germination, particularly affected by absent of OsRAV1 expression.

Fig. 8 Transcriptome analysis of DEGs regulated by OsRAV1. A Principal component analysis (PCA) based on the expressed genes. B Venn diagrams of the DEGs in OE and osrav1 seeds compared with NIP. 0 H and 24 H refer to the germination stages at 0 and 24 h, respectively. 0H1: DEGs exclusive to osrav1; 0H2: DEGs common between osrav1 and OE; 0H3: DEGs exclusive to OE in germinated seeds (0 h). 24H1: DEGs exclusive to osrav1; 24H2: DEGs common between osrav1 and OE; 24H3: DEGs exclusive to OE in germinated seeds (24 h). C-H KEGG enrichment analysis of DEGs exclusive to osrav1 (C and F) or OE (E and H), or common in both (D and G)

Gene ontology (GO) analysis of 0H1, 0H2, 0H3, 24H1, 24H2, 24H3 revealed enrichment in catalytic activity, metabolic process, response to stimuli, and stress (Additional file 1: Fig. S3). KEGG analysis highlighted enrichment in protein processing in endoplasmic reticulum and phenylpropanoid biosynthesis in 0H1 and 0H2 comparisons (Fig. 8C and D). However, the 0H3 exhibits significant enrichment in phenylpropanoid biosynthesis, rather than protein processing in the endoplasmic reticulum (Fig. 8E), indicating distinct seed status between OE lines and osrav1 mutants, formed during seed development and storage, lasted to even after a 3-day imbibition. Additional evidences are DEGs in 24H1 and 24H3 enriched in plant hormone signal transduction, whereas not for 24H2 (Fig. 8F-H; Additional file 1: Fig. S4A). These suggested that OsRAV1 regulates seed vigor (balance) through different hormonal pathways, in addition to crucial regulation of phenylpropanoid biosynthesis in the germination stage of rice seeds (Fig. 8F-H; Additional file 1: Fig. S4B).

Venn diagram analysis showed that 5525 genes were differentially expressed in 24 h germinated seeds compared to 0 h (Fig. 9A). To discover pathways that confer to the seed’s ability to germinate rapidly, we performed a cluster analysis of the genes. 2640 genes were higher expressed in 24 h germinated seeds compared to 0 h and then manually classified into two clusters (C1 and C2) (Fig. 9B). The genes of cluster C1 were down-regulated in osrav1 mutants, while those in cluster C2 were up-regulated in OE lines. Both C1 and C2 were mainly enriched in similar GO terms: response to stimulus, response to abiotic stimulus, and response to stress (Fig. 9C and D). KEGG analysis showed that C1 genes were involved in phenylpropanoid biosynthesis and plant hormone signal transduction, whereas C2 genes were significantly enriched in protein processing in the endoplasmic reticulum (Fig. 9E and F). MapMan analysis revealed that DEGs involved in abiotic stress, antioxidant, signaling, transcription factors, and defence pathways were active during seed germination (Additional file 1: Fig. S5). Further studying the functions of these genes will shed light on the molecular mechanisms of seed germination.

Fig. 9 Transcriptome analysis of DEGs in seeds germinating at 0 and 24 h. A Venn diagrams of DEGs in osrav1, OE, and NIP comparisons. B Expression profile of common DEGs (5525). OE: OE1 and OE2; osrav1: osrav1-1 and osrav1-2; with three biological replicates each. Clustering was drawn based on normalized FPKM. C, D Top 20 GO terms of cluster 1 (C1) and cluster 2 (C2). E, F KEGG enrichment analysis of C1 and C2

Discussion

Seed vigor is a complex trait encompassing seed storability, germination, seedling emergence and growth, and stress tolerance, influenced by various factors such as reserves accumulation and seed dormancy during development, seed deterioration during storage, and seed treatments and stress conditions during germination in rice. Developing new varieties with high seed vigor is crucial for direct seeding of rice. To achieve this goal, understanding the complex regulatory network of seed germination is prerequisite. Previously, AP2/ERF transcription factors have been reported to involve the balance of ABA and GA, seed vigor and stress-related processes in plants. ABI4 directly binds to NCED6 and GA2ox7, regulating ABA synthesis and GA degradation by controlling their transcription levels, which in turn regulates seed dormancy (Shu et al. 2013, 2016). Moreover, salt-induced ABI4 enhances RbohD expression, promotes ROS accumulation, thus results in cell membrane damage, and, consequently, a decrease in seed vigor (Luo et al. 2021). OsAP2-39 activates OsNCED1 transcription to enhance ABA synthesis, promoting GA degradation through the inhibition of OsEUI accumulation, therefore, enhances seed dormancy in rice (Yaish et al. 2010; Shu et al. 2013). Additionally, OsSAE1 promotes seed germination and salt tolerance in seedlings by suppressing OsABI5 expression. In this study, OsRAV1 positively regulated seed vigor, including initial germination rate and seedling establishment under salt stress (Figs. 5 and 6). Furthermore, transcriptome and metabolome analysis of seed aging treatments revealed that various AP2/ERF transcription factors likely serve as pivotal regulators within the complex network that governing seed vigor. Among them, OsRAV1 is highly induced and associated with positive regulation of OsbZIP23 (Wang et al. 2022). Under both natural and accelerated aging treatment, OE lines exhibited higher germination rates compared to NIP, while osrav1 mutants showed the opposite trend (Figs. 2 and 3). In plants, the growth-defense trade-off is commonplace. Readjustment of growth and adaptation to various stresses are crucial for survival under adverse environments. Previous studies have revealed associations among seed germination, longevity, and salt tolerance during germination phase. OsRAV1 is a candidate gene that positively regulates seed vigor and salt stress resistance.

Previous studies have shown that the indica (Xian) variety have higher seed vigor than japonica (Geng) varieties. To determine whether OsRAV1 contributes to the differences of seed vigor between Xian and Geng rice, allelic diversity analysis of OsRAV1 was conducted using 603 rice accessions, including both Xian and Geng accessions (Mansueto et al. 2017; Zhang et al. 2023a). Analysis of SNP data revealed that the germination rate of hap3 was significantly higher than that of other haplotypes under salt stress (Fig. 7B). Interestingly, Hap3 was predominantly found in Geng accessions and absent in Xian accessions. The genetic determinants of crop phenotypic diversity often involve natural mutations in promoters or coding sequences, which can modify transcriptional expression or protein functionality. We speculate that SNPs in OsRAV1 could affect its expression, and further investigation is needed to confirm its potential as a candidate gene for enhancing seed vigor, particularly in Geng accessions.

Omics analyses such as transcriptome, proteome, and metabolome analyses, have been employed to elucidate seed germination process in several crop species. Previous studies have identified a variety of biological processes involved in rice seed germination, including cell-wall metabolism, nucleotide degradation, amino acid biosynthesis, stress-response pathways, and more (Zhao et al. 2020b; Yang et al. 2020). However, these results were obtained using dry seeds, which inevitably undergo imbibition. Water uptake by mature dry seeds occurs in three phases: rapid phase (phase I), plateau phase (phase II), and a second rapid phase of water uptake (phase III). Seed imbibition and germination are overlapping processes; during phase II and phase III, the radicle protrudes through the seed coat (Zhao et al. 2020b; He et al. 2020b). It is equally important to understand genes responses from imbibed seeds to seedling establishment (radicle protrusion included).

In this study, we analyzed gene expression changes during the initial germination stage using RNA-Seq. Protein processing in the endoplasmic reticulum was mainly observed in 0H1 and 0H2, while phenylpropanoid biosynthesis was enriched in 0H3, with no significant protein processing pathways observed in the endoplasmic reticulum (Fig. 8C-E). These results indicate that OE lines achieved germination earlier, as confirmed by KEGG analyses of 24 h germinated seeds. DEGs in 24H1, 24H2, and 24H3 were all enriched in phenylpropanoid biosynthesis (Fig. 8F-H). Many pathways involved in seed germination are directly or indirectly related to phenylpropanoid biosynthesis (Foley et al. 2013). Phenylpropanoid biosynthesis continues to be enriched during seed germination and seedling establishment stages (Yang et al. 2020), and changes in phenylpropanoids-related metabolites contents have been observed in rice seeds under low-temperature stress(Yang et al. 2019). Phenolic acids, a major component of phenylpropanoids, play critical roles in defense against reactive oxygen species (ROS). In the Bidens pilosa L, Bruceine D inhibits seed germination by suppressing phenylpropanoid biosynthesis and inducing ROS accumulation (Tong et al. 2022). Lignin, a crucial phenylpropanoid compounds, acts as an inducible physical barrier that enhances plant seeds resistance to environmental stress during germination (Rao and Dixon 2017). SiMYB16 enhances salt tolerance in rice by regulating lignin biosynthesis (Yu et al. 2023). Interestingly, a recent report reveals that bZIP23-PER1A–mediated detoxification pathway enhances seed vigor in rice (Wang et al. 2022). The expression of OsbZIP23 and PER1A were significantly upregulated in osrav1 mutants, suggesting that the H2O2 content might be elevated (Additional file 1. Fig. S4C). We speculate that OsRAV1 enhances seed vigor through phenylpropane pathway.

ABA-inhibited seed germination is regulated by a core regulatory mechanism (Zhao et al. 2020a). Interestingly, DEGs unique to 24H1 and 24H3 were significantly enriched in plant hormone signal transduction, whereas shared DEGs in 24H2 were not (Fig. 8F-H; Additional file 1: Fig. S4A), indicating that OsRAV1 regulates seed vigor through different hormonal pathways. Several genes in the ABA signaling core are clearly regulated by OsRAV1, such as OsAPKs, OsBZIPs, OsABIs, and OsPYL. The expressions of OsbZIP23, OsbZIP72, OsABI5, and OsPP2C51, all were significantly upregulated in osrav1 seeds (Additional file 1: Fig. S4C), have essential role in ABA signaling, stress tolerance or seed vigor in rice (Bhatnagar et al. 2017; Wang et al. 2020, 2022; Li et al. 2022). On the contrary, the expressions of auxin related genes were significantly reduced in osrav1 seeds (Additional file 1: Fig. S4C), indicating that OsRAV1 affects seed germination through the crosstalk of auxin and ABA. Further studies should focus on interactions of OsRAV1 with upstream components of the auxin and ABA signaling pathway.

In most studies, rice seed vigor factors are mainly considered at the seed germination stage, with limited consideration of other stages. Seed storability, defined as the ability to remain viable during storage, is an important agronomic and physiological characteristic. Compared to natural aging, accelerated aging accelerates seed deterioration using high temperature and high humidity, significantly reducing processing time (Hay et al. 2018). In this study, seed germination rates of OE lines remained high under accelerated aging treatment (Fig. 2). In the accelerated aging method, seeds were suspended above either a saturated NaCl solution or water to generate a high-humidity environment (Hay et al. 2018; Wang et al. 2022). Would soaking seeds in an saturated NaCl solution achieve similar results? As expected, with increased aging time (9d), seeds lost their ability to germinate. This method represents another potential accelerated aging method for testing seed vigor, without requiring humidity control.

The endosperm is the major tissue for seed reserve accumulation in cereal grains, serving as a nutrient source for seed germination and subsequent seedling establishment. Recent evidence suggests that mature endosperm is essential for seedlings establishment; its removal impedes seedling cuticle formation, resulting in nonviable seedlings with developmental defects. This outcome is not due to lack of nourishment in the embryo (De Giorgi et al. 2021). Transcriptome analysis of embryo and endosperm at 0, 1, 3, 6, 9, 12, and 24 h post-imbibition revealed that differentially responsive genes are specific to embryo and endosperm. Embryo-specific DEGs are enriched in plant hormone signal transduction pathways, while endosperm-specific DEGs are enriched in biosynthesis pathways of phenylalanine, tyrosine, and tryptophan (Zhang et al., 2023). In this study, we observed distinct expression patterns of OsRAV1 between embryo and endosperm, highlighting tissue-specific regulatory roles. High OsRAV1 expression in endosperm of both dry seeds and seeds subjected to aging treatment suggests a significant role in stress resistance (storage). Upon water absorption at right temperature and initiation of germination, OsRAV1 expression levels in endosperm decrease gradually, while there is a significant increase (up to 9-fold in G-3 h) in embryos (Fig. 4D). At low temperature (4℃), OsRAV1 expression in embryo significantly increases (approximately 119-fold in I-3d), underscoring its crucial role in germination, particularly under challenging conditions (Fig. 4C). We speculate that OsRAV1 may regulate signaling communication between endosperm and embryo, playing a critical role in seed vigor and germination.

Conclusion

In conclusion, we have validated the AP2/ERF TF gene OsRAV1 is essential for seed vigor in rice. The Hap3 of OsRAV1 is a beneficial variant that increases rice seed germination under salt stress, and overexpression of OsRAV1 improves seed germination, vigor, and salt resistance, potentially making a significant contribution to the direct seeding cultivation of rice. The RNA-Seq results demonstrated that OsRAV1 may regulate seed germination through plant hormone signal transduction and phenylpropanoid biosynthesis. Furthermore, immersing seeds in a saturated NaCl solution at elevated temperature is an effective method for significantly accelerating the aging process.

Materials and Methods

Rice Varieties and Culture

The japonica rice variety Nipponbare (NIP) served as a wild-type plant and recipient for genetic transformation. Rice plants were cultivated at the farm of Yangzhou University, Jiangsu, China, during the rice seasons (May to October). Seed preparation and seedling nursery followed local practices as previously described (Gao et al. 2024). Seeds were harvested at their maturity stage and dried at 42℃ for 7 days to break seed dormancy.

Generation of Transgenic Plants

To generate OsRAV1 overexpression lines (OE), the coding region was amplified from NIP first-strand cDNA and inserted into the binary vector p1301UbiNOS under the maize ubiquitin promoter. Knockout osrav1 mutants were generated using CRISPR/Cas9 technology, targeting the sequence 5’-GCTCGTCCACGGACAAGCTGAGG-3’ of OsRAV1 in the pC1300-Ubi: Cas9 vector (Miao et al. 2020; Gu et al. 2022). The 2.0-kb OsRAV1 promoter sequence from NIP genomic DNA was inserted into the pCAMBIA1301 vector to drive expression of the GUS-encoding gene. All constructs were transformed into Agrobacterium tumefaciens EHA105 cells via chemical transformation and then subsequently into plants using A. tumefaciens-based method (Hiei et al. 1994). Primers used for constructing recombinant vectors are listed in Additional file 2: Table S1.

Seed Accelerated Aging

Seed accelerated aging followed established protocols with some modifications (Delouche and Baskin 1973; Wang et al. 2022). Seeds were subjected to saturated NaCl solution and aged in darkness at 42℃. We prepare ed the saturated NaCl solution and completed the aging process of the seeds in a 42℃ oven. First, we prepared an oversaturated NaCl solution in a glass bottle and placed it in the oven for 24 h, until undissolved crystals appeared at the bottom of the bottle, indicating the solution was saturated. Next, we transferred the solution to a 2.5 L conical flask and filled it to the brim to ensure it could submerge the seeds. We then placed the dry seeds in a mesh bag and immersed them in the solution. To prevent the seeds from floating, we blocked the mouth of the flask with a mesh bag and sealed it with a glass stopper. Prior to germination, seeds were rinsed with tap water and imbibed in distilled water at 4℃ for 3 days.

Seed Germination and Direct Seeding

For germination assays, twenty seeds per replicate of NIP, OE lines (OE1 and OE2), and osrav1 mutants (osrav1-1 and osrav1-2) were placed in 9 cm diameter Petri dishes with 12 mL distilled water at 28℃ for 5 days. NaCl concentrations 120 and 200mM were chosen when exposed to salt stress in Petri dishes. Germination was recorded when radicle protruded the seed coat (1 mm), germination index was calculated as ∑ (Gt/t), where Gt is the number of the germinated seeds on day t (He et al. 2019). Additionally, thirty seeds per replicate were sown at a depth of approximately 1 cm in soil at 28℃ for 7 days, with NaCl concentration of 0%, 0.25% and 0.5% (w/w). Root and shoot lengths and dry weight were measured. Three replications were conducted.

Expression Analysis and Histochemical Analysis of GUS Activity

Total RNA was extracted from dry seeds, seeds subjected to aging (3, 5, and 7 days), seeds undergoing imbibition (1–3 days), germinating seeds (1–4 days, after 3 days imbibition ), germinating seeds (3, 6, 12, 24, 48, and 72 h, without pre-imbibition), and various tissues (leaves, nodes, sheaths, and young panicles at the booting stage) using an RNA simple Total RNA kit (Tiangen, Beijing, China). Reverse transcrition into cDNA was performed using the Fast King One-step RT-qPCR kit (Tiangen, Beijing, China). RT-qPCR analysis was conducted as previously described (Liu et al. 2024). Primers used for RT-qPCR are listed in Additional file 2: Table S1. GUS activity was detected using the GUSblue kit (Huayueyang, Beijing, China) and samples were destained with pure ethanol prior to examination.

Subcellular Localization

The OsRAV1 coding sequence was PCR-amplified and cloned into pAN580 and pHG vectors to obtain pAN580-OsRAV1 and pHG-OsRAV1 constructs, respectively. Transient expression assays in rice protoplasts were performed as described previously (Chen et al. 2006; Gu et al. 2022). Agrobacterium tumefaciens GV3101 cells transformed with pHG empty vector or pHG-OsRAV1 constructs were used to infiltrate healthy Nicotiana benthamiana leaves. Fluorescent signals were visualized using a Zeiss LSM 710 laser scanning confocal microscope. The primers used for constructing recombinant vectors are listed in Additional file 2: Table S1.

Haplotype Analyses

Relative germination rates (ratio of saline-alkali stress to control) of 603 accessions of rice were obtain from a previous study (Zhang et al., 2023) (Additional file 3: Table S2). SNPs within the 1 kb region upstream of OsRAV1 were downloaded from the Rice SNP-Seek database (https://snpseek.irri.org/) (Mansueto et al. 2017). Haplotype analysis focused on variants present in at least 15 accessions.

RNA-seq and Analysis

Thirty tissue samples representing three OsRAV1 levels (NIP, OE1, OE2, osrav1-1, and osrav1-2) and two germination stages (0 and 24 h) with three biological replicates each were collected. Seeds (25 per replicate) were frozen in liquid N2 and stored at -80℃ until RNA extraction. RNA extraction, sequencing, and data analysis were conducted as previously described (Zhang et al. 2019). Differentially expressed genes (DEGs) (Padj<0.05 and fold change ≥ 2) were identified using DEGseq2 software. GO enrichment and KEGG pathway analysis were performed using Personal Gene Cloud (https://www.genescloud.cn). Heatmaps were generated using TBtools software with row scaling set to normalized (Chen et al. 2020).

Statistical Analysis

IBM SPSS Statistics version 26.0 software was used for statistical analysis. Differences were considered significant at p ≤ 0.05. Figures were prepared using OriginPro 2016 (OriginLab, Northampton, Massachusetts, USA) or Excel 2019.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Acknowledgements

We thank Professor Chen Chen from Yangzhou University for providing the pHG vector.

Author Contributions

YY, ZY, and YZ designed the research. GY and YY analyzed the data and prepared the manuscript. GY and ZX performed most of the experiments with the assistance of LX, LC, ZK, ZX, ZJ, DG, and HJ. All authors read and approved the final manuscript.

Funding

This work was financially supported by a grant from Jiangsu Provincial Key R&D Programme - Modern Agriculture (BE2022335), National Natural Science Foundation of China (31571608), Natural Science Foundation of Jiangsu (BK20201219), Yangzhou Modern Agriculture Project (YZ2022043) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (XKYCX18_083).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.

Abbreviations

ABA Abscisic Acid

DEGs Differentially expressed genes

GO Gene ontology; GUS:β-Glucuronidase

Hap Haplotype

KEGG Kyoto Encyclopedia of Genes and Genomes

PEG Polyethylene glycol

RGR Relative germination rate

RT-qPCR Quantitative real-time PCR

RNA-seq RNA sequencing

S-1Y Stored for one year

SNP single nucleotide polymorphism

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

Aung KM Oo WH Maung TZ Min M-H Somsri A Nam J Kim K-W Nawade B Lee C-Y Chu S-H Park Y-J Genomic landscape of the OsTPP7 gene in its haplotype diversity and association with anaerobic germination tolerance in rice Front Plant Sci 2023 14 1225445 10.3389/fpls.2023.1225445 37560030
Aung KM, Oo WH, Maung TZ, Min M-H, Somsri A, Nam J, Kim K-W, Nawade B, Lee C-Y, Chu S-H, Park Y-J (2023) Genomic landscape of the OsTPP7 gene in its haplotype diversity and association with anaerobic germination tolerance in rice. Front Plant Sci 14:1225445. 10.3389/fpls.2023.122544537560030 10.3389/fpls.2023.1225445
Bhatnagar N Min M-K Choi E-H Kim N Moon S-J Yoon I Kwon T Jung K-H Kim B-G The protein phosphatase 2 C clade A protein OsPP2C51 positively regulates seed germination by directly inactivating OsbZIP10 Plant Mol Biol 2017 93 4–5 389 401 10.1007/s11103-016-0568-2 28000033
Bhatnagar N, Min M-K, Choi E-H, Kim N, Moon S-J, Yoon I, Kwon T, Jung K-H, Kim B-G (2017) The protein phosphatase 2 C clade A protein OsPP2C51 positively regulates seed germination by directly inactivating OsbZIP10. Plant Mol Biol 93(4–5):389–401. 10.1007/s11103-016-0568-228000033 10.1007/s11103-016-0568-2
Chen S Tao L Zeng L Vega-Sanchez ME Umemura K Wang G A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice Mol Plant Pathol 2006 7 5 417 427 10.1111/j.1364-3703.2006.00346.x 20507457
Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang G (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice. Mol Plant Pathol 7(5):417–427. 10.1111/j.1364-3703.2006.00346.x20507457 10.1111/j.1364-3703.2006.00346.x
Chen C Chen H Zhang Y Thomas HR Frank MH He Y Xia R TBtools: an integrative Toolkit developed for interactive analyses of big Biological Data Mol Plant 2020 13 8 1194 1202 10.1016/j.molp.2020.06.009 32585190
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020) TBtools: an integrative Toolkit developed for interactive analyses of big Biological Data. Mol Plant 13(8):1194–1202. 10.1016/j.molp.2020.06.00932585190 10.1016/j.molp.2020.06.009
De Giorgi J Fuchs C Iwasaki M Kim W Piskurewicz U Gully K Utz-Pugin A Mène-Saffrané L Waridel P Nawrath C Longoni FP Fujita S Loubéry S Lopez-Molina L The Arabidopsis mature endosperm promotes seedling cuticle formation via release of sulfated peptides Dev Cell 2021 56 22 3066 3081e5 10.1016/j.devcel.2021.10.005 34706263
De Giorgi J, Fuchs C, Iwasaki M, Kim W, Piskurewicz U, Gully K, Utz-Pugin A, Mène-Saffrané L, Waridel P, Nawrath C, Longoni FP, Fujita S, Loubéry S, Lopez-Molina L (2021) The Arabidopsis mature endosperm promotes seedling cuticle formation via release of sulfated peptides. Dev Cell 56(22):3066–3081e5. 10.1016/j.devcel.2021.10.00534706263 10.1016/j.devcel.2021.10.005
Delouche JC Baskin CC Accelerated aging techniques for predicting the relative storability of seed lots Seed Sci Technol 1973 1 427 452
Delouche JC, Baskin CC (1973) Accelerated aging techniques for predicting the relative storability of seed lots. Seed Sci Technol 1:427–452
Foley ME Chao WS Horvath DP Doğramaci M Anderson JV The transcriptomes of dormant leafy spurge seeds under alternating temperature are differentially affected by a germination-enhancing pretreatment J Plant Physiol 2013 170 6 539 547 10.1016/j.jplph.2012.11.008 23261266
Foley ME, Chao WS, Horvath DP, Doğramaci M, Anderson JV (2013) The transcriptomes of dormant leafy spurge seeds under alternating temperature are differentially affected by a germination-enhancing pretreatment. J Plant Physiol 170(6):539–547. 10.1016/j.jplph.2012.11.00823261266 10.1016/j.jplph.2012.11.008
Gao Y Liu C Wang J Lv M Liu X Zhang X Zhou J Li X Wang Y Dong G Huang J Liang G Yang Z Zhou Y Yao Y Modified TAL expression in rice plant regulates yield components and grain quality in a N-rate dependent manner Field Crops Res 2024 306 109219 10.1016/j.fcr.2023.109219
Gao Y, Liu C, Wang J, Lv M, Liu X, Zhang X, Zhou J, Li X, Wang Y, Dong G, Huang J, Liang G, Yang Z, Zhou Y, Yao Y (2024) Modified TAL expression in rice plant regulates yield components and grain quality in a N-rate dependent manner. Field Crops Res 306:109219. 10.1016/j.fcr.2023.10921910.1016/j.fcr.2023.109219
Gu L Zhang Y Zhang M Li T Dirk LMA Downie B Zhao T ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A Plant Mol Biol 2016 90 1–2 157 170 10.1007/s11103-015-0403-1 26584560
Gu L, Zhang Y, Zhang M, Li T, Dirk LMA, Downie B, Zhao T (2016) ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A. Plant Mol Biol 90(1–2):157–170. 10.1007/s11103-015-0403-126584560 10.1007/s11103-015-0403-1
Gu H, Zhang K, Chen J, Gull S, Chen C, Hou Y, Li X, Miao J, Zhou Y, Liang G (2022) OsFTL4, an FT-like Gene, Regulates Flowering Time and Drought Tolerance in Rice (Oryza sativa L.). Rice (N Y) 15(1):47. 10.1186/s12284-022-00593-1
Han Q Chen K Yan D Hao G Qi J Wang C Dirk LMA Bruce Downie A Gong J Wang J Zhao T ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth Plant J 2020 104 1 268 282 10.1111/tpj.14922 32662115
Han Q, Chen K, Yan D, Hao G, Qi J, Wang C, Dirk LMA, Bruce Downie A, Gong J, Wang J, Zhao T (2020) ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth. Plant J 104(1):268–282. 10.1111/tpj.1492232662115 10.1111/tpj.14922
Hay FR Valdez R Lee J-S Sta. Cruz PC Seed longevity phenotyping: recommendations on research methodology J Exp Bot 2018 10.1093/jxb/ery358
Hay FR, Valdez R, Lee J-S, Sta. Cruz PC (2018) Seed longevity phenotyping: recommendations on research methodology. J Exp Bot. 10.1093/jxb/ery35810.1093/jxb/ery358
He Y Yang B He Y Zhan C Cheng Y Zhang J Zhang H Cheng J Wang Z A quantitative trait locus, qSE3, promotes seed germination and seedling establishment under salinity stress in rice Plant J 2019 97 6 1089 1104 10.1111/tpj.14181 30537381
He Y, Yang B, He Y, Zhan C, Cheng Y, Zhang J, Zhang H, Cheng J, Wang Z (2019) A quantitative trait locus, qSE3, promotes seed germination and seedling establishment under salinity stress in rice. Plant J 97(6):1089–1104. 10.1111/tpj.1418130537381 10.1111/tpj.14181
He Y Zhao J Yang B Sun S Peng L Wang Z Indole-3‐acetate beta‐glucosyltransferase OsIAGLU regulates seed vigour through mediating crosstalk between auxin and abscisic acid in rice Plant Biotechnol J 2020 18 9 1933 1945 10.1111/pbi.13353 32012429
He Y, Zhao J, Yang B, Sun S, Peng L, Wang Z (2020a) Indole-3‐acetate beta‐glucosyltransferase OsIAGLU regulates seed vigour through mediating crosstalk between auxin and abscisic acid in rice. Plant Biotechnol J 18(9):1933–1945. 10.1111/pbi.1335332012429 10.1111/pbi.13353
He Y Jia Z Defeng F Zhibo H Jiaming L Yufei Z Jinping C Jifeng Y Zhoufei W RNA-Seq study reveals AP2-Domain-containing signalling regulators involved in initial imbibition of seed germination in Rice Rice Sci 2020 27 4 302 314 10.1016/j.rsci.2020.05.006
He Y, Jia Z, Defeng F, Zhibo H, Jiaming L, Yufei Z, Jinping C, Jifeng Y, Zhoufei W (2020b) RNA-Seq study reveals AP2-Domain-containing signalling regulators involved in initial imbibition of seed germination in Rice. Rice Sci 27(4):302–314. 10.1016/j.rsci.2020.05.00610.1016/j.rsci.2020.05.006
Hiei Y Ohta S Komari T Kumashiro T Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA Plant J 1994 6 2 271 282 10.1046/j.1365-313X.1994.6020271.x 7920717
Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6(2):271–282. 10.1046/j.1365-313X.1994.6020271.x7920717 10.1046/j.1365-313X.1994.6020271.x
Huang J Cai M Long Q Liu L Lin Q Jiang L Chen S Wan J OsLOX2, a rice type I lipoxygenase, confers opposite effects on seed germination and longevity Transgenic Res 2014 23 4 643 655 10.1007/s11248-014-9803-2 24792034
Huang J, Cai M, Long Q, Liu L, Lin Q, Jiang L, Chen S, Wan J (2014) OsLOX2, a rice type I lipoxygenase, confers opposite effects on seed germination and longevity. Transgenic Res 23(4):643–655. 10.1007/s11248-014-9803-224792034 10.1007/s11248-014-9803-2
Huang C Zhao J Huang Q Peng L Huang Z Li W Sun S He Y Wang Z OsNAC3 regulates seed germination involving abscisic acid pathway and cell elongation in rice New Phytol 2024 241 2 650 664 10.1111/nph.19362 37908121
Huang C, Zhao J, Huang Q, Peng L, Huang Z, Li W, Sun S, He Y, Wang Z (2024) OsNAC3 regulates seed germination involving abscisic acid pathway and cell elongation in rice. New Phytol 241(2):650–664. 10.1111/nph.1936237908121 10.1111/nph.19362
Lee JS Velasco-Punzalan M Pacleb M Valdez R Kretzschmar T McNally KL Ismail AM Cruz PCS Sackville Hamilton NR Hay FR Variation in seed longevity among diverse Indica rice varieties Ann Bot 2019 124 3 447 460 10.1093/aob/mcz093 31180503
Lee JS, Velasco-Punzalan M, Pacleb M, Valdez R, Kretzschmar T, McNally KL, Ismail AM, Cruz PCS, Sackville Hamilton NR, Hay FR (2019) Variation in seed longevity among diverse Indica rice varieties. Ann Bot 124(3):447–460. 10.1093/aob/mcz09331180503 10.1093/aob/mcz093
Li Y Zhou J Li Z Qiao J Quan R Wang J Huang R Qin H SALT AND ABA RESPONSE ERF1 improves seed germination and salt tolerance by repressing ABA signaling in rice Plant Physiol 2022 189 2 1110 1127 10.1093/plphys/kiac125 35294556
Li Y, Zhou J, Li Z, Qiao J, Quan R, Wang J, Huang R, Qin H (2022) SALT AND ABA RESPONSE ERF1 improves seed germination and salt tolerance by repressing ABA signaling in rice. Plant Physiol 189(2):1110–1127. 10.1093/plphys/kiac12535294556 10.1093/plphys/kiac125
Li X Dong J Zhu W Zhao J Zhou L Progress in the study of functional genes related to direct seeding of rice Mol Breed 2023 43 6 46 10.1007/s11032-023-01388-y 37309311
Li X, Dong J, Zhu W, Zhao J, Zhou L (2023) Progress in the study of functional genes related to direct seeding of rice. Mol Breed 43(6):46. 10.1007/s11032-023-01388-y37309311 10.1007/s11032-023-01388-y
Liu S Liu W Lai J Liu Q Zhang W Chen Z Gao J Song S Liu J Xiao Y OsGLYI3, a glyoxalase gene expressed in rice seed, contributes to seed longevity and salt stress tolerance Plant Physiol Biochem 2022 183 85 95 10.1016/j.plaphy.2022.04.028 35569169
Liu S, Liu W, Lai J, Liu Q, Zhang W, Chen Z, Gao J, Song S, Liu J, Xiao Y (2022) OsGLYI3, a glyoxalase gene expressed in rice seed, contributes to seed longevity and salt stress tolerance. Plant Physiol Biochem 183:85–95. 10.1016/j.plaphy.2022.04.02835569169 10.1016/j.plaphy.2022.04.028
Liu X Gao Y Li R Zhang X Dong G Zhou J Zhou Y Yang Z Huang J Dai Q Yao Y Transcriptomic analysis of salt-tolerant and sensitive high‐yield japonica rice (Oryza sativa L.) reveals complicated salt‐tolerant mechanisms Physiol Plant 2024 176 2 e14275 10.1111/ppl.14275 38566267
Liu X, Gao Y, Li R, Zhang X, Dong G, Zhou J, Zhou Y, Yang Z, Huang J, Dai Q, Yao Y (2024) Transcriptomic analysis of salt-tolerant and sensitive high‐yield japonica rice (Oryza sativa L.) reveals complicated salt‐tolerant mechanisms. Physiol Plant 176(2):e14275. 10.1111/ppl.1427538566267 10.1111/ppl.14275
Luo X Dai Y Zheng C Yang Y Chen W Wang Q Chandrasekaran U Du J Liu W Shu K The ABI4-RbohD/VTC2 regulatory module promotes reactive oxygen species (ROS) accumulation to decrease seed germination under salinity stress New Phytol 2021 229 2 950 962 10.1111/nph.16921 32916762
Luo X, Dai Y, Zheng C, Yang Y, Chen W, Wang Q, Chandrasekaran U, Du J, Liu W, Shu K (2021) The ABI4-RbohD/VTC2 regulatory module promotes reactive oxygen species (ROS) accumulation to decrease seed germination under salinity stress. New Phytol 229(2):950–962. 10.1111/nph.1692132916762 10.1111/nph.16921
Mansueto L Fuentes RR Borja FN Detras J Abriol-Santos JM Chebotarov D Sanciangco M Palis K Copetti D Poliakov A Dubchak I Solovyev V Wing RA Hamilton RS Mauleon R McNally KL Alexandrov N Rice SNP-seek database update: new SNPs, indels, and queries Nucleic Acids Res 2017 45 D1 D1075 D1081 10.1093/nar/gkw1135 27899667
Mansueto L, Fuentes RR, Borja FN, Detras J, Abriol-Santos JM, Chebotarov D, Sanciangco M, Palis K, Copetti D, Poliakov A, Dubchak I, Solovyev V, Wing RA, Hamilton RS, Mauleon R, McNally KL, Alexandrov N (2017) Rice SNP-seek database update: new SNPs, indels, and queries. Nucleic Acids Res 45(D1):D1075–D1081. 10.1093/nar/gkw113527899667 10.1093/nar/gkw1135
McCouch SR Wright MH Tung C-W Maron LG McNally KL Fitzgerald M Singh N DeClerck G Agosto-Perez F Korniliev P Greenberg AJ Naredo MEB Mercado SMQ Harrington SE Shi Y Branchini DA Kuser-Falcão PR Leung H Ebana K Yano M Eizenga G McClung A Mezey J Open access resources for genome-wide association mapping in rice Nat Commun 2016 7 1 10532 10.1038/ncomms10532 26842267
McCouch SR, Wright MH, Tung C-W, Maron LG, McNally KL, Fitzgerald M, Singh N, DeClerck G, Agosto-Perez F, Korniliev P, Greenberg AJ, Naredo MEB, Mercado SMQ, Harrington SE, Shi Y, Branchini DA, Kuser-Falcão PR, Leung H, Ebana K, Yano M, Eizenga G, McClung A, Mezey J (2016) Open access resources for genome-wide association mapping in rice. Nat Commun 7(1):10532. 10.1038/ncomms1053226842267 10.1038/ncomms10532
Miao J Li X Li X Tan W You A Wu S Tao Y Chen C Wang J Zhang D Gong Z Yi C Yang Z Gu M Liang G Zhou Y OsPP2C09, a negative regulatory factor in abscisic acid signalling, plays an essential role in balancing plant growth and drought tolerance in rice New Phytol 2020 227 5 1417 1433 10.1111/nph.16670 32433775
Miao J, Li X, Li X, Tan W, You A, Wu S, Tao Y, Chen C, Wang J, Zhang D, Gong Z, Yi C, Yang Z, Gu M, Liang G, Zhou Y (2020) OsPP2C09, a negative regulatory factor in abscisic acid signalling, plays an essential role in balancing plant growth and drought tolerance in rice. New Phytol 227(5):1417–1433. 10.1111/nph.1667032433775 10.1111/nph.16670
Nakano T Suzuki K Fujimura T Shinshi H Genome-wide analysis of the ERF Gene Family in Arabidopsis and Rice Plant Physiol 2006 140 2 411 432 10.1104/pp.105.073783 16407444
Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF Gene Family in Arabidopsis and Rice. Plant Physiol 140(2):411–432. 10.1104/pp.105.07378316407444 10.1104/pp.105.073783
Rao X Dixon RA Brassinosteroid Mediated Cell Wall Remodeling in Grasses under Abiotic stress Front Plant Sci 2017 8 806 10.3389/fpls.2017.00806 28567047
Rao X, Dixon RA (2017) Brassinosteroid Mediated Cell Wall Remodeling in Grasses under Abiotic stress. Front Plant Sci 8:806. 10.3389/fpls.2017.0080628567047 10.3389/fpls.2017.00806
Sharoni AM Nuruzzaman M Satoh K Shimizu T Kondoh H Sasaya T Choi I-R Omura T Kikuchi S Gene Structures, classification and expression models of the AP2/EREBP Transcription Factor Family in Rice Plant Cell Physiol 2011 52 2 344 360 10.1093/pcp/pcq196 21169347
Sharoni AM, Nuruzzaman M, Satoh K, Shimizu T, Kondoh H, Sasaya T, Choi I-R, Omura T, Kikuchi S (2011) Gene Structures, classification and expression models of the AP2/EREBP Transcription Factor Family in Rice. Plant Cell Physiol 52(2):344–360. 10.1093/pcp/pcq19621169347 10.1093/pcp/pcq196
Shu K Zhang H Wang S Chen M Wu Y Tang S Liu C Feng Y Cao X Xie Q ABI4 regulates primary seed dormancy by regulating the Biogenesis of Abscisic Acid and Gibberellins in Arabidopsis PLoS Genet 2013 9 6 e1003577 10.1371/journal.pgen.1003577 23818868
Shu K, Zhang H, Wang S, Chen M, Wu Y, Tang S, Liu C, Feng Y, Cao X, Xie Q (2013) ABI4 regulates primary seed dormancy by regulating the Biogenesis of Abscisic Acid and Gibberellins in Arabidopsis. PLoS Genet 9(6):e1003577. 10.1371/journal.pgen.100357723818868 10.1371/journal.pgen.1003577
Shu K Chen Q Wu Y Liu R Zhang H Wang P Li Y Wang S Tang S Liu C Yang W Cao X Serino G Xie Q ABI4 mediates antagonistic effects of abscisic acid and gibberellins at transcript and protein levels Plant J 2016 85 3 348 361 10.1111/tpj.13109 26708041
Shu K, Chen Q, Wu Y, Liu R, Zhang H, Wang P, Li Y, Wang S, Tang S, Liu C, Yang W, Cao X, Serino G, Xie Q (2016) ABI4 mediates antagonistic effects of abscisic acid and gibberellins at transcript and protein levels. Plant J 85(3):348–361. 10.1111/tpj.1310926708041 10.1111/tpj.13109
Tong Y Yi S-C Liu S-Y Xu L Qiu Z-X Zeng D-Q Tang W-W Bruceine D may affect the phenylpropanoid biosynthesis by acting on ADTs thus inhibiting Bidens pilosa L. seed germination Ecotoxicol Environ Saf 2022 242 113943 10.1016/j.ecoenv.2022.113943 35999761
Tong Y, Yi S-C, Liu S-Y, Xu L, Qiu Z-X, Zeng D-Q, Tang W-W (2022) Bruceine D may affect the phenylpropanoid biosynthesis by acting on ADTs thus inhibiting Bidens pilosa L. seed germination. Ecotoxicol Environ Saf 242:113943. 10.1016/j.ecoenv.2022.11394335999761 10.1016/j.ecoenv.2022.113943
Wang Y Hou Y Qiu J Wang H Wang S Tang L Tong X Zhang J Abscisic acid promotes jasmonic acid biosynthesis via a ‘SAPK10-bZIP72‐AOC’ pathway to synergistically inhibit seed germination in rice (Oryza sativa) New Phytol 2020 228 4 1336 1353 10.1111/nph.16774 32583457
Wang Y, Hou Y, Qiu J, Wang H, Wang S, Tang L, Tong X, Zhang J (2020) Abscisic acid promotes jasmonic acid biosynthesis via a ‘SAPK10-bZIP72‐AOC’ pathway to synergistically inhibit seed germination in rice (Oryza sativa). New Phytol 228(4):1336–1353. 10.1111/nph.1677432583457 10.1111/nph.16774
Wang W-Q Xu D-Y Sui Y-P Ding X-H Song X-J A multiomic study uncovers a bZIP23-PER1A–mediated detoxification pathway to enhance seed vigor in rice Proc Natl Acad Sci USA 2022 119 9 e2026355119 10.1073/pnas.2026355119 35217598
Wang W-Q, Xu D-Y, Sui Y-P, Ding X-H, Song X-J (2022) A multiomic study uncovers a bZIP23-PER1A–mediated detoxification pathway to enhance seed vigor in rice. Proc Natl Acad Sci USA 119(9):e2026355119. 10.1073/pnas.202635511935217598 10.1073/pnas.2026355119
Wang F Xu H Zhang L Shi Y Song Y Wang X Cai Q He W Xie H Zhang J The lipoxygenase OsLOX10 affects seed longevity and resistance to saline-alkaline stress during rice seedlings Plant Mol Biol 2023 111 4–5 415 428 10.1007/s11103-023-01334-8 36867321
Wang F, Xu H, Zhang L, Shi Y, Song Y, Wang X, Cai Q, He W, Xie H, Zhang J (2023) The lipoxygenase OsLOX10 affects seed longevity and resistance to saline-alkaline stress during rice seedlings. Plant Mol Biol 111(4–5):415–428. 10.1007/s11103-023-01334-836867321 10.1007/s11103-023-01334-8
Yaish MW El-kereamy A Zhu T Beatty PH Good AG Bi Y-M Rothstein SJ The APETALA-2-Like transcription factor OsAP2-39 controls key interactions between Abscisic Acid and Gibberellin in Rice PLoS Genet 2010 6 9 e1001098 10.1371/journal.pgen.1001098 20838584
Yaish MW, El-kereamy A, Zhu T, Beatty PH, Good AG, Bi Y-M, Rothstein SJ (2010) The APETALA-2-Like transcription factor OsAP2-39 controls key interactions between Abscisic Acid and Gibberellin in Rice. PLoS Genet 6(9):e1001098. 10.1371/journal.pgen.100109820838584 10.1371/journal.pgen.1001098
Yamasani MR Pandu VR Kalluru S Bommaka RR Bandela R Duddu B Komeri S Kumbha D Vemireddy LR Haplotype analysis of QTLs governing early seedling vigor-related traits under dry-direct-seeded rice (Oryza sativa L.) conditions Mol Biol Rep 2023 50 10 8177 8188 10.1007/s11033-023-08714-9 37555871
Yamasani MR, Pandu VR, Kalluru S, Bommaka RR, Bandela R, Duddu B, Komeri S, Kumbha D, Vemireddy LR (2023) Haplotype analysis of QTLs governing early seedling vigor-related traits under dry-direct-seeded rice (Oryza sativa L.) conditions. Mol Biol Rep 50(10):8177–8188. 10.1007/s11033-023-08714-937555871 10.1007/s11033-023-08714-9
Yang M Yang J Su L Sun K Li D Liu Y Wang H Chen Z Guo T Metabolic profile analysis and identification of key metabolites during rice seed germination under low-temperature stress Plant Sci 2019 289 110282 10.1016/j.plantsci.2019.110282 31623771
Yang M, Yang J, Su L, Sun K, Li D, Liu Y, Wang H, Chen Z, Guo T (2019) Metabolic profile analysis and identification of key metabolites during rice seed germination under low-temperature stress. Plant Sci 289:110282. 10.1016/j.plantsci.2019.11028231623771 10.1016/j.plantsci.2019.110282
Yang J Su L Li D Luo L Sun K Yang M Gu F Xia A Liu Y Wang H Chen Z Guo T Dynamic transcriptome and metabolome analyses of two types of rice during the seed germination and young seedling growth stages BMC Genomics 2020 21 1 603 10.1186/s12864-020-07024-9 32867689
Yang J, Su L, Li D, Luo L, Sun K, Yang M, Gu F, Xia A, Liu Y, Wang H, Chen Z, Guo T (2020) Dynamic transcriptome and metabolome analyses of two types of rice during the seed germination and young seedling growth stages. BMC Genomics 21(1):603. 10.1186/s12864-020-07024-932867689 10.1186/s12864-020-07024-9
Yang B, Chen M, Zhan C, Liu K, Cheng Y, Xie T, Zhu P, He Y, Zeng P, Tang H, Tsugama D, Chen S, Zhang H, Cheng J (2022) Identification of OsPK5 involved in rice glycolytic metabolism and GA/ABA balance for improving seed germination via genome-wide association study. J Exp Bot 73(11):3446–3461. 10.1093/jxb/erac071
Yin G Xin X Fu S An M Wu S Chen X Zhang J He J Whelan J Lu X Proteomic and Carbonylation Profile Analysis at the critical node of seed ageing in Oryza sativa Sci Rep 2017 7 1 40611 10.1038/srep40611 28094349
Yin G, Xin X, Fu S, An M, Wu S, Chen X, Zhang J, He J, Whelan J, Lu X (2017) Proteomic and Carbonylation Profile Analysis at the critical node of seed ageing in Oryza sativa. Sci Rep 7(1):40611. 10.1038/srep4061128094349 10.1038/srep40611
Yu Y Guo D-D Min D-H Cao T Ning L Jiang Q-Y Sun X-J Zhang H Tang W Gao S-Q Zhou Y-B Xu Z-S Chen J Ma Y-Z Chen M Zhang X-H Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway Plant Physiol Biochem 2023 195 310 321 10.1016/j.plaphy.2022.11.032 36657296
Yu Y, Guo D-D, Min D-H, Cao T, Ning L, Jiang Q-Y, Sun X-J, Zhang H, Tang W, Gao S-Q, Zhou Y-B, Xu Z-S, Chen J, Ma Y-Z, Chen M, Zhang X-H (2023) Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway. Plant Physiol Biochem 195:310–321. 10.1016/j.plaphy.2022.11.03236657296 10.1016/j.plaphy.2022.11.032
Zhang Z Huang, Mo, Lv, Gao, Chen, Yin, Ju, Dong Z Yang, Li W Huang Y Transcriptomic and Co-expression Network Profiling of Shoot Apical Meristem Reveal Contrasting Response to Nitrogen Rate between Indica and Japonica Rice subspecies Int J Mol Sci 2019 20 23 5922 10.3390/ijms20235922 31775351
Zhang Z, Huang, Mo, Lv, Gao, Chen, Yin, Ju, Dong Z, Yang, Li W, Huang Y (2019) Transcriptomic and Co-expression Network Profiling of Shoot Apical Meristem Reveal Contrasting Response to Nitrogen Rate between Indica and Japonica Rice subspecies. Int J Mol Sci 20(23):5922. 10.3390/ijms2023592231775351 10.3390/ijms20235922
Zhang Y Li D Dirk LMA Downie AB Zhao T ZmAGA1 hydrolyzes RFOs late during the lag phase of seed germination, shifting Sugar metabolism toward seed germination over seed aging tolerance J Agric Food Chem 2021 69 39 11606 11615 10.1021/acs.jafc.1c03677 34553917
Zhang Y, Li D, Dirk LMA, Downie AB, Zhao T (2021) ZmAGA1 hydrolyzes RFOs late during the lag phase of seed germination, shifting Sugar metabolism toward seed germination over seed aging tolerance. J Agric Food Chem 69(39):11606–11615. 10.1021/acs.jafc.1c0367734553917 10.1021/acs.jafc.1c03677
Zhang G Bi Z Jiang J Lu J Li K Bai D Wang X Zhao X Li M Zhao X Wang W Xu J Li Z Zhang F Shi Y Genome-wide association and epistasis studies reveal the genetic basis of saline-alkali tolerance at the germination stage in rice Front Plant Sci 2023 14 1170641 10.3389/fpls.2023.1170641 37251777
Zhang G, Bi Z, Jiang J, Lu J, Li K, Bai D, Wang X, Zhao X, Li M, Zhao X, Wang W, Xu J, Li Z, Zhang F, Shi Y (2023a) Genome-wide association and epistasis studies reveal the genetic basis of saline-alkali tolerance at the germination stage in rice. Front Plant Sci 14:1170641. 10.3389/fpls.2023.117064137251777 10.3389/fpls.2023.1170641
Zhang H Chen G Xu H Jing S Jiang Y Liu Z Zhang H Wang F Hu X Zhu Y Transcriptome analysis of rice embryo and endosperm during seed germination Int J Mol Sci 2023 24 10 8710 10.3390/ijms24108710 37240056
Zhang H, Chen G, Xu H, Jing S, Jiang Y, Liu Z, Zhang H, Wang F, Hu X, Zhu Y (2023b) Transcriptome analysis of rice embryo and endosperm during seed germination. Int J Mol Sci 24(10):8710. 10.3390/ijms2410871037240056 10.3390/ijms24108710
Zhang Y Song X Zhang W Liu F Wang C Liu Y Dirk LMA Downie AB Zhao T Maize PIMT2 repairs damaged 3-METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor Plant J 2023 115 1 220 235 10.1111/tpj.16225 36999611
Zhang Y, Song X, Zhang W, Liu F, Wang C, Liu Y, Dirk LMA, Downie AB, Zhao T (2023c) Maize PIMT2 repairs damaged 3-METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor. Plant J 115(1):220–235. 10.1111/tpj.1622536999611 10.1111/tpj.16225
Zhao H Nie K Zhou H Yan X Zhan Q Zheng Y Song C ABI5 modulates seed germination via feedback regulation of the expression of the PYR/PYL/RCAR ABA receptor genes New Phytol 2020 228 2 596 608 10.1111/nph.16713 32473058
Zhao H, Nie K, Zhou H, Yan X, Zhan Q, Zheng Y, Song C (2020a) ABI5 modulates seed germination via feedback regulation of the expression of the PYR/PYL/RCAR ABA receptor genes. New Phytol 228(2):596–608. 10.1111/nph.1671332473058 10.1111/nph.16713
Zhao J He Y Li X Weng X Feng D Ying J Wang Z An integrated RNA-Seq and physiological study reveals gene responses involving in the initial imbibition of seed germination in rice Plant Growth Regul 2020 90 2 249 263 10.1007/s10725-019-00567-2
Zhao J, He Y, Li X, Weng X, Feng D, Ying J, Wang Z (2020b) An integrated RNA-Seq and physiological study reveals gene responses involving in the initial imbibition of seed germination in rice. Plant Growth Regul 90(2):249–263. 10.1007/s10725-019-00567-210.1007/s10725-019-00567-2
Zhao J He Y Huang S Wang Z Advances in the Identification of Quantitative Trait Loci and genes involved in seed Vigor in Rice Front Plant Sci 2021 12 659307 10.3389/fpls.2021.659307 34335643
Zhao J, He Y, Huang S, Wang Z (2021) Advances in the Identification of Quantitative Trait Loci and genes involved in seed Vigor in Rice. Front Plant Sci 12:659307. 10.3389/fpls.2021.65930734335643 10.3389/fpls.2021.659307
Zhou T, Yu D, Wu L, Xu Y, Duan M, Yuan D (2024) Seed storability in Rice: physiological bases, Molecular mechanisms, and application to breeding. Rice Sci S1672630824000209. 10.1016/j.rsci.2024.02.011
