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. 2024 Oct 11;14:23818. doi: 10.1038/s41598-024-74795-6

Role of total polyphenol content in seed germination characteristics of spring barley varieties amidst climate change

Ivana Jovanović 1, Nicole Frantová 1,✉, Jhonny E Alba-Mejía 1, Lenka Porčová 2, Vratislav Psota 3, Jana Asszonyi 1, Radim Cerkal 1, Tomáš Středa 1,4
PMCID: PMC11470085  PMID: 39394377

Abstract

The amount of total polyphenol content (TPC) in the grain could provide insights into the conditions during maturation and might also serve as an indicator of the grain’s ability to germinate in the malting process or as seeds in the field. Varieties with higher natural TPC content might exhibit better germination parameters both in the field and in the malt house. This study investigates the relationship between TPC and seed germination characteristics i.e. seed vigour in four spring barley varieties over two years, considering diverse environmental conditions and exposure to drought conditions. The evaluation of seed germination characteristics in barley, with a focus on the root length and average diameter under drought conditions (−0.5 MPa) and suboptimal temperature (10 °C), was conducted. Drought conditions were induced using polyethylene glycol (PEG 6000). After durations of seven and fourteen days, the germinated seeds from the Petri dishes were scanned and subjected to analysis using WinRHIZO software following the metrics: Len 7, Len 14 (root length after seven and fourteen days in cm) and AvgD 7, AvgD 14 (root diameter after seven and fourteen days in mm). The findings support our initial hypothesis, indicating a variety-specific relationship between seed germination characteristics and increased TPC, where higher germination parameters might be associated with elevated TPC levels in some barley varieties.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-74795-6.

Keywords: Drought conditions, Environment, Genotype, Seed germination characteristics, Spring barley, TPC

Subject terms: Plant sciences, Environmental sciences

Introduction

Climate change is poised to have adverse impacts on global food supplies, challenging research into enhancing agricultural productivity in the face of worsening climate conditions to ensure global food security1. Despite conventional breeding efforts, some crops remain susceptible to climate-related factors during certain development stages2. These stages of the plant life cycle include germination, seedling growth, pollination, flowering, and seed development1,3. To maintain a steady food supply, there will be an increasing necessity for high-yielding crops that can provide stable yields under adverse environmental conditions.

How climate change impacts seed vigour is thoroughly described by Finch–Savage and Bassel4, explaining how the sowing time is selected based on climate and seasons because of the critical need to ensure optimal environmental conditions for seed germination and growth. Even if optimal conditions for sowing can be chosen, the subsequent conditions affecting crop establishment cannot. Robust seeds with enhanced seed vigour can moderate these challenges by ensuring consistent seedling growth across various environmental conditions. Seed vigour, which includes factors like germination speed and root growth, is critical for seed longevity, successful and uniform plant establishment in the field, and early stress tolerance. Although the term “seed vigour” is often used to describe the potential for rapid, uniform emergence and development of normal seedlings under diverse field conditions, its definition can vary depending on the research context and specific parameters being measured. In this study, we focus on root growth as a critical component of seed vigour. To maintain clarity and neutrality, we refer to these parameters as “seed germination characteristics.”

Increasing emphasis on seed quality has influenced the food technology chain, including the brewing industry. Currently, the seed industry uses seed vigour to assess seed quality. High seed vigour is linked to the potential for enhanced growth and productivity in agricultural production5. This research takes a closer look at one of the key crops used in the Czech Republic’s brewing industry, particularly malting barley. The renewed attention to barley grain is due to its nutritional value characterized by low fat content and a rich array of complex carbohydrates (predominantly starch), proteins, essential minerals, vitamins (α-tocopherol), and a variety of antioxidants, especially phenolic compounds6,7. Phenolic compounds are particularly important in the brewing process because they contribute to the formation of haze in beer, which is a critical quality parameter for brewers. Therefore, understanding the relationship between seed germination characteristics and TPC in barley varieties used for malting can help optimize both the brewing process and the quality of the final product.

The role of phenolic compounds in seed physiology, including their impact on seed germination characteristics, is significant. According to Rodríguez et al.8, phenolic compounds can limit oxygen supply to the embryo by oxygen fixation, resulting from the oxidation of phenolic compounds. This mechanism plays a crucial role in regulating seed dormancy and germination, particularly under stress conditions. The hulls of barley grains impose dormancy by limiting oxygen diffusion to the embryo, with the efficiency of this mechanism increasing at higher temperatures. In species where removal of hulls is possible through threshing, this process improves germination by enhancing oxygen availability and altering hormone balances such as abscisic acid (ABA) and gibberellin (GA). However, in barley, where hulls removal is not possible by threshing, the presence of hulls remains a significant factor in dormancy. Additionally, phenolic compounds can act as germination inhibitors themselves or through their regulatory role on oxygen levels, affecting the overall dormancy and germination process.

The total polyphenol content in germinated seeds and sprouts can undergo changes during germination while having a distinct impact on bound phenolics9. It has been observed that, in certain species, an increase in TPC is associated with enhanced seed germination characteristics10,11. The quantity and composition of TPC in barley grain, like many other compounds, depends on the genetic characteristics of the variety, growing, and climatic conditions. Basařová et al.12 have noted that as the protein content of the grain increases, the polyphenol content decreases. This observation has led to the realization of an experiment aimed at identifying the relationship between environmental conditions and the TPC content in grains of various malting barley varieties. Barley grains can produce various metabolites and chemicals in response to stress conditions like drought. Some of these compounds include polyphenols such as flavonoids13–15, phenolic acids16, tannins17. These polyphenols are known for their antioxidant properties and may protect the plant from oxidative stress. Other secondary metabolites such as terpenoids18, alkaloids19, and other phenolic compounds can play a role in defence mechanisms against stress.

A significant challenge in researching seed germination characteristics is their sensitivity to strong variations influenced by the year. While this aspect cannot be overlooked, when examined alongside environmental and genetic factors, its impact can be mitigated. The results section will illustrate how these factors affect seed germination characteristics differently each year, with environmental, genetic influences, and their interaction being particularly prominent. Therefore, the aims of this study are to determine and explain the relationship between seed germination characteristics and TPC in four spring barley varieties grown under diverse conditions in the Czech Republic. We examined the following hypotheses: (H1) Elevated seed germination characteristics among various spring barley varieties are associated with higher total polyphenols content. (H2) The ranking of barley varieties in terms of seed germination characteristics and TPC is similar across different environments.

Results

Annual examination of correlation trait patterns in spring barley varieties through principal component analysis

To explore correlation patterns in the relationships between total polyphenol content and seed germination characteristics across the years 2021 and 2022, principal component analysis (PCA) was conducted. In 2021, eigenvalues of 3.74 corresponding to 73% of the total variance for PC1 and 1.04 corresponding to 93% of the total variance for PC2 were obtained. The traits loading heavily on PC1 were primarily related to seed germination characteristics, such as AvgD 7 and AvgD 14. These traits had higher eigenvalues, suggesting that they explained most of the variance captured by PC1. In contrast, polyphenols contributed more heavily to PC2, indicating that their influence was distinct from germination characteristics and acted as an independent factor influencing the dataset.

Shifting to 2022, eigenvalues of 3.73 corresponding to 73% of the total variance for PC1 and 1.00 corresponding to 92% of the total variance for PC2 were observed, indicating a continued but slightly diminished influence of polyphenols compared with 2021. One likely explanation is the variation in environmental conditions between the 2 years, which may have affected both polyphenol synthesis and its relationship with germination traits. Environmental stressors such as temperature, rainfall, or soil conditions can influence polyphenol production differently from year to year, potentially explaining the diminished influence observed in 2022. Nonetheless, polyphenols still contributed to shaping the relationship examined, particularly evident with variables AvgD 7 and AvgD 14. These trait patterns emphasize the dynamic and varying explanatory power of polyphenols in influencing germination characteristics traits over the analysed time span (Fig. 1). Moreover, due to the significant influence of annual variations on all traits, reflecting the strong effect of the year, all analyses were conducted separately.

Fig. 1.

Fig. 1

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The biplot illustrates the grouping of variables based on Principal Component Analysis (PCA) for the datasets of 2021 (A), and 2022 (B). The variables represent trait values measured across four different spring barley varieties grown in three localities: 1 –Polyphenols refers to TPC (total polyphenol content), 2 - Length 7 refers to length after 7 days, Length 14 refers to length after 14 days, AvgD 7 refers to average diameter after 7 days, and AvgD 14 refers to average diameter after 14 days. The lines (or vectors) represent the original variables (traits) projected into the reduced-dimensional PCA space. These lines show how each variable contributes to the principal components (PC1, PC2) and how they are related to each other. The direction and length of the lines convey specific information about the relationships between the variables.

Correlations among traits

After confirming through PCA analysis that the year effect significantly influenced all traits, separate correlation analyses were conducted for each year, enhancing the ability to identify connections between traits across different varieties and environments. While certain varieties consistently demonstrated correlation patterns across years and environments, others showed inconsistencies, with correlations shifting from negative to positive across consecutive years.

In 2021, using a sample size of n = 12, the Spearman correlation coefficient’s critical value at α = 0.05 was determined to be rs = 0.578. Particularly, the variety Bojos displayed a strong positive correlation (rs = 0.767) between TPC and seed germination characteristics, suggesting that increased TPC corresponded with vigorous seeds, especially when seed germination characteristics were represented as Len 7. Conversely, KWS Amadora exhibited a negative correlation (rs = − 0.617) when seed germination characteristics were represented as Len 14, implying its decrease as TPC increased. The correlation patterns varied further with AvgD 7, showing a negative correlation with Bojos (rs = − 0.633), while Overture demonstrated a positive correlation (rs = 0.603), indicating increased seed germination characteristics with higher TPC levels. Similarly, the relationship between TPC and seed germination characteristics represented as AvgD 14 exhibited a positive correlation (rs = 0.667) with the Overture variety. Interestingly, in the Bojos variety, these results suggest that higher TPC levels may stimulate root elongation, while root diameter remains unaffected (Supplementary Table 1).

In 2022, employing the Pearson correlation coefficient with a sample size of n = 12, the critical value at α = 0.05 was determined to be r = 0.576. Interestingly, only one variety, KWS Amadora, exhibited a strong negative correlation (r = − 0.892), indicating a consistent decrease in seed germination characteristics with increasing TPC. Especially, no correlations were found among localities in both observation years. The correlation analyses reveal interesting variations in the responses of different varieties to changes in total polyphenol content across the two years (Supplementary Table 2).

Effect of the environment on seed germination characteristics and TPC

The results from 2021 (Fig. 2) (Supplementary Table 3), demonstrated the impact of the environment on the relationship between seed germination characteristics and TPC among barley varieties, as indicated by significant statistical differences. In terms of seed germination characteristics, significant differences in both Len 7 and AvgD 7 suggest that environmental factors significantly influence early root development. The significant differences between Čáslav and Lednice in Len 7 (p = 0.003) and AvgD 7 (p = 0.014) emphasize the environmental impact on this trait. Among the observed localities, Čáslav and Lednice exhibited a significant difference in seed germination characteristics represented by AvgD 7 (p = 0.007) in 2022, while no statistical significances in the Len 7 parameter were found in the same year (Supplementary Table 4).

Fig. 2.

Fig. 2

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Relationship between germination characteristics, represented by AvgD 7 (average diameter after 7 days), Len 7 (length after 7 days) (A), and total polyphenol content (TPC) (B) for the 2021 dataset. For germination characteristics, asterisks indicate significant differences across localities for AvgD 7 and Len 7. For TPC, asterisks indicate significant differences between varieties. Bars represent median values. Asterisks (*) denote significant differences at p < 0.05.

However, the variation in this relationship across different localities and the absence of a consistent ranking of barley varieties in terms of seed germination characteristics and TPC across environments provide a detailed view on our second hypothesis (H2). While a similar ranking of barley varieties in terms of seed germination characteristics and TPC content across different environments was expected, the data suggest that the expression of these traits is highly environment-dependent, complicating a straightforward interpretation of H2.

Effect of genotype on seed germination characteristics and TPC

The distinct TPC levels between KWS Amadora and Laudis 550 (p = 0.005) indicated a significant genotype effect on total polyphenol content in 2021 (Fig. 3) (Supplementary Table 3). This observation supports our first hypothesis (H1), suggesting that elevated seed germination characteristics, as indicated by root parameters, is associated with higher TPC, although this association appears to be variety-specific rather than consistent across all tested varieties. In the following year, 2022, the significant differences continued to be observed (Fig. 3). For seed germination characteristics represented by AvgD 14, a significant difference was observed between the varieties KWS Amadora and Overture (p = 0.016), indicating variability in root development over time. Additionally, the analysis of total polyphenol content revealed a significant difference between KWS Amadora and Laudis 550 (p = 0.009), reaffirming the impact of genotype on total polyphenol content (Supplementary Table 4).

Fig. 3.

Fig. 3

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Relationship between germination characteristics, represented by AvgD 7 and AvgD 14 (average diameter after 7 and 14 days) (C), and total polyphenol content (TPC) (D) for 2022 dataset. For germination characteristics in AvgD 7 chart, asterisks indicate significant differences across localities. In AvgD 14 chart, asterisks indicate significant differences between varieties based on post-hoc test results, as they do for TPC. Bars represent median values for each variety. Asterisks (*) denote significant differences at p < 0.05.

These findings illustrate the combined impact of genotype and environment on both seed germination characteristics and TPC, shedding light on the adaptive responses of barley to its environment and genetic background. Detailed analysis showed that the contribution effect of genotype and environment was significant for TPC, with genotype being more pronounced (45%). On the other hand, for germination characteristics represented by AvgD 7, the effect of the environment (26%) was higher compared with AvgD 14, where the effect of genotype (25%) prevailed. The G x E interaction effect was not significant.

Discussion

This study examined the relationship between total polyphenol content (TPC) and seed germination characteristics i.e. seed vigour, focusing on four diverse spring barley varieties over two years under contrasting environmental conditions. Our findings highlight the significant influence of genetic background on TPC, evidenced by distinct levels observed between varieties such as KWS Amadora and Laudis 550. This genotype effect supports our initial hypothesis (H1), suggesting a correlation between elevated seed germination characteristics, reflected through root parameters, and increased TPC. Additionally, the impact of the environment on seed germination characteristics, demonstrated by differences in root development (Len 7 and AvgD 7) across various localities, aligns with our second hypothesis (H2) but also adds complexity to its interpretation. The absence of a consistent ranking of barley varieties in terms of seed germination characteristics and TPC across different environments suggests a highly context-dependent expression of these traits.

Significant statistical differences in TPC and seed germination characteristics, as seen in the different responses of varieties across years, illustrate the environmental modulation of these traits. For instance, differences in TPC and seed germination characteristics between Čáslav and Lednice in 2021 and 2022 emphasize the environment’s role (such as rainfall and air temperature patterns) in this relationship. These findings align with broader research on plant reactions to environmental stress, such as the impact of drought stress on wheat genotypes, which significantly reduces germination rate, seed vigour, and growth parameters like root length, as a crucial trait for plant adaptation to adverse environmental conditions20.

Yearly variability in environmental effects on root diameter

Due to a strong year effect, all analyses were performed separately. The primary rationale behind this was to explore the fixed effects of genotype, environment, and G × E interactions more comprehensively, including examining the percentage contribution of these fixed effects to the studied relationships. We found that the environmental effect on root diameter after seven days was significant in certain years, particularly in 2022. The environment, understood as the agricultural field conditions, affected root diameter even under similar agricultural practices. However, this effect was not consistent across all years and varieties. These findings suggest that environmental conditions during the ripening stage of grains may influence germination characteristics in subsequent generations, indicating that ripening conditions impact germination. The effect of genotype, however, cannot be overlooked, as the G × E interaction was significant in 2022. This interaction suggests that the performance of different genotypes in terms of TPC varies depending on the environmental conditions they experience. Certain genotypes may show higher or lower TPC depending on the environment. In practical terms, this highlights the importance of selecting genotypes suited to specific environmental conditions to optimize TPC. For breeders and growers, this means that cultivar selection strategies cannot rely solely on genotype performance in a single environment. Instead, a multi-environment approach is essential to identify genotypes that consistently perform well across diverse environments or to match specific genotypes to particular environmental niches. This G x E interaction also emphasize the need for region-specific recommendations in crop management.

Influence of phenolic compounds on germination characteristics in barley varieties under varied environmental conditions

The role of phenolic compounds and their impact on germination characteristics was discussed in the introduction of this study. Based on the findings, it appears that the observed variations in germination characteristics and TPC among different barley varieties indicate that each variety may have a unique polyphenol profile influenced by environmental conditions. This implies that the duration of exposure to stress plays a significant role. Similar to the effects of elevated temperatures, drought stress during seed development can significantly reduce germination characteristics i.e. seed vigour. Previous studies have shown that drought stress during seed filling adversely affects seed vigour in soybean21–23, while also accelerating the maturation of caryopses in barley24, leading to decreased starch accumulation but a significant increase in protein content in the endosperm. Reduced starch availability may limit the energy reserves necessary for proper germination and early seedling development. Furthermore, drought stress environments often results in reduced performance for traits such as weight of grains, which is critical indicator of seed vigour25. The lower grain weight, in particular, reflects poor seed filling, directly impacting seed vigour and the ability of seedlings to establish and thrive under adverse conditions.

This variability indicates that while TPC could potentially serve as an indicator of seed vigour, its reliability is highly dependent on the specific variety and environmental context. While higher polyphenol content generally correlates with increased antioxidant capacity and better survival under stressful conditions, cases where higher seed vigour is associated with reduced polyphenol content can occur due to several reasons. Some varieties might naturally exhibit higher seed vigour while maintaining a lower polyphenol content due to their genetic characteristics26. This association often arises from allele variations within genes responsible for polyphenol synthesis pathways27,28. Agro-environmental conditions, such as the specific field conditions where the barley samples were grown, can significantly impact polyphenol synthesis, with a higher polyphenol content under less nitrogen content in soil29. Certain environmental stressors or optimal growth conditions might influence the plant’s prioritization of energy allocation, leading to changes in polyphenol production30. Some varieties could have mechanisms that optimize seed vigour by channelling resources away from polyphenol synthesis toward other processes vital for seed development and germination31. Varieties prioritizing traits for enhanced vigour, such as better germination rates or stress tolerance, might sacrifice polyphenol content as part of their overall survival strategy under harsh conditions such as wrong cropping patterns, low soil nutrients, or an unbalanced microbial community32. It is important to note that the effect or preceding crop on polyphenol content is important too. Varieties may adjust their polyphenol production based on the prevailing environmental cues during the specific growing season33. The influence of the above-mentioned factors was limited in our study by using identical agricultural techniques across all locations and years. Emphasized are thus the effects of environmental conditions, including yearly variations, and the variety on the observed traits.

Future research should focus on expanding the genotypic range by incorporating more barley varieties and conducting detailed genetic profiling to understand the genetic basis of polyphenol synthesis and seed germination characteristics. Longitudinal and multi-location studies are necessary to capture the long-term environmental effects and yearly variations on these traits. Controlled stress experiments, including varying the duration and timing of stress exposure, will help elucidate critical periods for intervention. Detailed polyphenol analysis and functional studies should identify influential phenolic compounds, while exploring the impact of agronomic practices and soil health on polyphenol synthesis and seed germination characteristics. Interdisciplinary collaborations with ecologists and agronomists, as well as predictive modelling, will provide a holistic approach to developing resilient barley varieties.

Conclusions

The results highlight the significant influence of both genetic background and environmental conditions on total polyphenol content (TPC) and seed germination characteristics. The significant genotype-environment interactions emphasize the need for a tailored approach in selecting barley varieties that perform consistently under specific environmental conditions, particularly for optimizing TPC. Incorporating TPC, along with other morphological traits such as root length, seedling biomass, and grain weight, may provide a targeted strategy to enhance both seed vigour and adaptability in barley, ultimately improving its performance and resilience across varying environmental conditions.

While TPC alone may not be a definitive predictor of seed vigour, it remains a valuable biomarker for future research and may play an important role in optimizing barley cultivation and processing.

Materials and methods

Plant material and growing conditions

To observe effects of variations in environmental factors on grain composition, three localities (Čáslav, Uherský Ostroh, and Lednice) with intensive cultivation of barley specifically grown for malt production were selected. Their altitude ranges from 171 to 260 m above sea level, with differing soil conditions, air temperatures, rainfall (Table 1). Four high-quality malting varieties of spring barley (Hordeum vulgare, L.) Overture, KWS Amadora, Bojos and Laudis 550 were grown. The Bojos and Laudis 550 varieties met the requirements set out in the application for the protected geographical indication (PGI) ‘České pivo’34. Therefore, the Research Institute of Brewing and Malting recommended them for the production of beer under the PGI ‘České pivo’. The cultivation process followed common agricultural practices during the growing seasons of 2021, and 2022. The Central Institute for Supervising and Testing in Agriculture, in cooperation with the Research Institute of Brewing and Malting, oversaw the cultivation process and provided the seeds.

Table 1.

Basic characteristics of the localities used in this study.

Localities GPS Soil type* Soil group Altitude (m) Average annual precipitation sum (mm) Average annual air temperature (°C)
Čáslav 49.9109° N, 15.3908° E Phaeozem Chernozems 260 583 9.7
Uherský Ostroh 48.9856° N, 17.3899° E Kambisol Kambisols 196 651 9.8
Lednice 48.7999° N, 16.8034° E Chernozem Chernozems 171 514 10.3
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Soil type and soil group are based on the World Reference Base for Soil Resources (WRB) classification35. Average annual precipitation sum, and air temperature are represented for the period (1991–2020) based on the long-term data obtained from the Czech Hydrometeorological Institute34.

Data from the climatological stations of the Czech Hydrometeorological Institute36 were used to evaluate weather conditions of the years (Lednice, data from station Strážnice for Uherský Ostroh, Čáslav), as outlined in Table 2. From the perspective of the possible influence on the quality of seeds, the course of weather during the filling and ripening stage in grains has a fundamental impact. In this context, there is a significant difference between the meteorological conditions in May and June 2021 and 2022. The year 2021 was characterized by significantly lower temperatures and significantly higher precipitation totals during May. This could affect the quality of seeds during the grain filling period - including a lower content of photosynthates. In contrast, conditions in June were temperature-wise comparable in both years, with limited precipitation totals in 2021. Meteorological conditions in May 2022 (drought) and in June 2022 (higher precipitation totals) could lead to biochemical differences in the grain compared with 2021.

Table 2.

Overview of meteorological conditions in years 2021–2022, represented as the heating map (and average values for each month for the period 1991–2020).

graphic file with name 41598_2024_74795_Tab1_HTML.jpg

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Determination of germination characteristics

The germination characteristics of the harvested seeds were assessed after they had broken dormancy, i.e., three months post-harvest. Selected barley seeds represented four different varieties and three localities. The first step involved sorting the seeds (in the sieving fraction over 2.5 mm)37, and then they were immersed in a 3% sodium hypochlorite solution for 10 min, followed by washing with distilled water three times.

Germination of barley seeds is an important characteristic for both seed and malting quality. While this parameter is evaluated under optimal conditions for germination (laboratory temperatures and optimal moisture), it differs from germination in the soil and malting38.

The evaluation of germination characteristics in barley, with a focus on germination under drought conditions (−0.5 MPa) and suboptimal temperature (10 °C), was carried out. The control variant underwent a standard germination test conducted according to the ISTA standard method, with no significant differences found. Drought conditions at -0.5 MPa were induced using polyethylene glycol (PEG 6000) based on the methodology of Michel and Kaufmann39. Additionally, six seeds of each variety were placed in separate 90 mm diameter plastic sterile Petri dishes, each containing 8 ml of PEG 6000 solution. The experiment involved three repetitions of each variety at each locality to ensure accuracy. This quantity of seeds was most suitable for ease of manipulation on the limited scanning area of the scanner, meeting the technical requirements. The use of PEG in the Petri dishes, without filter paper, was considered a valuable technique, as filter paper could potentially interfere with the integrity of the root structure during sample manipulation. Petri dishes were labelled and enclosed in plastic bags to prevent evaporation. Subsequently, the Petri dishes were placed in a climate-controlled chamber (in darkness conditions) set at 10 °C. After durations of seven and fourteen days, the germinated seeds from the Petri dishes were scanned and subjected to analysis using WinRHIZO (Régent Instruments Inc., Quebec, Canada), version 2020 Arabidopsis. To assess germination characteristics under drought in the Petri dishes, the following metrics were employed: Len 7, Len 14, AvgD 7, and AvgD 14 (root length (cm) and average diameter of roots (mm) after seven and fourteen days).

Preparation of the extract for TPC evaluation

Twenty grams of barley grains from each variety and locality were grounded finely on a 1 mm sieve barley grinder (each sample in three repetitions). After that, 5 g of ground barley was weighed into a 200 ml tall beaker. Then, 100 ml of 75% dimethylformamide solution was added, according to the EBC method 9.1140. Shortly afterward, the mixer head was immersed in the beaker and mixed for ninety seconds. The mixture was supposed to be left from 12 to 15 min and the whole procedure was repeated three times in each sample. After the last mixing, the solution was poured into a centrifuge tube. Samples were centrifuged at 3000 rpm for 10 min. From the centrifuged solution 25 ml of the solution was pipetted into a 100 ml volumetric flask and made up to the mark line with deionized water. The solution was poured into a centrifuge tube and for the last time samples were centrifuged at 15,000 rpm for 30 min. The pure solution has been used for the determination of total polyphenols content.

Preparation of a blank solution

Twenty ml of extract was pipetted into a 50 ml volumetric flask. After that, 0.10 ml ammonium solution was added. The solution was mixed thoroughly and made up to the mark line with deionized water. Then it was let to stand for 10 min and the clarity of the solution was observed.

Determination of total polyphenol content (TPC)

Twenty ml of extract was pipetted into a 50 ml volumetric flask, then 16 ml of ethylenediaminetetraacetic sodium hydroxide (CMC/EDTA) was added. Then, 0.10 ml of ammonium solution was added, made up to the mark line with deionized water and mixed thoroughly. Just before the measurement, a solution of ammonium ferric citrate in an amount of 0.10 ml was added and the content was shaken. The measurements were carried out with the requirement that each sample undergo three measurements within a 10-min window. This protocol was implemented to avoid potential colour changes in the sample resulting from the introduction of an ammonium ferric citrate solution over extended periods, which could potentially affect the recorded results. Consequently, it is crucial to strictly follow the measurement guidelines and complete the measurements within 10 min of introducing the ammonium ferric citrate solution. The absorbance was measured spectrophotometrically in a 10 mm cuvette at a wavelength of 525 nm against a blank solution.

Calculation and evaluation of TPC

The calculation was performed according to the formula for calculating the content of polyphenols in the original solution:

Inline graphic

where Pp is a polyphenols content in the original solution (%).

Ah is the absorbance of the main solution (%).

Ak is the absorbance of the blank solution (at 525 nm wavelength).

Afterward, it was essential to perform a recalculation of the sample’s dry matter, which was carried out using the following formula:

graphic file with name M2.gif

where Ps is a polyphenols content in dry matter of the sample (%).

V is the content of the water in barley seeds.

To obtain the water content of the barley seeds, it was necessary to determine the percentage of seeds moisture for each variety.

Statistical analyses

All statistical analyses were performed using the Python (3.10.12), using the libraries SciPy (1.12.0)41, statsmodels, scikit-learn, and matplotlib. All data were tested for normality using the Shapiro-Wilk test42. For datasets not exhibiting a normal distribution, multiple Kruskal-Wallis one-way analyses of variance were utilized43. In cases of normal distribution, a two-way MANOVA was conducted to assess the significant differences in total polyphenol content and germination characteristics across four different genotypes at three distinct localities. To identify the specific traits, varieties, and localities that demonstrated significant differences, separate ANOVAs were conducted for each parameter that exhibited a normal distribution after MANOVA. This step was essential for identifying precise differences. Subsequently, Tukey’s HSD test was used for the 2022 data which met a normal distribution criteria, and Dunn’s post-hoc test was used for the 2021 data which did not meet a normal distribution criteria with applied significance (p < 0.05) within the traits. The Tukey’s HSD test was used to assess the percentage contributions of genotype, environment, and genotype-environment interaction (G x E) to the observed relationship, with each effect’s contribution calculated using partial eta squared (η2)42,43.

Principal Component Analysis (PCA) was conducted to group and relate traits for each year. The Spearman correlation coefficient (rs) was used for datasets not exhibiting normal distribution, while the Pearson correlation coefficient (r) was applied to datasets with a normal distribution. This approach ensures accurate analysis of the relationships between traits. Graphical representations (Figs. 1, 2 and 3) were generated using the Plotly Python Graphing Library (5.19.0). Principal Component Analysis (PCA) biplot visualizes the multivariate relationships among total polyphenol content (TPC) and germination characteristics (such as root length and average root diameter) across different barley varieties and localities. The PCA biplot reduces the dimensionality of the data, making it easier to identify clustering patterns and correlations between variables. Meanwhile, the box plots represent median values across multiple categories. These box plots highlight the variations in TPC and seed germination characteristics (root length, average root diameter) among different barley varieties across localities. Box plots were chosen for their simplicity, yet they provide an effective visual representation of the results.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (205.9KB, pdf)

Acknowledgements

We would like to express our gratitude to The Central Institute for Supervising and Testing in Agriculture and the Research Institute of Brewing and Malting for overseeing the cultivation process and providing the seeds.

Author contributions

IJ: Conceptualization, Methodology, Formal analysis, Investigation, Visualisation, Writing—Original Draf. NF: Conceptualization, Formal analysis, Writing—Review & Editing. JEAM: Methodology, Writing—Review & Editing. LP: Writing—Review & Editing. VP: Methodology, Writing—Review & Editing, Funding acquisition. JA: Methodology. RC: Writing—Review & Editing, Funding acquisition. TS: Conceptualization, Writing—Review & Editing, Supervision, Funding acquisition.

Funding

The study was prepared with the financial support of the research project QL24010109 and the institutional support MZE-RO1923 of the Ministry of Agriculture of the Czech Republic. Moreover, the additional funding for this article was provided by Mendel University in Brno, which supported both the research and publication efforts.

Data availability

Data is provided within the manuscript and as a supplementary information file (doi.org/10.5281/zenodo.11518623).The Supplementary Materials include all statistical results from the correlation analysis and ANOVA/Kruskal-Wallis tests, including both significant and non-significant findings, detailing the dynamics of the traits.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

The original online version of this Article was revised: The Funding section in the original version of this Article was omitted. The Funding section now reads: "The study was prepared with the financial support of the research project QL24010109 and the institutional support MZE-RO1923 of the Ministry of Agriculture of the Czech Republic. Moreover, the additional funding for this article was provided by Mendel University in Brno, which supported both the research and publication efforts."

Publisher’s note

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

Change history

2/3/2025

A Correction to this paper has been published: 10.1038/s41598-025-86339-7

References

  • 1.Reed, R. C., Bradford, K. J. & Khanday, I. Seed germination and vigor: ensuring crop sustainability in a changing climate. Heredity. 128, 450–459 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Středa, T., Středová, H., Chuchma, F., Kučera, J. & Rožnovský, J. Smart method of agricultural drought regionalization: a winter wheat case study. Contrib. Geophys. Geod. 49, 25–36 (2019). [Google Scholar]
  • 3.Wing, I. S., De Cian, E. & Mistry, M. N. Global vulnerability of crop yields to climate change. J. Environ. Econ. Manag. 109, 102462 (2021). [Google Scholar]
  • 4.Finch-Savage, W. E. & Bassel, G. W. Seed vigour and crop establishment: extending performance beyond adaptation. J. Exp. Bot. 67, 567–591 (2016). [DOI] [PubMed] [Google Scholar]
  • 5.Wen, D. et al. Rapid evaluation of seed vigor by the absolute content of protein in seed within the same crop. Sci. Rep. 8, 5569 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Baik, B. K. & Ullrich, S. E. Barley for food: characteristics, improvement, and renewed interest. J. Cereal Sci. 48, 233–242 (2008). [Google Scholar]
  • 7.Arigò, A., Česla, P., Šilarová, P., Calabrò, M. L. & Česlová, L. Development of extraction method for characterization of free and bonded polyphenols in barley (Hordeum vulgare L.) grown in Czech Republic using liquid chromatography-tandem mass spectrometry. Food Chem. 245, 829–837 (2018). [DOI] [PubMed] [Google Scholar]
  • 8.Rodríguez, M. V., Barrero, J. M., Corbineau, F., Gubler, F. & Benech-Arnold, R. L. Dormancy in cereals (not too much, not so little): about the mechanisms behind this trait. Seed Sci. Res. 25, 99–119 (2015). [Google Scholar]
  • 9.Gan, R. Y. et al. Bioactive compounds and bioactivities of germinated edible seeds and sprouts: an updated review. Trends Food Sci. Technol. 59, 1–14 (2017). [Google Scholar]
  • 10.Burguieres, E., McCue, P., Kwon, Y. I. & Shetty, K. Effect of vitamin C and folic acid on seed vigour response and phenolic-linked antioxidant activity. Bioresour Technol. 98, 1393–1404 (2007). [DOI] [PubMed] [Google Scholar]
  • 11.Chloupek, O. et al. Better bread from vigorous grain? Czech J. Food Sci. 26, 402–412 (2008). [Google Scholar]
  • 12.Basařová, G. et al. Sladařství: Teorie a Praxe výroby Sladu (Havlíček Brain Team, 2015).
  • 13.Piasecka, A. et al. Drought-related secondary metabolites of barley (Hordeum vulgare L.) leaves and their metabolomic quantitative trait loci. Plant. J. 89, 898–913 (2017). [DOI] [PubMed] [Google Scholar]
  • 14.Mikyška, A., Dušek, M. & Čejka, P. Influence of barley variety and growing locality on the profile of flavonoid polyphenols in malt. Kvas Prum. 65, 149–157 (2019). [Google Scholar]
  • 15.Mikyška, A. & Jurková, M. Study on the effect of malt and decoction mashing on polyphenols and antiradical power of wort. Kvas Prum. 70, 846–854 (2024). [Google Scholar]
  • 16.Fayez, K. A. & Bazaid, S. A. Improving drought and salinity tolerance in barley by application of salicylic acid and potassium nitrate. J. Saudi Soc. Agric. Sci. 13, 45–55 (2014). [Google Scholar]
  • 17.Singh, S., Gupta, A. K. & Kaur, N. Influence of drought and sowing time on protein composition, antinutrients, and mineral contents of wheat. Sci. World J. 485751 (2012). (2012). [DOI] [PMC free article] [PubMed]
  • 18.Vaughan, M. M. et al. Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance. Plant. Cell. Environ. 38, 2195–2207 (2015). [DOI] [PubMed] [Google Scholar]
  • 19.Yahyazadeh, M., Meinen, R., Hänsch, R., Abouzeid, S. & Selmar, D. Impact of drought and salt stress on the biosynthesis of alkaloids in Chelidonium majus L. Phytochemistry. 152, 204–212 (2018). [DOI] [PubMed] [Google Scholar]
  • 20.Kızılgeçi, F., Tazebay, N., Namlı, M., Albayrak, Ö. & Yıldırım, M. The drought effect on seed germination and seedling growth in bread wheat (Triticum Aestivum L). Int. J. Agric. Environ. Food Sci. 1, 33–37 (2017). [Google Scholar]
  • 21.Samarah, N. H., Mullen, R. E. & Anderson, I. Soluble sugar contents, germination, and vigor of soybean seeds in response to drought stress. J. New. Seeds. 10, 63–73 (2009). [Google Scholar]
  • 22.Wijewardana, C., Reddy, K. R., Krutz, L. J., Gao, W. & Bellaloui, N. Drought stress has transgenerational effects on soybean seed germination and seedling vigor. PLoS One. 14, e0214977 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tuladhar, P., Sasidharan, S. & Saudagar, P. Role of phenols and polyphenols in plant defense response to biotic and abiotic stresses. in Biocontrol Agents and Secondary Metabolites: Applications and Immunization for Plant Growth and Protection (ed Jogaiah) 419–441 (Woodhead Publishing, (2021).
  • 24.Li, F. et al. Novel insights into the effect of drought stress on the development of root and caryopsis in barley. PeerJ. 8, e8469 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Abdelghany, A. M., Lamlom, S. F. & Naser, M. Dissecting the resilience of barley genotypes under multiple adverse environmental conditions. BMC Plant. Biol. 24, 16 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Alfaro, S. et al. Influence of genotype and harvest year on polyphenol content and antioxidant activity in murtilla (Ugni molinae Turcz) fruit. J. Soil. Sci. Plant. Nutr. 13, 67–78 (2013). [Google Scholar]
  • 27.Han, Z. et al. Association mapping for total polyphenol content, total flavonoid content and antioxidant activity in barley. BMC Genom. 19, 81 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yang, X. J., Dang, B. & Fan, M. T. Free and bound phenolic compound content and antioxidant activity of different cultivated blue highland barley varieties from the Qinghai-Tibet Plateau. Molecules. 23, 879 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Heimler, D., Romani, A. & Ieri, F. Plant polyphenol content, soil fertilization and agricultural management: a review. Eur. Food Res. Technol. 243, 1107–1115 (2017). [Google Scholar]
  • 30.Pérez-Ochoa, M. L. et al. Effects of annual growth conditions on phenolic compounds and antioxidant activity in the roots of Eryngium montanum. Plants. 12, 3192 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kołton, A., Długosz-Grochowska, O., Wojciechowska, R. & Czaja, M. Biosynthesis regulation of folates and phenols in plants. Sci. Hortic. (Amsterdam). 291, 110561 (2022). [Google Scholar]
  • 32.Fan, D., Zhao, Z., Wang, Y., Ma, J. & Wang, X. Crop-type-driven changes in polyphenols regulate soil nutrient availability and soil microbiota. Front. Microbiol. 13, 964039 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Brahmi, F. et al. Impact of growth sites on the phenolic contents and antioxidant activities of three Algerian Mentha species (M. Pulegium L., M. Rotundifolia (L.) Huds., and M. Spicata L). Front. Pharmacol. 13, 886337 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Commission Regulation. Publication of an application pursuant to article 6(2) of Council Regulation (EC) 510/2006 on the protection of geographical indications and designations of origin for agricultural products and foodstuffs. Off J. Eur. Union. 85, 13–16 (2008). [Google Scholar]
  • 35.FAO. World Reference base for soil Resources (Food and Agriculture Organization of the United Nations (FAO), 2015).
  • 36.Horáková, V., Dvořáčková, O. & Nečas, M. Seznam doporučených odrůd - Obilniny (Ústřední kontrolní a zkušební ústav zemědělský, 2022).
  • 37.Analytica, E. B. C. Sieving test of barley method 3.11.1. (2010).
  • 38.Ullmannová, K., Středa, T. & Chloupek, O. Use of barley seed vigour to discriminate drought and cold tolerance in crop years with high seed vigour and low trait variation. Plant. Breed. 132, 295–298 (2013). [Google Scholar]
  • 39.Michel, B. E. & Kaufmann, M. R. The osmotic potential of polyethylene glycol 6000. Plant. Physiol. 51, 914–916 (1973). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Analytica, E. B. C. Total polyphenols in beer by spectrophotometry (IM) method 9.11. (2010).
  • 41.Kruskal, W. H. & Wallis, W. A. Use of ranks in One-Criterion Variance Analysis. J. Am. Stat. Assoc. 47, 583–621 (1952). [Google Scholar]
  • 42.McDonald, J. H. Handbook of Biological Statistics: One-way ANOVA (Sparky House Publishing, 2014).
  • 43.Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods. 17, 261–272 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (205.9KB, pdf)

Data Availability Statement

Data is provided within the manuscript and as a supplementary information file (doi.org/10.5281/zenodo.11518623).The Supplementary Materials include all statistical results from the correlation analysis and ANOVA/Kruskal-Wallis tests, including both significant and non-significant findings, detailing the dynamics of the traits.

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