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Article

Raffinose Priming Improves Seed Vigor by ROS Scavenging, RAFS, and α-GAL Activity in Aged Waxy Corn

by
Min Zhu
†,
Ru Xiao
†,
Tong Yu
,
Tao Guo
,
Xuemei Zhong
,
Jianzhou Qu
,
Wanli Du
and
Wei Xue
*
College of Agronomy, Specialty Corn Institute, Shenyang Agricultural University, Shenyang 110866, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(12), 2843; https://doi.org/10.3390/agronomy14122843
Submission received: 4 November 2024 / Revised: 22 November 2024 / Accepted: 26 November 2024 / Published: 28 November 2024
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

:
Raffinose family oligosaccharides (RFOs) are known to benefit plants under stress conditions; however, the role of exogenous raffinose in seed germination remains poorly understood. In this study, we investigated the potential role of raffinose in promoting seed germination and elucidated the underlying mechanisms. The results showed that artificial aging significantly reduced the germination rate and vigor of waxy corn seeds. Conversely, exogenous raffinose significantly enhanced the germination of these artificially aged seeds. Exogenous raffinose significantly reduced the levels of reactive oxygen species (O2− and H2O2) and enhanced the activity of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Additionally, the levels of α-galactosidase (α-GAL) and raffinose synthase (RAFS) were significantly elevated in raffinose-treated aged seeds. These findings suggest that exogenous raffinose induces the expression of α-GAL and RAFS, thereby providing energy and reducing excessive reactive oxygen species (ROS), which in turn promotes the germination of artificially aged seeds. This study provides a theoretical foundation for enhancing seed vigor and extending seed longevity in crop management.

1. Introduction

Seed aging is a complex and irreversible process associated with internal factors including membrane damage, DNA integrity loss, mitochondrial dysregulation, protein damage, and disrupted antioxidative machinery [1,2,3], as well as external factors like storage conditions [4]. According to the “free radical theory of aging”, a biological theory about the aging process, which suggests that many of the changes and damages that occur during aging are caused by free radicals, the loss of seed vigor is caused by excessive production of reactive oxygen species (ROS) and disruption of antioxidative machinery [5]. The content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) are significantly increased, and antioxidant enzymes activities and gene expression are reduced in aged seeds, thereby reducing seed vigor and seed germination, even affecting seedling growth and development [3,4,6].
Raffinose family oligosaccharides (RFOs) are a class of soluble oligosaccharides widely present in plants. They include raffinose, stachyose, and verbascose, among which raffinose is commonly found in all plants [7,8]. In recent years, it has been found that RFOs can act as an osmo protectant and stabilizer for cells, directly or indirectly participating in the scavenging of ROS, protecting cellular metabolism, improving seed germination, promoting seed vigor, and extending seed longevity [9]. After being absorbed by the seed, raffinose leads to the activation of signal transduction pathways (with the production of plant hormones and signaling molecules), further activating antioxidant response elements (AREs) (activation of Nrf2-like transcription factors) and increasing the expression of antioxidant enzyme genes, thereby enhancing the activity of antioxidant enzymes to reduce the level of ROS. Raffinose, the first member of the RFOs, is a trisaccharide synthesized by raffinose synthase (RAFS) using sucrose and galactoside as substrates. Studies have found that RAFS is a key enzyme in the synthesis pathway of RFOs, regulating the synthesis and accumulation of raffinose in plants [10]. Raffinose synthase in plants promotes the rapid accumulation of raffinose, thereby enhancing the seed vigor of Arabidopsis, maize, and rice [7,11]. Currently, there is only one raffinose synthase gene (ZmRS) which has been cloned in the corn genome, and it only has the function of synthesizing raffinose [12]. α-galactosidase (α-GAL) is distributed in various plant organs such as seeds, seedlings, and leaves, which participate in RFO unloading, seed development and germination, leaf development, and senescence processes. Hence, the hydrolytic action of α-GAL on RFOs is also important for seed vigor, which provides energy for seed germination [13].
Seed priming is a physiological method that encompasses the controlled hydration of seeds for the commencement and regulation of pre-germinative metabolic processes and activation of antioxidant enzymes while preventing radicle emergence, which leads to a high rate of germination and further good development in seedling growth and even crop yield under biotic and abiotic stress conditions [12,14]. Previous studies have demonstrated that using fructan oligosaccharide [15], salicylic acid [16], lipo chitooligosaccharide [17,18], and gibberellins [6] as priming agents significantly enhanced the germination rate of rice, maize, chicory, and other crop seeds under abiotic stress. This was associated with an increase in the activity of antioxidant enzymes in the seeds and a reduction in the accumulation of ROS and MDA; thus, seed vigor and germination rate improved. Under low-temperature stress, seed germination parameters and antioxidant enzyme activities of waxy corn seeds treated with melatonin is significantly increased, while H2O2 and MDA content is reduced, resulting in a significant improvement of seed vigor [19]. After priming treatment with 30% PEG-6000 for 24 h, the vigor of aged soybean seeds was enhanced by coordinating carbon metabolism, ROS scavenging, hormone signal transduction, and lignin synthesis [4].
Waxy corn (Zea mays L. sinensis Kulesh) is a plant, which originated in Southwest China and had better palatability and digestibility than field maize. Seed germination ability is one of the most important parameters for waxy corn production. However, seed aging can reduce germination rate and seed vigor, adversely affecting the growth and yield of waxy corn. Therefore, improvement of the seed vigor of waxy corn is becoming increasingly important. The main objectives of this study were as follows: (1) to determine whether raffinose improves the seed germination and seed vigor of waxy corn; (2) to elucidate the protective role of raffinose on the aged seeds of waxy corn by evaluating ROS and antioxidant enzyme activities; and (3) to examine the effects of raffinose on α-GAL and RAFS and the expression levels of ZmRS. It is hypothesized that exogenous raffinose will mitigate the effects of seed aging in waxy corn by reducing ROS levels and enhancing raffinose synthase activity, thereby improving seed vigor and longevity.

2. Materials and Methods

2.1. Experimental Materials and Treatments

Waxy corn inbred lines SYKN167 and SD88, provided by Specialty Corn Institute of Shenyang Agricultural University, were used in this study.
Seeds were artificially aged following a modified version of Bhattacharyya [20]’s protocol. Waxy corn seeds placed in mesh bags were completely submerged in a sealed thermostatic water bath (SENXIN, Shanghai, China) at 58 °C (±1 °C) for 30 min, based on prior experiments. After aging, the seeds were cooled at room temperature and stored following restoration to their original moisture content at 4 °C.
Both non-aged and artificially aged seeds were sterilized with 0.1% NaClO2 for 10 min, rinsed three times with distilled water, and subjected to priming with 20 mL of 13.5 mM (based on previous experiments) raffinose at 25 °C in darkness for 12 h. The seeds were then rinsed with distilled water three times and dried with filter paper to remove surface moisture, allowing them to return to their original water content at room temperature for later use.
After priming, twenty seeds were placed in petri dishes lined with two layers of filter paper and incubated in darkness for 7 days at 25 °C. All the experiments were laid out in a completely randomized design with three biological replications. The seed germination was recorded daily and determined the seedling length on the 7th day. Seeds were considered to be germinated when radicle length exceeded 2 mm. The germinated seeds were collected at 24 h, 48 h, 72 h, and 96 h, and stored at −80 °C. In addition to monitoring seed germination, the materials collected at these four time points were used for the following analyses: seed vigor assessment, determination of antioxidant enzyme activities, histochemical detection, and quantification of superoxide anion and hydrogen peroxide. Based on the results from these four time points, samples from 24 h and 72 h were selected for the determination of sucrose synthase activity and α-galactosidase activity. The experiment included four treatments:
  • Non-aged seeds + distilled water (CK);
  • Non-aged seeds + raffinose solution (CK + RAF);
  • Artificially aged seeds + distilled water (AA);
  • Artificially aged seeds + raffinose solution (AA + RAF).

2.2. Determination of Seed Germination Indexes

Germination rate (GR) was determined on the 7th day [19].
Germination Rate (%) (GR) = (Total number of seeds germinated on the 7th day/Total seeds) × 100%.
Germination Index (GI) = ∑ (Gt/Dt), where Gt is the number of seeds germinated on day Dt.

2.3. Seed Vigor Test

Ten seeds for each treatment were split longitudinally along the embryo, soaked in 20 mL 0.1% 2, 3, 5-triphenyltetrazolium chloride (TTC) solution at 35 °C for 30 min in the dark, rinsed with distilled water, and photographed to observe the staining intensity [21].

2.4. Determination of Antioxidant Enzyme Activities

A total of 0.1 g fresh embryo samples, equivalent to the fresh embryo weight of two seeds, were ground in 500 μL of 50 mM cool phosphate buffer [pH 7.0, containing 1% (w/v) PVP]. The homogenate was centrifuged at 15,000× g for 20 min at 4 °C.
Superoxide dismutase (SOD) activity was measured using the nitro blue tetrazolium (NBT) method [22]. The reaction system comprised 50 μL of enzyme solution and 1.5 mL of a reaction mixture that included 14.5 mM methionine, 0.1 mM EDTA, 5 mM nitroblue tetrazolium (NBT), and 5 mM riboflavin. Then, the mixture was exposed to light for 10 min and OD560 was determined rapidly.
Peroxidase (POD) activity was determined by the method of Castro [23]. The reaction mixture was contained 50 μL of enzyme extract, 1 mL of sodium acetate buffer (100 mM, pH 5.4), 0.5 mL of 0.25% (w/v) guaiacol solution, and 50 μL of 0.75% (w/v) H2O2 solution. The absorbance at 470 nm was measured for the mixture using a multifunctional microplate reader for 4 min.
Catalase (CAT) activity was measured according to the method of Havir [24]. A total of 100 μL of enzyme extract was mixed with 300 μL of 10 mM H2O2, and the absorbance at 240 nm was measured using a multifunctional microplate reader for 4 min, with readings taken every minute.

2.5. Histochemical Detection and Quantification of Superoxide Anion and Hydrogen Peroxide

H2O2 was detected histochemically by diaminobenzidine (DAB) staining, and O2− by nitroblue tetrazolium (NBT) staining [21]. Seeds were individually immersed in 20 mL of 1% DAB solution at 30 °C for 12 h and 20 mL of 0.02% NBT solution for 6 h, and then photographed. H2O2 content was quantified using a detection kit (Solarbio, Beijing, China), and O2− content was determined using a kit from Leagene (Beijing, China), following the manufacturer’s instructions.

2.6. Determination of Raffinose Synthase Activity

Approximately 0.1 g of fresh maize embryos were ground into powder in liquid nitrogen. The powder was homogenized in PBS, centrifuged, and the supernatant was collected to obtain the protein solution. Raffinose synthase (RAFS) activity was measured using an ELISA detection kit (Meimian, Yancheng, China), following the manufacturer’s instructions.

2.7. Determination of α-Galactosidase Activity

α-galactosidase activity was determined based on the method of Feurtad [25], with modifications. Approximately 0.1 g of fresh maize embryos were ground into powder with liquid nitrogen, and 200 μL 0.1 M Hepes-0.5 M NaCl buffer (pH 8) was added for 5 min for enzyme extraction. The mixture was then centrifuged for 15 min, and the supernatant was collected as the enzyme extract. The reaction system [75 μL McIlvaine buffer (pH 4.5), 15 μL 10 mM p-Nitrophenyl-α-D-galactopyranoside (substrate) (Meryer (Shanghai, China), Product # M86238-25G, Purity: AR), 60 μL enzyme extract] was incubated at 37 °C for 15 min, after which the reaction was terminated by adding 75 μL 0.2 M Na2CO3. Absorbance was measured at 405 nm.
The α-galactosidase activity (U/g) was calculated as:
α-GAL (U/g) = X × V1 × V/(V2 × W × T)
where X: The pNP concentration determined based on the standard curve; V1: total reaction volume; V: extract volume; V2: sample volume; W: sample weight; T: reaction time.

2.8. Quantitative Real-Time PCR (qRT-PCR)

The total RNA was extracted using a Plant RNA Rapid Extraction Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). Reverse transcription was performed with MonScriptTM RTIII All-in-One Mix, and qRT-PCR was conducted with MonAmpTM ChemoHS qPCR Mix, following the manufacturers’ instructions. Primer sequences are shown in Table 1. The 2–ΔΔCt method was used to calculate the relative expression level.

2.9. Statistical Analysis

The data were expressed as the means ± standard deviations (SD) and were analyzed using SPSS (version 25.0, SPSS Inc., Chicago, IL, USA). One-way ANOVA was performed with the Least Significant Difference (LSD) method at a significance level of p < 0.05. Multiple comparisons were performed using Duncan’s test. Plotting was performed using Microsoft Excel 2019 (Microsoft, Redmond, WA, USA).

3. Results

3.1. Promotive Effect of Raffinose on Germination of Seeds

To investigate the effect of exogenous raffinose (RAF) on the germination indices of waxy corn seeds, seeds treated with CK, CK + RAF (13.5 mM), AA, and AA + RAF (13.5 mM) were subjected to germination treatment. The results showed that exogenous raffinose improved the germination rate of both SYKN167 and SD88, while artificial aging (AA) significantly reduced germination, decreasing by 9.80% and 20.45%, respectively. However, exogenous RAF significantly mitigated this reduction, increasing the germination rate of artificially aged seeds by 7.84% in SYKN167 and 20.45% in SD88. Similarly, the germination index followed the same pattern as the germination rate (Figure 1A).
Exogenous RAF also promoted seedling and mesocotyls length in artificially aged seeds, increasing by 18.89% and 5.50% (p > 0.05) in SYKN167, while in SD88, it increased by 8.08% and 28.78% and respectively (Figure 1B). In addition, exogenous RAF treatment enhanced germination and the elongation of mesocotyls and roots in both lines compared to the CK, whereas artificial aging had a significantly inhibitory effect (Figure 1C).

3.2. Effect of Raffinose Priming on Seed Vigor

TTC staining was used to assess seed vigor, as TTC reduction to red TTF (triphenylmethyl) indicates viable tissue. Therefore, the vigor of seeds can be determined based on the intensity of staining of the embryo. In both SYKN167 and SD88, artificial aging weakened TTC staining compared to CK, whereas exogenous RAF treatment enhanced staining, indicating improved seed vigor (Figure 2).

3.3. Effect of Raffinose on Antioxidant Enzyme Activity

Antioxidant enzymes are activated under stress to scavenge excess ROS. As shown in Figure 3A, exogenous RAF significantly increased POD activity in both SYKN167 and SD88 compared to CK. In SYKN167, POD activity increased by 12.39%, 12.12%, 17.26%, and 26.57%, while in SD88, it increased by 10.57%, 5.47%, 5.42%, and 25.87% at 24, 48, 72, and 96 h, respectively. In contrast, the artificial aging markedly decreased the POD activity. However, RAF treatment notably mitigated these reductions, with SYKN167 showing increases of 35.15%, 23.09%, 13.80%, and 32.70%, and SD88 showing increases of 20.27%, 13.81%, 21.50%, and 62.62%, respectively (Figure 3A). Similar trends were observed for SOD and CAT activities, both of which were significantly decreased by artificial aging but enhanced by exogenous RAF priming (Figure 3B,C).

3.4. Effect of Raffinose on Reactive Oxygen Species Levels During Waxy Corn Seed Germination

ROS function as signaling molecules to regulate seed germination and excessive ROS accumulation has a toxic effect on seeds. Artificial aging led to higher accumulations of the superoxide anion (O2−) and hydrogen peroxide (H2O2), as revealed by NBT and DAB staining. However, exogenous RAF treatment significantly reduced the levels of these reactive oxygen species (ROS) in both SYKN167 and SD88 (Figure 4A and Figure 5A). Quantification of ROS during germination further supported these findings. Artificial aging significantly increased O2− content, with SYKN167 showing increases of 27.46%, 5.57%, 18.63%, 8.03%, and SD88 showing increases of 61.03%, 9.47%, 65.39%, and 29.64% at 24, 48, 72, and 96 h, respectively. RAF treatment, however, significantly reduced O2− levels. In SYKN167, O2− content decreased by 63.14%, 50.32%, 24.93%, and 123.56%, while in SD88, it decreased by 7.59%, 29.98%, 71.82%, and 68.53% (Figure 4B). H2O2 levels followed a similar trend (Figure 5B).

3.5. Changes in Raffinose Synthase Activity During Germination

RAFS is the main enzyme involved in the biosynthetic pathway of RFOs. The activity of RAFS was examined during seed germination under various conditions. Exogenous RAF significantly increased RAFS activity compared to CK, with SYKN167 showing increases of 83.20% at 24 h and 13.13% at 72 h, and SD88 showing increases of 26.28% and 9.47%, respectively. Artificial aging, however, greatly reduced RAFS activity. Exogenous RAF treatment restored RAFS activity in artificially aged seeds, with SYKN167 showing increases of 25.49%, and 17.42%, and SD88 showing increases of 23.48%, and 9.35% (p > 0.05) at 24 and 72 h, respectively (Figure 6A). Additionally, the transcript levels of ZMRS, a gene encoding raffinose, were significantly upregulated in raffinose-primed seeds compared to CK (Figure 6B).

3.6. Changes in α-Galactosidase Activity During Germination

α-GAL activity, which hydrolyzes RFOS to provide energy for seed growth, was also measured. Artificial aging significantly decreased α-GAL activity in both SYKN167 and SD88, with SYKN167 showing reductions of 3.20% (p > 0.05) and 62.63%, and SD88 showing reductions of 36.56%, and 41.70% at 24 and 72 h, respectively. In contrast, exogenous RAF treatment significantly increased α-GAL activity in artificially aged seeds, with SYKN167 showing increases of 128.01%, and 24.02%, and SD88 showing increases of 17.27% and 20.95% at 24 and 72 h, respectively (Figure 7).

4. Discussion

In this study, it was found that after exogenous raffinose treatment, the germination rate of waxy corn seeds was improved (Figure 1), the activity of antioxidant enzymes and raffinose synthase was enhanced (Figure 3 and Figure 6), the level of reactive oxygen species (ROS) was reduced (Figure 4 and Figure 5), and the activity of α-galactosidase was increased (Figure 7), thereby improving seed vigor.
Seed vigor is a crucial indicator of seed quality and is closely related to processes such as the germination, development, storage, and deterioration of seeds. Previous studies have found that artificial aging treatments inhibit seed germination and further weaken seed vigor [3,26]. Many studies have shown that treating seeds with exogenous substances can promote seed germination and increase seed vigor [27,28]. Moreover, overexpression of ZmGolS2/ZmRS in Arabidopsis seeds significantly enhanced seed vigor [7]. Consistent with previous studies, in this experiment exogenous RAF priming treatment promoted the germination of waxy corn inbred seeds (SYKN167, SD88) and increased seed vigor. However, artificial aging significantly reversed this positive effect. Germination rate, seedling length, mesocotyl length, and seed vigor of the artificially aged seeds were significantly improved using exogenous RAF priming treatment, which is consistent with the results of the above experiments (Figure 1 and Figure 2). These findings suggest that exogenous RAF promotes the germination and growth of waxy corn seeds.
Antioxidant enzymes are the first line of defense against ROS attack and are activated under stress to scavenge excess ROS [29,30]. It has been shown that the activities of CAT and POD in chickpea seeds gradually decreased with the extension of aging time in accelerated aging conditions [6]. However, after the expression of SiGols6 in Arabidopsis thaliana, the content of active oxygen decreased, while the activities of SOD and POD increased [31]. It was also found that the mitochondrial SOD activity of aged oat seeds primed by ASC, GSH, or ASC + GSH was significantly higher than that of the control [32]. The same results were obtained in the present experiment as in the above experiment, in which the SOD, CAT, and POD activities in the seed embryos of different waxy corn inbred lines after artificial aging treatment were significantly lower than those in the CK, but the activity of these enzymes was significantly enhanced after treatment with RAF (Figure 3). These results indicate that exogenous RAF alleviates the adverse effects of artificial aging treatment by boosting antioxidant enzyme activities in both inbred lines.
In normal seeds, the production and scavenging of ROS are in a dynamic equilibrium [33]. During seed storage, due to adverse environmental conditions, ROS such as O2− and H2O2 can be accumulated, leading to the oxidation of cellular components, lipid peroxidation of membranes, and damage to the integrity of cellular organelle membranes. This causes irreversible damage to seed cells, resulting in a decrease in germination rate and a loss of vigor [34,35,36,37,38]. This experiment showed that after artificial aging treatment, the content of H2O2 and O2− in the SYKN167 and SD88 inbred lines was significantly higher than that in the CK treatment, and after treating waxy corn seeds with exogenous RAF, the levels of H2O2 and O2− were significantly decreased (Figure 4 and Figure 5), while the RAFS activity was elevated.
RFOs have been found to act as antioxidants to counteract the accumulation of reactive oxygen species (ROS) under stress conditions [39,40]. Raffinose is the only RFO present in maize, and raffinose synthase (RAFS) is the main enzyme involved in the biosynthetic pathway of RFO. Raffinose is formed by the addition of galactose units from galactinol to sucrose in a reaction catalyzed by RAFS. Currently, there is only one raffinose synthase gene (ZmRS) which has been cloned in the corn genome, and it only has the function of synthesizing raffinose [7]. The overexpression of galactinol synthase and raffinose synthase in transgenic Arabidopsis plants led to an increase in the concentrations of galactinol and raffinose. This resulted in the enhancement of ROS scavenging capacity and improvement of tolerance to oxidative stress [41,42]. Under oxidative damage caused by methyl viologen (MV), overexpression of RS5 showed a protective effect against oxidative stress [43]. In this experiment, it was found that the RAFS enzyme activity of the SYKN167 and SD88 inbred lines was positively promoted after exogenous RAF priming treatment (Figure 6A). Similarly, the expression of the ZMRS was significantly downregulated in artificially aged seeds, but the downregulation was significantly alleviated after exogenous RAF priming treatment (Figure 6B). This strongly suggested that exogenous RAF, similar to gene overexpression, contributed to enhancing ZmRS expression levels and RAFS activity, thereby scavenging artificially aged seed ROS activity, enhancing antioxidant enzyme activity, and further promoting seed germination (Figure 8).
Additionally, exogenous raffinose treatment can not only eliminate the detrimental effects of ROS, but also provide the necessary energy for seed germination. α-GAL is involved in many physiological and biochemical processes such as RFO unloading, seed development and germination, and senescence [13]. RFOs is hydrolyzed into sucrose and D-galactose under the action of alkaline α-GAL [44,45], which may be integrated into the cell membranes or cell walls of growing buds and root tips, providing energy and thus enhancing seed germination vigor [46]. The activity of AGAL and α-GAL in chickpeas increased during early germination and seed maturation [47]. Treatment of pea seeds with DGJ, a specific inhibitor of α-GAL, on the other hand, completely blocked RFO catabolism, while germination was drastically reduced compared to the control [44,45,46,47,48]. In the present study, we showed that artificial aging treatment significantly reduced the α-GAL activity in SYKN167 and SD88 inbred seeds, inhibiting the catabolism of raffinose, thereby reducing the seed germination rate. However, after treating the artificially aged seeds of SYKN167 and SD88 inbred lines with exogenous RAF, the α-GAL activity in the embryo significantly increased (Figure 7). These demonstrated that exogenous RAF priming treatment further affected seed vigor by modulating the activity of α-GAL (Figure 8). Indeed, as demonstrated, raffinose holds significant research importance for enhancing seed vigor and crop improvement.
While molecular evidence has established that raffinose family oligosaccharides (RFOs) contribute positively to plant stress tolerance and seed vitality, a comprehensive understanding of their underlying molecular mechanisms remains scarce. Furthermore, research into how exogenous raffinose modulates plant stress resistance and seed vigor is virtually nonexistent. Consequently, investigating the effects of exogenous raffinose on the vigor across a variety of crops is of paramount importance for future studies.

5. Conclusions

This study provides a new perspective on RFOs in seed vigor improvement. This study investigated the effect of exogenous raffinose (13.5 mM) priming treatment on seed vigor of waxy corn after artificial aging. Exogenous RAF significantly improved the germination rate of artificially aged seeds, increasing it by 7.84% (p < 0.05, Duncan’s test) in SYKN167 and by 20.45% (p < 0.05, Duncan’s test) in SD88. The results demonstrated that artificial aging significantly inhibited seed germination and reduced seed vigor. However, exogenous raffinose priming mitigated these effects by enhancing seed vigor through the regulation of RAFS activity and ZMRS gene expression. This process reduced ROS levels, increased antioxidant enzyme activity, and boosted α-GAL activity, which provided the energy required for seed germination (Figure 8). In actual production, seeds can be treated with 13.5 mM raffinose to induce a response that enhances germination rates and the early growth of seedlings. These findings offer valuable insights into the role of exogenous raffinose in seed aging and could serve as a theoretical foundation for utilizing raffinose to improve seed vigor in crop management strategies. Further research is needed to explore the physiological mechanisms by which raffinose treatment affects the vigor of different crops under various environmental conditions.

Author Contributions

Conceptualization, M.Z. and W.D.; methodology, W.X. and J.Q.; validation, T.Y. and T.G.; investigation, R.X. and M.Z.; resources, M.Z. and W.X.; data curation, M.Z., R.X. and T.G.; writing—original draft preparation, R.X. and M.Z.; writing—review and editing, M.Z., R.X. and W.X.; visualization, X.Z. and T.Y.; supervision, X.Z., J.Q. and W.D.; project administration, W.X.; funding acquisition, W.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Basic Scientific Research Project of the Education Department of Liaoning Province (LJKZ0675, LJKMZ20221015, LJ212410157006) and the Special Program for Science and Technology of Liaoning Province (2024JH5/10400012).

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effect of exogenous raffinose on germination of waxy corn seeds. (A) The germination rate and germination index of the seeds on the 7th day of germination; (B) The mesocotyledon and seedling length on the 7th day of seed germination. (C) The germination status of the seeds at 24 h and 72 h. CK: non-aged seeds + distilled water, CK + RAF: non-aged seeds + 13.5 mM raffinose, AA: artificially aged seeds + distilled water, AA + RAF: artificially aged seeds + 13.5 mM raffinose. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same variety based on the Duncan test (p < 0.05).
Figure 1. Effect of exogenous raffinose on germination of waxy corn seeds. (A) The germination rate and germination index of the seeds on the 7th day of germination; (B) The mesocotyledon and seedling length on the 7th day of seed germination. (C) The germination status of the seeds at 24 h and 72 h. CK: non-aged seeds + distilled water, CK + RAF: non-aged seeds + 13.5 mM raffinose, AA: artificially aged seeds + distilled water, AA + RAF: artificially aged seeds + 13.5 mM raffinose. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same variety based on the Duncan test (p < 0.05).
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Figure 2. Effect of raffinose priming on seed vigor of waxy corn.
Figure 2. Effect of raffinose priming on seed vigor of waxy corn.
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Figure 3. Effect of raffinose on the antioxidant enzyme activity of waxy corn. (A) POD activity during seed germination; (B) SOD activity during seed germination; (C) CAT activity during seed germination. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
Figure 3. Effect of raffinose on the antioxidant enzyme activity of waxy corn. (A) POD activity during seed germination; (B) SOD activity during seed germination; (C) CAT activity during seed germination. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
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Figure 4. Changes in superoxide anion levels in exogenous raffinose-treated waxy corn seeds. (A) NBT staining status of seeds; (B) O2− content during seed germination. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
Figure 4. Changes in superoxide anion levels in exogenous raffinose-treated waxy corn seeds. (A) NBT staining status of seeds; (B) O2− content during seed germination. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
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Figure 5. Changes in hydrogen peroxide levels in exogenous raffinose-treated waxy corn seeds. (A) DAB staining status of seeds; (B) H2O2 content during seed germination. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
Figure 5. Changes in hydrogen peroxide levels in exogenous raffinose-treated waxy corn seeds. (A) DAB staining status of seeds; (B) H2O2 content during seed germination. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
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Figure 6. Changes in the raffinose synthase activity of waxy corn. (A) RAFS enzyme activity during seed germination; (B) relative expression of the ZMRS gene. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
Figure 6. Changes in the raffinose synthase activity of waxy corn. (A) RAFS enzyme activity during seed germination; (B) relative expression of the ZMRS gene. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
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Figure 7. Changes in the α-galactosidase activity of waxy corn. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
Figure 7. Changes in the α-galactosidase activity of waxy corn. Data represent means ± SD of three replicates. The different letters indicate significant differences between different treatments within the same germination time based on the Duncan test (p < 0.05).
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Figure 8. Mechanism of raffinose in improving the seed vigor of aged waxy corn.
Figure 8. Mechanism of raffinose in improving the seed vigor of aged waxy corn.
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Table 1. Primer sequence information.
Table 1. Primer sequence information.
GeneForward Primer SequenceReverse Primer Sequence
β-actinCATGGAGAACTGGCATCACACCTTCTGCGTCATTTTCTCTCTGTTGGC
ZmRSGAGCTCTACGATGGTTTGCACTCCCCCATCCACAGCGAGTTGTAGGC
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MDPI and ACS Style

Zhu, M.; Xiao, R.; Yu, T.; Guo, T.; Zhong, X.; Qu, J.; Du, W.; Xue, W. Raffinose Priming Improves Seed Vigor by ROS Scavenging, RAFS, and α-GAL Activity in Aged Waxy Corn. Agronomy 2024, 14, 2843. https://doi.org/10.3390/agronomy14122843

AMA Style

Zhu M, Xiao R, Yu T, Guo T, Zhong X, Qu J, Du W, Xue W. Raffinose Priming Improves Seed Vigor by ROS Scavenging, RAFS, and α-GAL Activity in Aged Waxy Corn. Agronomy. 2024; 14(12):2843. https://doi.org/10.3390/agronomy14122843

Chicago/Turabian Style

Zhu, Min, Ru Xiao, Tong Yu, Tao Guo, Xuemei Zhong, Jianzhou Qu, Wanli Du, and Wei Xue. 2024. "Raffinose Priming Improves Seed Vigor by ROS Scavenging, RAFS, and α-GAL Activity in Aged Waxy Corn" Agronomy 14, no. 12: 2843. https://doi.org/10.3390/agronomy14122843

APA Style

Zhu, M., Xiao, R., Yu, T., Guo, T., Zhong, X., Qu, J., Du, W., & Xue, W. (2024). Raffinose Priming Improves Seed Vigor by ROS Scavenging, RAFS, and α-GAL Activity in Aged Waxy Corn. Agronomy, 14(12), 2843. https://doi.org/10.3390/agronomy14122843

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