1 Introduction

PHS is a crucial concern worldwide as it considerably affects global agricultural production, economy, and food quality [1, 2]. It is one of the most damaging abiotic stresses for wheat (Triticum aestivum L.) and other cereals globally [3]. It consists of the germination of physiologically mature seeds in spikes prior to or during harvest due to high humidity or wet (longer period of precipitation) [3, 4]. PHS engenders drastic reduction in wheat grain yield and end-use quality and occasions an annual global loss of about $1 billion [1, 4,5,6]. In China, PHS represents the most limiting factor in about 83% of major wheat planting areas, principally in the Southwestern Winter Wheat Zone (SWWZ), Middle and Lower Yangtze River Valleys Winter Wheat Zone (MLWZ), and Northeastern Spring Wheat Zone (NSWZ) [4, 4,5, 10, 13,22,23,26]. However, information related to GISs is lacking. Hence, identifying potential GISs may offer valuable metabolic resources to deepen understanding of PHS tolerance mechanisms and for biochemical control of PHS.

Germination inhibitors or GISs are widely spread compounds in the plant kingdom that can inhibit or delay the germination of plant reproductive materials (seeds, spores, etc.) [27, 28]. Including polyphenols, alkaloids, cyanides, amino acids, essential oils, etc., GISs can be categorized into two groups, germination retarders and germination destructors, depending on their impacts on seed morphology, structure, and physiology [27]. Germination retarders are interconnected to seed dormancy, and their exogenous application at a proper dosage can extend the dormancy period of non-dormant seeds [27]. Studies in many weed and crop species have shown that most polyphenols (phenolic acids, flavonoids, lignans, etc.) possess germination retardation capacity [29,30,31,32,33,34,35]. Particularly, proanthocyanidins (flavonoids) can interact synergically with ABA to enhance seed dormancy [32, 36]. Unfortunately, only some phenylethyl alcohols (2-phenylethyl alcohol, 1-phenylethyl alcohol, 4-vinylphenol and their derivatives), dihydroactinidiolide, and tetrahydroactinidiolide were identified as GISs in wheat husks [37, 38]. Thus, the need to identify potential germination inhibitory polyphenols in wheat.

Metabolomics analysis is a novel functional genomics tool that aids in disclosing the DAMs (differentially accumulation of metabolites) within cells and organs of the same or different genotypes at a given time [6, 39]. Global metabolomics via MS (mass spectrometry) has allowed the identification of thousands of DAMs associated with a specific treatment [6, 40, 41]. The quantification of metabolites allows an extensive overview of the functional status of the plant organ, which can help assess the functions of genes [6]. Moreover, a general metabolomics approach offers powerful tools to examine the temporal regulation of diverse metabolic pathways [9, 42]. Therefore, we speculated that a comparative metabolomics analysis of polyphenols of PHS susceptible and resistant genotypes might help uncover potential candidate GISs.

In this study, based on PHS tests of a PHS resistant (Lincang Hulled Wheat, LHW) and PHS highly susceptible (Yunmai53, Yun53), we found that the resistance capability of LHW was associated with endogenous factors. Accordingly, we performed the polyphenols and phytohormones profiling of physiologically matured spikes and shells of the two varieties and unveiled key DAPs and variations in plant hormones’ levels. Our findings may enhance the molecular understanding of PHS resistance and trigger biochemical control of PHS in wheat.

2 Materials and methods

2.1 Plant materials and growing conditions

Two contrasting wheat varieties, Lincang Hulled Wheat (LHW, PHS-resistant) and Yunmai53 (Yun53, PHS highly susceptible), were used in this study. They were offered by the Yunnan Academy of Agricultural Sciences, Kunming, Yunnan, China. The materials were planted in the experimental base of the Academy located at ** for pre-harvest sprouting resistance in white winter wheat. Theor Appl Genet 125:793–805" href="/article/10.1007/s42452-023-05464-y#ref-CR46" id="ref-link-section-d155476480e1501">46]. In the present study, we found that the HWR of the PHS-resistant variety LHW was higher than that of the highly-susceptible variety Yun53. This result shows that PHS does not correlate with the water absorption capacity of seeds but repose principally on endogenous factors, including phytohormones and GISs.

To identify potential polyphenols with germination inhibitory properties, we carried out metabolomics analysis of polyphenols in the spikes and shells of the two varieties. We identified 214 phenolic compounds, most of which exhibited higher relative content in LHW compared to Yun53. Conformingly, the analysis revealed that the TPC of LHW spikes and shells was significantly higher than that of Yun53, respectively. These results indicate that higher phenolics content may strongly correlate with PHS tolerance, and wheat genotypes with higher TPC might resist PHS. Higher polyphenol content was also associated with seed dormancy in carob [31]. The 214 metabolites were principally classified into phenolic acids (42.52%), flavones (23.83%), and flavonoid carbonosides (16.36%). These results suggest that improving phenolic acids and flavonoid biosynthesis in develo** wheat spikes may confer PHS tolerance. We identified 180 DAPs, including 168 up-regulated between the two varieties. Functional characterization assigned these DAPs primary in tyrosine metabolism, biosynthesis of secondary metabolites, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, and isoflavonoid biosynthesis. These findings confirm that the phenylpropanoid pathway is highly induced during LHW develo** spikes than in Yun53. High polyphenols content is also associated with variation in grain colors [40], and grain color is genetically linked to PHS resistance in wheat [8, 13]. Supportively, Lang et al. found that the transcription factor MYB10-D promotes PHS resistance by stimulating ABA and flavonoid biosynthesis in wheat [47]. Furthermore, we filtered out twenty-four most up-regulated DAPs as potential candidate GISs. These metabolites could serve as metabolic markers for future functional studies toward the complete dissection of the regulatory network of PHS tolerance in wheat. Moreover, the effects of each of these key DAPs on wheat seed germination need to be investigated for application in the biochemical control of PHS in wheat and other crops. It is demonstrated that some phenolic compounds may possess germination-inhibitory properties [35, 48, 49]. For instance, caffeic acid, p-coumaric acid, chlorogenic acid, fumaric acid, ferulic acid, gallic acid, hydrocinnamic acid, p-hydroxybenzoic acid, vanillic acid, p-vanillin, pyrocatechol, and proanthocyanidins germination inhibitory effects have been proved in many weed and crop species [27, 29, 32,33,34, 50].

PHS is primarily influenced by seed dormancy. Seed dormancy and germination are governed by the ABA/GA ratio [18, 19, 51]. The unbalance of this ratio in favor of ABA and GA promotes seed dormancy and germination, respectively [18, 19, 51]. Consistency, we found that the content of ABA in LHW was significantly higher than in Yun53, while that of GA was significantly higher in Yun53 than in LHW. These results confirm the key regulatory function of the interplay between ABA and GA for PHS regulation through the control of seed dormancy or germination. ABA and polyphenols may function synergically to enhance seed dormancy and therefore prevent PHS in wheat. It is demonstrated that ABA and flavonoids interact to promote seed dormancy [32, 36]. ABA might also stimulate polyphenols’ biosynthesis and accumulation in LHW during spike development in order to enhance its resistance to PHS. In grape berry, exogenous application of ABA has significantly promoted flavonoid biosynthesis [52]. We also uncovered that the content of ethylene in the highly-susceptible variety Yun53 was significantly higher than in LHW, indicating the plant hormone ethylene promotes seed germination via negative regulation of ABA biosynthesis or signaling. Corroboratively, it is demonstrated that ethylene is a negative regulator of seed dormancy via negative control of ABA biosynthesis and signaling and interplay between other phytohormones and signal molecules [53, 54]. Compared to Yun53, the content of JA and SA in LHW was significantly higher, suggesting they may involve in promoting seed dormancy. The specific role of JA and SA in PHS resistance needs further investigation. Currently, it is reported that JA is not a critical regulator of germination and dormancy in wheat; however, it could cause seed dormancy loss by modulating the ABA/GA ratio [21]. Taken together, our findings show that further studies are required to deepen our understanding of the molecular mechanisms underlying PHS tolerance in wheat.

5 Conclusions

In summary, this study combined polyphenols and phytohormones profiling analyses to unveil the importance of phenolic compounds, ABA, JA, and SA, in PHS resistance in wheat. The PHS resistance capability of LHW is associated with its higher content of diverse phenolic compounds, ABA, JA, and SA. Tyrosine metabolism, biosynthesis of secondary metabolites, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, and isoflavonoid biosynthesis were the main pathways significantly induced in LHW. Twenty-four key up-regulated (Log2FC ≥ 8) DAPs in LHW were identified as potential candidate GISs. Our findings provide key resources for more insights into the molecular mechanisms governing PHS resistance in wheat. Moreover, they offer metabolic markers to enhance PHS control in wheat.