UVB Stress Induced Changes in Germination and Carbohydrate Mobilization in Chenopodium Quinoa Willd. Seeds

Abstract

Chenopodium quinoa Willd. (quinoa) is a very promising crop due to its nutraceutical properties and strong tolerance to extreme conditions, including high UVB. However, the physiological mechanisms underlying its adaptation to high UVB are still unclear, especially during germination as its traditional sowing consists of either broadcasting or continuous stream distribution in furrows. We evaluated the response of germinating quinoa seeds to acute UVB radiation, looking at the mobilization of starch reserves as well as the utilization of starch and free sugars. Biometric and physiological traits were evaluated in control (0 W m⁻²) and UVB (3.4 W m⁻²)-exposed seeds during a 24 h treatment starting with seed imbibition. Quinoa exposed to UVB showed a delay in germination and strong reduction in root elongation. Although the negative effect of UVB on germination was fully recovered at 48 h of imbibition, that on root elongation was irreversible, especially with a longer exposure time. Further analysis showed low differences in the concentration of free sugars, except at 2 and 24 h of treatment. Furthermore, starch mobilization in UVB-treated seeds was strongly reduced compared to control. This was associated with the amylolytic activity analysis, which showed strong reductions in both α- and β-amylase activities during the whole treatment, indicating that UVB strongly reduced enzyme activation for the mobilization and use of starch reserves. Overall, these data suggest that quinoa seeds can regulate the expression of genes encoding enzymes involved in reserve mobilization, in order to resist to acute UVB radiation and maintain seed viability.

1. Introduction

Solar radiation is the primary source of energy for metabolism, growth, and development in plants. Depletion of the stratospheric ozone layer, a component of climate change, has motivated studies on the effects of ultraviolet B (UVB) radiation on the growth and yield of agricultural crops [1,2]. Although UVB represents only a fraction of the solar spectrum, with wavelengths between 280 and 315 nm, its high energy has a strong impact on living organisms. In plants, depending on the wavelength, fluence rate, exposure duration, and interactions with other factors, UVB radiation can induce stress and photomorphogenic responses, which are not mutually exclusive [3]. In general, UVB radiation may reduce photosynthesis, damage proteins and DNA, and alter membrane integrity, while tolerant species are able to activate antioxidant systems, synthetize protective secondary metabolites, and adjust their metabolism to minimize oxidative stress [4,5,6,7,8,9]. These adaptive responses contribute to the survival of plants in high-UVB environments.

Intense UVB radiation is a common characteristic of the Andean region due to its high elevation and thin ozone layer [10]. Moreover, the Andes have extended dry periods and freezing temperatures during the dry season. These climatic conditions represent a challenging environment where certain plant species, such as quinoa, have evolved and adapted to these extreme conditions [11]. In this study, we performed laboratory-controlled experiments to investigate the mechanisms underlying UVB tolerance, providing a model to understand how quinoa can cope with such stress factors during germination.

Quinoa (Chenopodium quinoa Willd.) is an herbaceous plant native to the Andean region of South America. It has been cultivated as a staple food for thousands of years by the Andean populations and, in the last two decades, has received worldwide interest due to its high protein and well-balanced amino acid contents [12,13]. In addition, it has been reported that quinoa shows strong tolerance to salinity [14], drought [15], and high UVB radiation [16]. However, the physiological mechanisms and associated signaling pathways behind its adaptation to high UVB are still unclear, especially during germination.

In the Andean highlands, the traditional sowing of quinoa consists of either broadcasting or distributing the seeds as a continuous stream in furrows after preparing the ground by manual or motorized ploughing [17]. Consequently, during the first stage of germination, seeds and seedlings are exposed to sunlight and high UVB levels. Seed germination is the most important growth stage that determines plant establishment. Germination begins with the uptake of water through seed imbibition, followed by embryo expansion and ending with radicle emergence [18]. During this stage, an increase in respiration occurs and several hydrolytic enzymes, such as α-amylase, are synthesized or activated to break down the storage reserves that are used to sustain the growing embryo [18,19,20]. Therefore, we hypothesized that UVB radiation may compromise seed viability by altering the energy reserve mobilization and utilization pathways, as UVB can damage the protein structure and alter several signaling mechanisms in plant cells [21,22].

The aim of this study was to investigate for the first time the effects of UVB radiation on the mobilization of starch reserves in quinoa seeds during germination. Specifically, we addressed the following research questions: (i) does UVB affect the germination percentage and radicle elongation? and (ii) does UVB alter carbohydrate metabolism—including starch content, soluble sugars, and amylase activities—during early germination?

2. Results and Discussion

Developmental and growth data included germination (Figure 1A) and radicle elongation (Figure 1B), as well as representative pictures showing the phenotypes (Figure 1C). Germination begins with the uptake of moisture by the seeds followed by elongation of the embryonic axis or radicle. Under control conditions, quinoa seeds exhibited rapid germination when exposed to moisture, with radicle protrusion within 6 h after imbibition in 50% of seeds (Figure 1A), and reached an average root length of 3.6 ± 1.8 mm at 12 h and a maximum of 13 ± 2.69 mm at 24 h (Figure 1B). In contrast, UVB-treated seeds started germinating at 9 h after imbibition in 56% of seeds (Figure 1A) and reached an average root length of 1.82 ± 1.07 mm at 12 h and a maximum of 5.2 ± 0.61 mm at 24 h (Figure 1B). Delayed germination in UVB-treated seeds was clearly observed, with significant differences at each time point for treated seeds in comparison with the control (Figure 1A). Similarly, the root elongation data showed a length reduction in the UVB-treated seeds, with significant differences in comparison with the control (Figure 1B,C). These findings were consistent with the observations of Mousavi et al. [23], who reported UVB-induced inhibition of germination in the Scrophularia striata Lizan ecotype. Although no prior data are available regarding the effects of UVB on quinoa germination, similar reductions in germination rate and root length have been documented under other abiotic stresses, such as salinity [24].

To evaluate the resilience of quinoa seeds following UVB exposure, seeds were transferred to darkness after treatment to allow for recovery of normal metabolic processes. After the recovery period, the germination rate and radicle elongation of UVB-treated seeds were determined. The data showed full recovery of germination (100%) within 48 h after imbibition (HAI) in every sampled dish, comparable to that in control seeds (Figure 2A). This result confirmed that seeds which did not germinate during UVB exposure remained viable, as they germinated completely after transfer to recovery conditions. Interestingly, this recovery was observed even in seeds subjected to prolonged UVB exposure (24 h) (Figure 2A), indicating a high level of UVB tolerance in germinating quinoa seeds. However, radicle growth was strongly affected by the UVB treatment, with reduced recovery at higher UVB exposure times (Figure 2C). In fact, the root length in control seeds (0 h of UVB) reached an average of 13 ± 2.69 mm at 24 HAI and 40.43 ± 7.61 mm at 48 HAI, while UVB-treated seeds (24 h of UVB) reached only 5 ± 0.5 mm at 24 HAI and 12 ± 3.94 mm at 48 HAI (Figure 2B). This may be due to incomplete recovery of normal metabolic pathways.

During radicle protrusion, sucrose and other sugars in the grain are used as early energy resources before the mobilization of starch reserves commences [19]. In dry quinoa seeds, sucrose is present at approximately 3 g per 100 g seed DW [19], supporting its role prior to starch hydrolysis. In this study, control and UVB-treated seeds showed similar patterns in their content of soluble sugars over the experiment, with significant differences between treatments at specific time points (Figure 3). In detail, 2 h-control samples contained significantly higher level of glucose (Figure 3A), sucrose (Figure 3B), fructose (Figure 3C), and pooled sugars (Figure 3D) than UVB-treated samples at the same time point. Contrarily, 24 h-UVB samples showed significantly higher accumulation of glucose (Figure 3A), fructose (Figure 3C), and pooled sugars (Figure 3D) than their respective controls, while no significant differences were observed in sucrose (Figure 3B). These results indicate that UVB exposure could prevent the utilization of glucose and fructose by the embryo. Similar results have previously been reported by Prado et al. [24] during quinoa germination under salinity stress, demonstrating that the reduction in germination and growth could be due to the inefficient use of energy sources under abiotic stresses.

The initial starch content in dry seeds was 368 mg g FW⁻¹, and the level started to decrease after 3 h, regardless of treatment (Figure 4). In detail, the starch contents in control and UVB-treated samples did not show significant differences within the first 6 h of treatment. Beyond this time point, UVB-treated seeds exhibited a slight reduction in starch content compared to their respective controls, yet consistently retained significantly higher starch levels (Figure 4). This sustained retention of starch in UVB-exposed samples was probably due to the reduced activity of starch-degrading enzymes, as previously reported in quinoa and other species under different abiotic stress conditions [25,26,27].

To evaluate the effect of UVB on the mobilization of starch reserves, we analyzed α-amylase (EC 3.2.1.1) and β-amylase (EC 3.2.1.2) activities. These enzymes, in coordination with maltase (EC 3.2.1.3) and debranching enzyme (EC 3.2.1.68), catalyze the breakdown of starch into glucose, which is subsequently transported to sites of demand (mainly after its conversion into sucrose) [28]. Zymography was performed by incubating gels in starch solution, revealing amylase activity as light bands after staining. Reverse images of gels were analyzed to quantify the band intensities as % of activity (relative activity with respect to the highest intensity band of control seeds). The activity of β-amylase, at a low level (4.6%), was present in dry seeds (Figure 5). The presence of β-amylase activity in dry seeds is consistent with previous reports showing that certain hydrolytic enzymes are stored in an inactive form within mature seeds [29,30]. These enzymes are pre-synthesized during seed development and remain stable until imbibition, when they are rapidly activated to support the mobilization of stored reserves. Our data showed that β-amylase activity was increased at 3 HAI (60%) in UVB-treated seeds compared to control, and was maintained at approximately 50% for the rest of the experiment (Figure 5).

The evaluation of α-amylase activity of UVB-exposed samples showed a general reduction in enzyme activity at each time point compared to the control (Figure 6). α-amylase activity was already present (7.6%) in dry seeds, as reported by Hager et al. [19]. In UVB-exposed samples, α-amylase activity showed little reduction at 1 HAI (4.6%) while increasing to 50.6% at 6 HAI; however, it remained significantly lower than that in the control during the experiment (Figure 6), as reported for Vigna radiata under enhanced UVB [31]. This reduced α-amylase activity may partially explain the slower starch consumption in UVB-treated seeds.

The multiple correlation (MC) analysis (Table S1) indicated that α-amylase induction was partially correlated with germination in both UVB-treated and control seeds, with a slightly stronger correlation in control seeds. In contrast, β-amylase induction showed only a partial correlation with time and root length in UVB-treated seeds, while no correlation was observed in the control group. Additionally, a moderate correlation between α- and β-amylase induction was found under both treatment conditions.

Taken together, these findings support the hypothesis that quinoa seed germination under UVB exposure is delayed due to the reduced and postponed activation of starch-degrading enzymes. Germination regulation in quinoa seeds is tightly linked to energy metabolism, involving factors such as α-amylase activity, endogenous hormones, and availability of soluble sugars. Recently, Zeng et al. [32] analyzed the regulatory roles of various endogenous hormones during quinoa seed germination. Their study demonstrated that hormone levels fluctuated continuously throughout the germination process, suggesting that multiple hormones contribute to its regulation. Furthermore, the pattern of hormonal regulation appears to differ depending on UVB exposure, suggesting a complex interplay between light stress and hormonal signaling.

3. Materials and Methods

3.1. Plant Material and Growth Conditions

Seeds of quinoa (Chenopodium quinoa Willd.) variety “Réal,” originally from Bolivia, were commercially obtained from Priméal (Peaugres, France) (https://www.primeal.bio/en, accessed on 18 August 2025). For each replicate, 40 dried seeds were selected and placed in Petri dishes without lids (9 cm diameter) containing a filter paper moistened with 10 mL of distilled water. The dishes were covered with either UV-blocking (referred to as control) or UV-transmitting (referred to as UVB) plastic filter discs (G. Valota SpA, Bergamo, Italy) throughout the experiment, as previously reported [11]. Each treatment (control and UVB) was performed in three biological replicates (n = 40 seeds per replicate). The experimental conditions were as described in Mariotti et al. [33]. In detail, the dishes were placed in a growth chamber with a temperature of 22 ± 1 °C and relative humidity of 75%. Distilled water was supplied as needed to prevent dryness, maintaining the initial 10 mL throughout the experiment.

3.2. UVB Radiation Treatment

UVB radiation was applied using three Philips TL 20W/01RS UV-B Narrowband lamps (Koninklijke Philips Electronics, Eindhoven, The Netherlands) with emission peak at 311 nm. The UVB exposure level was set by adjusting the distance (15 cm) between the lamps and dishes, and was measured using an UVB meter (Skye Instruments Ltd., Powys, UK). The irradiation levels registered under the filter discs were control = 0 W m⁻² and UVB = 3.4 W m⁻². The experiment lasted 24 h and samples were collected at specific time points (0, 1, 2, 3, 6, 9, 12, and 24 h).

For recovery, another set of dishes containing control and UVB-treated seeds were transferred to control conditions at a temperature of 22 ± 1 °C and relative humidity of 75%. Sampling was performed at 24 and 48 h after imbibition (HAI).

3.3. Biometric Analysis

Biometric analysis included the monitoring of germination and root length. Germination percentage was calculated as the number of germinated seeds relative to the total seeds per dish. For each treatment (control and UVB), three biological replicates were performed (n = 40 seeds per replicate). Root length was quantified from digital images of seedlings using the ImageJ software version 1.53t (National Institutes of Health, Bethesda, MD, USA). In total, 40 individual roots were measured per replicate. Measurements were taken immediately after treatment at specific time points, and after recovery. No separate viability test was carried out on ungerminated seeds; however, their viability was confirmed during the recovery experiment, as all seeds eventually germinated within 48 h after imbibition under control conditions.

An additional set of dishes not used for testing of biometric traits were prepared, and fresh samples (100 mg) were collected immediately after treatment and at specific time points. Samples were immediately frozen in liquid nitrogen and stored at −80 °C for further biochemical analyses.

3.4. Extraction and Determination of Soluble Sugars

Soluble sugars were extracted from frozen homogenized samples and assayed through coupled enzymatic assay methods, as described by Huarancca Reyes et al. [34]. Briefly, extracts in 5.5% HClO₄ were centrifuged at 13,000× g for 10 min, and the supernatant was neutralized with 1 M K₂CO₃. The resulting supernatant after centrifugation was then collected to assay glucose, fructose, and sucrose following the enzymatic method that involves measuring the increase in absorbance at 340 nm. The accuracy of this method was tested using standards with known amounts of carbohydrates. The quantity of soluble sugars is expressed as μmol hexose equivalent g FW⁻¹. Three biological replicates were considered for this analysis.

3.5. Extraction and Determination of Starch

Starch was extracted from frozen homogenized samples and determined according to the I₂-KI method, as previously reported [35]. Briefly, samples were boiled in 10 mM KOH and neutralized with 1 M HCl. The resulting supernatant after centrifugation (13,000× g, 15 min) was used for starch quantification by adding fresh iodine solution (0.13% I₂ and 0.3% KI in distilled water). The absorbance was measured at 595 nm, and the content is expressed as mg starch g FW⁻¹. Three biological replicates were performed for this analysis.

3.6. Protein Extraction and Zymogram of Amylolytic Activity

Protein extraction was performed by grinding frozen samples in 100 mM HEPES-KOH pH 7.5 and centrifugation at 13,000× g for 15 min. The supernatant was collected and divided in two aliquots for enzymatic assays of α-amylase and β-amylase activity, respectively. Protein quantification was performed with Bradford’s standard assay using bovine serum albumin as a standard [36].

The total amylolytic activity, which was evaluated using the crude extract, mainly consisted of β-amylase (pH 5) as the incubation of starch digestion was short and no bands were revealed at higher pH on the isoelectric focusing (IEF) gel [35]. For α-amylase (pH 8), an aliquot of crude extract (20 µL) was heated at 70 °C for 15 min with 10 mM CaCl₂ to inactivate β-amylase, debranching enzyme, and α-glucosidase [35]. IEF was performed using the Criterion Precast Gel System (Bio-Rad, Hercules, CA, USA), following the instructions provided by the manufacturer. A total of 10 µg protein was loaded in each well of precast Criterion IEF 1.0 mm thick polyacrylamide gels pH 3–10 (Bio-Rad, Hercules, CA, USA). The gels were run in 1× IEF anode buffer (7 mM phosphoric acid) and 1× IEF cathode buffer (20 mM lysine, 20 mM arginine) for 1 h at 100 V and 1.5 h at 300 V, sequentially. The activity staining was performed as reported [35]. Briefly, the gels were incubated in a soluble starch solution (1% in 50 mM Na-acetate buffer pH 5.2 containing 10 mM CaCl₂) for 2 h, washed in the same buffer, and then incubated for 20 min (β-amylase gel) or 40 min (α-amylase gel). The gels were stained with fresh iodine solution (1.3% I₂ and 3% KI in distilled water) for 1 min, followed by a water-rinse. The ImageJ software version 1.53t (National Institutes of Health, Bethesda, MD, USA) was used to quantify the intensity of bands. Three biological replicates were considered for this analysis.

3.7. Statistical Analysis

Values are presented as mean ± standard deviation. After performing the Shapiro–Wilk test for determination of normality, data were subjected to Student’s t-test for biometric and physiological traits determined immediately after treatment, and one-way analysis of variance (ANOVA) followed by Tukey’s test for traits after recovery. Significant differences among means were evaluated at the level of p < 0.05. The software STATISTICA for Windows version 14.0 (Stat-Soft, Inc., Tulsa, OK, USA) was used for all computations. The multiple correlation (MC) analysis was conducted using the Excel software.

4. Conclusions

Quinoa seeds exposed to UVB radiation exhibited a general delay in germination, although this was fully compensated at 48 h after imbibition (HAI). Notably, UVB-treated seeds showed a marked reduction in root elongation, particularly under prolonged exposure, which appeared to cause irreversible damage—likely due to the failure to restore normal metabolic pathways. Biochemical analyses revealed that starch mobilization was significantly impaired in UVB-treated seeds compared to controls. This finding was consistent with the amylolytic activity assays, which indicated strong decreases in both α- and β-amylase activity throughout the treatment period.

Overall, the study demonstrated that UVB exposure delays quinoa seed germination primarily by impairing starch mobilization and utilization. This inhibition, in turn, contributes to reduced root growth. The observed temporal delay may reflect a seed survival strategy, whereby germination is postponed until more favorable environmental conditions arise.

In future research, it would be valuable to examine how UVB radiation affects the expression of genes involved in starch metabolism and root development during quinoa seed germination. Additionally, quantifying the levels of key plant growth regulators could shed light on the hormonal mechanisms underlying UVB-induced germination delays.