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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2026 Mar 6;27(5):2436. doi: 10.3390/ijms27052436

Involvement of the Light Signalling Components HY5 and BIC1,2 and SPA1 in Plant Responses to Elevated Daytime UV-B Doses

Pavel Pashkovskiy 1, Anna Abramova 1,*, Mikhail Vereshchagin 1, Vladimir V Kuznetsov 1, Vladimir D Kreslavski 2
Editors: Gábor Kocsy, Zsolt Gulyás
PMCID: PMC12985706  PMID: 41828652

Abstract

Plants respond to ultraviolet B radiation (280–320 nm) with an integrated reaction that includes the reception of the acting stress factor, followed by the generation of reactive oxygen species and damage to macromolecules and membrane structures, as well as changes in cellular metabolism and the formation of protective systems. However, the involvement of key UV-B–related signalling components such as HY5, SPA1 and BIC1 or BIC2 proteins in physiological, biochemical and molecular responses remains insufficiently understood. The effects of 8, 16 and 24 h of UV-B exposure (within an 8 h photoperiod over three days) on the net photosynthetic rate (Pn), chlorophyll fluorescence parameters Y(II) and Fv/Fm, reflecting the functional state of PSII, nonphotochemical quenching (NPQ), pigment contents (Chl(a+b), carotenoids, anthocyanins and UV-absorbing pigments (UAPs) and the expression of key light-induced genes in wild-type Arabidopsis thaliana and spa1, bic1,2 and hy5 mutants were studied. UV-B irradiation resulted in a gradual reduction in the Pn, Y(II), Fv/Fm values and Chl(a+b) but caused a marked increase in the anthocyanin and UAP contents and only minor changes in the carotenoid content. The hy5 mutant presented the lowest net photosynthetic rate (Pn), chlorophyll fluorescence parameters, and chlorophyll and carotenoid contents under all the UV-B exposures. In addition, the accumulation of anthocyanins and UAPs during UV-B treatment was consistently the lowest in hy5. After any UV-B exposure, the highest accumulation of UAPs and anthocyanins was observed in the spa1 mutant, whereas the highest Pn values were detected after 24 h in bic1,2. One of the reasons for the reduced photosynthetic activity and antioxidant capacity in hy5 may be the lower expression levels of CHS and PAL in this variety than in the other genotypes. Our results indicate that HY5 is required to maintain antioxidant responses and photosynthetic performance under repeated daytime UV-B exposure (16.8 kJ m−2 per day). In contrast, BIC1, BIC2, and SPA1 also contribute to UV-B tolerance, but through distinct regulatory mechanisms and to a lesser extent.

Keywords: A. thaliana, antioxidants, light signalling, recurrent stress, stepwise acclimation, UV-B radiation

1. Introduction

UV-B radiation (280–320 nm) is an important environmental factor that has both stress and signalling effects on plants. Despite its low proportion of solar radiation, UV-B has high biological activity: it causes the formation of reactive oxygen species (ROS) and damage to nucleic acids, proteins, and membranes. Moreover, plants can use UV-B as a regulator of development and physiological adaptation [1,2]. This is provided by a specialized signalling system that activates protective and metabolic reactions.

The key receptor for UV-B signalling is UVR8 (UV resistance locus 8), a homodimeric protein that undergoes monomerization upon UV-B exposure and subsequently accumulates in the nucleus, where it initiates a signalling cascade through interaction with the E3 ubiquitin ligase COP1 (Constitutive photomorphogenic 1) [3]. The attachment of UVR8 to COP1 disrupts the interaction of COP1 with SPA (SUPPRESSOR OF PHYA-105) family proteins, resulting in the stabilization and accumulation of the transcription factor HY5 (ELONGATED HYPOCOTYL5) and its homologue HYH [4,5]. HY5 is known as a universal regulator of photomorphogenesis under a wide range of light conditions, including visible light and UV-B, and is involved in the activation of many genes that control the biosynthesis of pigments (including flavonoids), antioxidant accumulation, photomorphogenesis, and photoprotective mechanisms [5,6].

SPA family proteins play a central role in regulating COP1 activity by degrading light-dependent transcription factors in the dark [7]. However, accumulating evidence suggests that their functions in UV-B signalling are much broader. SPA1-SPA4 isoforms exhibit distinct functional specializations in response to UV-B exposure, with SPA1 and SPA3 primarily involved in the response to low-intensity UV-B, whereas SPA2 and SPA4 are activated at high intensities of repeated UV-B [8]. In addition, SPA proteins are involved in the posttranslational regulation of HY5, a key transcription factor in photomorphogenesis. They control stomatal stability by influencing phosphorylation processes and are believed to coordinate light-dependent signalling pathways with the regulation of the stomatal apparatus and water exchange. This range of functions highlights the importance of SPA proteins as integrators of diverse light signals [9].

UV-B signalling is also regulated by negative feedback mechanisms. The interaction of UVR8 with the RUP1 and RUP2 proteins (REPRESSORS OF UV-B PHOTOMORPHOGENESIS) promotes the restoration of its dimeric state and the completion of the signalling response [10]. BIC1 and BIC2 (BLUE LIGHT INHIBITORS OF CRYPTOCHROMES) proteins play important roles in the regulation of the UV-B response, as they are specific inhibitors of cryptochrome 1 (CRY1) activity [10]. BIC1 and BIC2 suppress the induction of the RUP2 gene under blue light, thereby affecting the efficiency of UVR8 redimerization. Genetic studies have indicated that the BIC1 and BIC2 proteins function as negative regulators of photoreceptor signalling, including pathways that interact with UVR8-mediated UV-B responses; loss of BIC1/BIC2 alters UVR8 redimerization and downstream gene regulation, which is consistent with the increased sensitivity of bic1,2 mutants to UV-B–related stress compared with that of wild-type plants [11].

The transcription factor HY5 represses the UVR8- and cryptochrome-mediated signalling pathways by inducing the expression of the RUP1, RUP2, BIC1 and BIC2 genes [5]. BIC1 enhances the activity of COP1 toward HY5 in the presence of SPA proteins, whereas BIC2 is capable of directly binding to the promoters of HY5 target genes by competing with HY5 for cis-regulatory elements. These observations indicate that BIC proteins are involved in fine-tuning the photomorphogenetic response at both the posttranslational regulation and transcription levels [12].

The current model reflects the action of a complex signalling network that integrates interactions between UVR8, HY5, SPA, and BIC proteins. This network not only ensures plant resistance to UV-B but also supports photosynthetic productivity by regulating the balance among photoprotective mechanisms, metabolism, and water homeostasis [3]. However, despite significant progress, our understanding of these processes remains incomplete.

In particular, the mechanisms underlying the integration of signals from UVR8, HY5, BIC1, 2 and SPA1 under conditions of changing light and accompanying stresses have not been sufficiently studied. How their functional interactions coordinate photoprotective processes with photosynthetic activity under UV-B irradiation is unclear [13].

Thus, several key questions remain unanswered; in particular, how are the regulators HY5, BIC1,2 and SPA involved in the integration of light signals and the formation of adaptive responses of plants to repeated daytime UV-B irradiation, and how do their interactions ensure the maintenance of photochemical and antioxidant balance in plants in a changing environment?

2. Results

2.1. Chlorophyll Fluorescence Parameters

Initially, during the experiment, the portion of UV-B available in the spectrum had no significant effect on the parameters reflecting the PSII state, Fv/Fm (0.82) and Y(II) (0.5–0.6), of the WT and bic1,2 (Figure 1).

Figure 1.

Figure 1

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Effects of cumulative UV-B exposure on chlorophyll a fluorescence parameters in the leaves of A. thaliana WT, bic1,2, hy5 and spa1 mutants. The fluorescence parameters Fv/Fm (A), Y(II) (B) and NPQ (C) are shown. Different lowercase letters indicate statistically significant differences among genotypes within the same time point (p ≤ 0.05; one-way ANOVA followed by Tukey’s HSD test). The mean values ± SDs are shown (n = 5).

Initially, the values of Y(II) and Fv/Fm in the hy5 mutant were lower than those in the other mutants and the WT, whereas the NPQ level was greater in the spa1 mutant (Figure 1). The values of Fv/Fm in the WT and other mutants ranged from 0.77 to 0.82. Moreover, the initial Fv/Fm value in the hy5 mutant was 0.70, which suggests PSII impairment in this mutant. After 16 h of irradiation, the Y(II) and Fv/Fm values of the WT and all the mutants decreased compared with the initial values of these parameters, and this trend was maintained after 24 h of UV-B exposure (Figure 1A,B). By the end of the UV-B treatment, the Fv/Fm values of the WT and mutants were within 0.61–0.75 for hy5. In contrast, the NPQ values increased during irradiation, with the highest level of NPQ observed in the spa1 mutant (Figure 1C).

2.2. Net Photosynthesis, Stomatal Conductance and Transpiration

Initially, the Pn, Tr and GS values in the hy5 mutant were lower than those in the other variants, whereas in the spa1 mutant, the Pn and Tr values were lower than those in the WT and bic1,2 mutant (Figure 2). After 16 h, all these parameters decreased in the WT and all the mutants compared with their initial levels, but the smallest reduction was observed in the hy5 mutant. After 24 h, this trend persisted, and the hy5 mutant consistently presented the lowest Pn, GS and Tr values.

Figure 2.

Figure 2

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Effects of cumulative UV-B exposure on gas exchange parameters in the leaves of A. thaliana WT, bic1,2, hy5 and spa1 plants. The net photosynthetic rate (Pn) (A), transpiration rate (Tr) (B) and stomatal conductance (GS) (C) are shown. Different lowercase letters indicate statistically significant differences among genotypes within the same time point (p ≤ 0.05; one-way ANOVA followed by Tukey’s HSD test). The data are presented as mean values ± SD (n = 5).

2.3. Contents of Photosynthetic and Other Pigments

Initially, the highest values of Chl(a+b) and carotenoid contents were detected in the spa1 mutant, and the lowest values were detected in the hy5 mutant (Figure 3A). The pigment contents in bic1,2 and WT plants were intermediate. After irradiation, the contents of Chl decreased after 8 h and 16 h of irradiation, but the reduction was slight. After 24 h of UV-B treatment, the pigment content was the same as that after 16 h. The carotenoid content was constant after irradiation, excluding that of the hy5 mutant, where only a slight decrease was observed.

Figure 3.

Figure 3

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Effects of cumulative UV-B exposure on the contents of photosynthetic pigments in the leaves of A. thaliana WT, bic1,2, hy5 and spa1 mutants. The chlorophyll (a + b) content (A) and carotenoid content (B) are shown. Different lowercase letters indicate statistically significant differences among genotypes within the same time point (p ≤ 0.05; one-way ANOVA followed by Tukey’s HSD test). The data are presented as mean values ± SD (n = 4).

Initially, the values of the Chl a/Chl b and Chl(a+b)/Car ratios were equal in all the variants (Figure 4). After irradiation, the values of Chl a/Chl b decreased with increasing exposure time. The Chl(a+b)/Car ratios after 24 h of UV-B exposure were almost the same as those in the initial variants, but the ratio in the spa1 mutant was higher than that in hy5.

Figure 4.

Figure 4

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Effects of UV-B exposure on the Chl a/Chl b (A) and Chl(a+b)/Car (B) ratios in the leaves of A. thaliana WT and bic1,2, hy5, and spa1 mutants. Different lowercase letters indicate statistically significant differences among genotypes within the same time point (p ≤ 0.05; one-way ANOVA followed by Tukey’s HSD test). The data are presented as mean values ± SD (n = 4).

Initially, the highest anthocyanin and UV-absorbing pigment (UAP) contents were detected in the spa1 mutant, and the lowest values were detected in the hy5 mutant. The pigment contents in bic1,2 and WT plants were intermediate. After irradiation, as the plants were irradiated with UV-B, the anthocyanin content increased 2–3-fold in all the variants and UAPs, approximately 2-fold, excluding the hy5 mutant, where the increase was insignificant (Figure 5).

Figure 5.

Figure 5

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Effects of UV-B exposure on the accumulation of the anthocyanin Anth (A) and UV-B–absorbing pigments UAPs (B) in the leaves of A. thaliana WT and bic1,2, hy5 and spa1 mutants. Different lowercase letters indicate statistically significant differences among genotypes within the same time point (p ≤ 0.05; one-way ANOVA followed by Tukey’s HSD test). The mean values ± SDs are shown (n = 5).

2.4. Gene Expression

UV-B exposure resulted in statistically significant, genotype-specific differences in the gene expression of bic1, 2, hy5 and spa1 at each analysed time point (Table 1).

Table 1.

Relative expression levels of light-responsive genes in A. thaliana spa1, hy5 and bic1,2 mutants after 24 h of cumulative UV-B exposure applied over three consecutive 8 h photoperiods. For each gene, the expression values were normalized to those of Actin1 and Tubulin3 as reference genes. The expression level in the WT at the corresponding time point was set to 1.0. Different letters indicate significant differences among genotypes within the same time point (p ≤ 0.05; one-way ANOVA followed by Tukey’s HSD test). The mean values ± SDs are shown (n = 3). The colour scale represents relative expression levels: white corresponds to the WT (1.0), blue indicates a decrease of at least twofold relative to the WT, and red indicates a twofold or greater increase compared with the WT.

Genotype bic1,2 hy5 spa1 bic1,2 hy5 spa1 bic1,2 hy5 spa1 bic1,2 hy5 spa1
Genes/Time (h) 0 8 16 24
COP1 0.14 c 1.07 a 0.45 b 0.86 a 0.56 c 0.65 b 1.51 b 1.94 a 1.24 c 0.32 a 0.21 b 0.11 c
DET1 1.74 b 4.31 a 0.57 c 0.14 c 1.01 a 0.45 b 3.23 b 2.23 c 4.54 a 1.40 a 0.13 b 0.18 b
RBCL 0.97 b 4.57 a 0.45 c 0.55 a 0.24 b 0.16 c 1.25 b 1.57 a 0.38 c 1.36 a 0.05 b 0.02 b
CAB1 3.41 a 2.67 b 2.20 c 3.09 c 4.91 a 3.45 b 1.74 a 0.29 c 0.72 b 0.24 b 0.32 a 0.16 c
HY5 1.95 a 0.31 b 0.28 b 2.48 b 0.52 c 3.37 a 3.75 b 0.11 c 4.16 a 1.95 b 0.11 c 3.18 a
PAL 2.63 b 2.78 a 1.16 c 0.43 c 1.39 b 2.40 a 2.84 b 1.38 c 3.46 a 1.42 b 0.22 c 2.03 a
GPX6 0.87 b 2.24 a 0.13 c 0.12 c 0.39 b 2.14 a 6.88 a 0.81 c 5.56 b 0.26 a 0.12 b 0.13 b
APX3 1.45 a 0.52 c 1.24 b 6.28 a 2.48 c 4.81 b 1.23 c 2.19 b 8.12 a 0.36 c 0.46 b 5.27 a
CHS 1.23 b 0.36 c 2.51 a 1.23 b 0.60 c 2.53 a 1.21 b 0.23 b 2.00 a 0.92 b 0.32 c 2.23 a
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At 0 h, COP1 expression was significantly lower in bic1,2 and spa1 than in hy5 (Table 1). In contrast, DET1 transcript levels were markedly higher in hy5 than in the other genotypes. Moreover, RBCL expression was elevated in hy5 (approximately 4.6-fold greater than that in the WT) and significantly reduced in spa1, whereas the expression of bic1,2 did not differ from that in the WT (Table 1). The expression of HY5 was significantly greater in bic1,2 (approximately twofold) than in hy5 and spa1. Among the phenylpropanoid-related genes, PAL expression was higher in bic1,2 and hy5 than in spa1, whereas CHS expression was highest in spa1 and lowest in hy5. The expression of APX3 was significantly greater in bic1,2 than in hy5, whereas GPX6 did not differ significantly among the genotypes (Table 1).

After 8 h of UV-B exposure, HY5 expression increased significantly in spa1 and exceeded that in bic1,2 and hy5 (approximately 3.4-fold greater than that in the WT; Table 1). At this time point, PAL and CHS expression were also highest in spa1, whereas PAL expression was significantly reduced in bic1,2. The expression of antioxidant-related genes showed genotype-specific responses: APX3 expression increased strongly in bic1,2 and spa1 (up to approximately sixfold and fivefold, respectively), whereas GPX6 expression was significantly elevated only in spa1 (Table 1). The expression of photosynthesis-related genes differed among the genotypes, with RBCL and CAB1 transcript levels being lower in spa1 and hy5 than in bic1,2 at this time point.

After 16 h of UV-B exposure, HY5 expression remained significantly greater in spa1 and bic1,2 than in hy5 (Table 1). The expression of PAL and CHS was highest in spa1, intermediate in bic1,2, and significantly reduced in hy5. Moreover, APX3 expression reached its maximum in spa1 (more than eightfold greater than that in the WT), whereas GPX6 expression strongly increased in both bic1,2 and spa1 (Table 1). In contrast, RBCL expression was significantly lower in spa1 than in bic1,2 and hy5, whereas CAB1 expression was lowest in hy5.

After 24 h of UV-B exposure, the expression of photosynthesis-related genes strongly depended on genotype (Table 1). RBCL and CAB1 transcript levels were markedly reduced in hy5 and spa1, whereas bic1,2 maintained significantly increased expression of both genes. The expression of HY5 remained elevated in spa1 but was strongly reduced in hy5. Among the defense-related genes, CHS and PAL expression were highest in spa1, intermediate in bic1,2, and significantly reduced in hy5. The expression of APX3 remained high in spa1 but declined in bic1,2 and hy5, whereas GPX6 expression was low and did not differ significantly among the genotypes at this time point.

3. Discussion

Under natural light conditions, plants are exposed to UV-B mainly during the day, simultaneously with photosynthetically active radiation. This irradiation regime is fundamentally different from experimental systems with isolated UV-B and requires coordinated functioning of the photosynthetic apparatus and light-dependent signalling cascades. In accordance with current concepts, photomorphogenic responses to UV-B are initiated by the UVR8 receptor and are implemented through the HY5 transcription factor, whereas proteins of the SPA and BIC families act as negative regulators of signal intensity and duration [8,14,15,16,17]. The data obtained in this study allow us to experimentally separate the contributions of these components at the physiological, biochemical, and transcriptional levels during repeated daytime UV-B exposure.

UV-B is known to reduce the rate of photosynthetic activity by suppressing Rubisco activity, limiting CO2 diffusion, and disrupting the functioning of photosystems [18]. In line with these data, in our experiment, all the genotypes presented decreases in the rates of photosynthesis and gas exchange parameters (Figure 2). However, in bic1,2 and spa1, this decrease was less pronounced than that in hy5, indicating a higher functional stability of the photosynthetic apparatus with weakened regulation of the UVR8–HY5 cascade. These differences were not accompanied by a pronounced decrease in chlorophyll or carotenoid content (Figure 3), which is consistent with the concept of a functional, rather than structural, damage to the photosynthetic apparatus under moderate UV-B [19].

One of the key mechanisms of protection of the photosynthetic apparatus is the enhancement of nonphotochemical fluorescence quenching (NPQ), which allows for the dissipation of excess energy as heat and prevents photodamage to PSII [20]. Notably, increased NPQ may reflect either adaptive photoprotective energy dissipation or sustained excitation pressure under prolonged UV-B exposure. In WT, bic1,2, and spa1, UV-B treatment led to a decrease in Y(II) accompanied by an increase in NPQ (Figure 1), indicating a shift toward enhanced nonphotochemical energy dissipation as a compensatory response to reduced photochemical efficiency. This coordinated adjustment was associated with relatively stable Fv/Fm values, which is consistent with the effective protection of PSII. In contrast, in hy5, NPQ induction was markedly attenuated, whereas the decreases in Fv/Fm and Y(II) were more pronounced, indicating a limited capacity to dissipate excess excitation energy and preserve PSII functionality. These data are consistent with the previously demonstrated role of HY5 in the regulation of NPQ and cyclic electron transport as components of photoprotection [21] and confirm that the absence of HY5 leads to increased sensitivity of PSII to UV-B. In WT and bic1,2, NPQ induction occurred in the absence of strong accumulation of UV-screening pigments, suggesting that acclimatory adjustment in these genotypes relied predominantly on transcriptionally mediated reprogramming rather than maximal reinforcement of photophysical energy dissipation. Under low, acclimatory UV-B doses, increased NPQ capacity is frequently associated with improved stabilization of PSII activity and increased resilience of the photosynthetic apparatus [22,23,24]. Therefore, limited NPQ reinforcement during repeated exposure cycles may constrain long-term functional stability, particularly when sustained energy dissipation is needed to prevent PSII overexcitation.

In contrast, the spa1 mutant exhibited a coordinated photoprotective response under the applied daytime UV-B regime. Together, elevated basal and inducible levels of anthocyanins and other UV-B–absorbing phenylpropanoids, strong activation of antioxidant pathways, and pronounced NPQ induction indicate that both screening and energy-dissipating mechanisms are reinforced. Such integration of pigment-based shielding, ROS detoxification, and regulated nonphotochemical quenching is characteristic of the acclimatory UV-B responses described previously [25,26]. Mechanistically, the loss of SPA1 function is expected to stabilize HY5 and related transcriptional regulators, thereby promoting the sustained activation of UVR8-dependent protective gene expression [27,28,29]. This enhanced downstream signalling capacity likely underpins the maintenance of PSII photochemistry and gas exchange during recurrent irradiation. These findings support the established role of HY5 as a central integrator, linking UVR8 perception to the sustained regulation of energy dissipation and UV-screening pigment biosynthesis, together with the transcriptional control of stress-responsive pathways, thereby reinforcing its importance in UV-B acclimation under repeated daytime exposure. A prominent manifestation of this regulatory function is the control of phenylpropanoid-derived UV screening compounds. The accumulation of anthocyanins and other UV-absorbing phenolic compounds is considered an important shielding mechanism that reduces UV-B penetration into the mesophyll [30]. In our study, spa1 presented the highest accumulation of anthocyanins and UV-absorbing compounds, whereas hy5 presented low levels throughout the experiment (Figure 5). These differences are directly correlated with the expression of the phenylpropanoid pathway genes PAL and CHS (Table 1), confirming the central role of HY5 in the transcriptional activation of these genes [5,14].

In parallel, UV-B induced antioxidant mechanisms aimed at limiting the accumulation of reactive oxygen species [2]. In spa1 and bic1,2, the induction of APX3 and GPX6 was observed (Table 1), whereas in hy5, this induction was weak or transient. Thus, the biochemical and transcriptional data are consistent and indicate a disruption in the coordination of antioxidant defences in the absence of HY5.

At the gene expression level, fundamentally different strategies of adaptation to UV-B were revealed. In spa1, UV-B irradiation was accompanied by a stable increase in HY5 expression at all time points (Table 1), which is consistent with the role of SPA proteins as components of the COP1–SPA complex in regulating HY5 stability [8,31]. The increase in HY5 correlated with the activation of PAL, CHS, APX3, and GPX6, but in the late phase, it was accompanied by a significant decrease in the expression of the photosynthesis-related genes RBCL and CAB1 (Table 1). These findings indicate transcriptional redistribution toward protective programs during prolonged UV-B exposure, which is consistent with the concept of a switch in COP1 function from repressor to signalling upon the activation of UVR8 [8].

In bic1,2, the absence of early induction of PAL and CHS with increasing HY5 for 8–16 h suggests that BIC proteins primarily regulate the amplitude and kinetics of the UVR8 signal rather than the ability of HY5 to activate transcription [10,32]. Furthermore, the maintenance of RBCL and CAB1 expression at WT levels for up to 24 h (Table 1) indicates the priority maintenance of photosynthetic function with a compensatory increase in antioxidant defense. The maintenance of RBCL and CAB1 transcript levels suggests transcriptional support of the photosynthetic apparatus; however, posttranscriptional regulation and enzyme activity were not assessed in this study. Therefore, we cannot exclude the possibility that protein abundance, Rubisco activity, or LHCII stability may differ from transcript patterns, and direct measurements of protein levels and enzymatic activity will be needed to fully elucidate the mechanism underlying this response.

In the hy5 mutant, UV-B did not induce a robust protective transcriptional response. The suppression of PAL, CHS, APX3, and GPX6 expression was accompanied by a pronounced decrease in the expression of RBCL and CAB1 in the late phase (Table 1), indicating the simultaneous weakening of both the protective and photosynthetic programs. The concurrent reduction in protective pigment biosynthesis and photosynthesis-related gene expression suggests that HY5 acts as a central coordinator of both processes. The observed phenotype may reflect not only direct transcriptional effects of HY5 on these target genes but also indirect consequences of impaired redox homeostasis and altered signalling crosstalk under UV-B exposure. These results support a central role of HY5 in coordinating UV-B–induced responses and photoprotection under the specific conditions tested, although the contribution of additional HY5-independent pathways cannot be excluded. [33,34]. Notably, HY5 has a close homologue, HYH (HY5 HOMOLOG), which shares overlapping DNA-binding specificity and partially redundant regulatory functions. Previous studies in A. thaliana have demonstrated that HYH can compensate, at least in part, for the loss of HY5, particularly under UV-B and high-light conditions. This partial functional redundancy may explain why the hy5 mutant, although clearly hypersensitive, does not exhibit a complete collapse of UV-B–induced transcriptional responses. However, the compensation appears insufficient to sustain full activation of phenylpropanoid biosynthesis and photosynthetic stability under repeated UV-B exposure. Functional overlap between HY5 and HYH has also been reported in other photomorphogenic contexts, suggesting that partial redundancy within the bZIP transcription factor network may buffer extreme phenotypes in single mutants. Nevertheless, HY5 remains the dominant regulator of UVR8-dependent transcriptional reprogramming, and the loss of HY5 cannot be fully compensated for by HYH [35].

Future experiments combining protein-level analyses, measurements of Rubisco activity, and assessments of the chloroplast redox state would help clarify whether the reduction in photosynthetic performance in hy5 is a direct transcriptional consequence or a secondary effect of insufficient photoprotection.

The novelty of this study lies in its focus on repeated daytime UV-B exposure as a model of cumulative acclimation rather than a single stress event. This approach allows us to examine how photoprotective responses are maintained over successive exposure cycles under conditions that more closely resemble natural light regimes. By comparing hy5, bic1,2, and spa1 mutants under the same irradiation schedule, we were able to distinguish how alterations in different components of the UVR8 signalling pathway affect NPQ regulation, phenylpropanoid accumulation, and UV-B–responsive gene expression. The results revealed that enhanced UVR8 signalling (bic1,2), reduced COP1–SPA repression (spa1), and loss of HY5 function led to distinct patterns of physiological and transcriptional adjustment. The combined analysis of physiological parameters, pigment accumulation, and transcript levels therefore helps clarify how perturbations within the UVR8 signalling pathway are associated with differences in photosynthetic performance during repeated UV-B exposure.

The transcription factor HY5 has been shown to be essential for the coordinated activation of phenylpropanoid and antioxidant pathways, maintaining the photochemical stability of PSII and the transcriptional activity of photosynthesis-related genes during prolonged UV-B exposure. In the absence of HY5, both protective (CHS, PAL, APX3, and GPX6) and photosynthetic (RBCL and CAB1) genes are suppressed, leading to functional destabilization of the photosynthetic apparatus.

Distinct adaptation strategies of spa1 and bic1,2 mutants are revealed under repeated daytime UV-B radiation exposure. The spa1 mutant is characterized by increased HY5-dependent activation of the phenylpropanoid and antioxidant pathways, accompanied by suppression of photosynthesis-related gene expression during prolonged UV-B exposure. In contrast, bic1,2 maintains the transcriptional activity of the photosynthetic apparatus with delayed induction of antioxidant defense.

Taken together, the obtained data suggest that proteins of the SPA and BIC families do not replace HY5 but rather modulate the kinetics and direction of the UV-B signalling response, determining the balance between enhancing photoprotection and maintaining photosynthetic function. The presented scheme (Figure 6) integrates the obtained results into a modern model of UV-B signalling and highlights the multilayered nature of plant adaptation to daytime UV-B stress. This model does not imply independent or isolated functioning of these regulatory layers; rather, it reflects a dynamically interconnected network in which extensive cross-regulation and reciprocal signal exchange occur among perception, signal transduction, and physiological responses.

Figure 6.

Figure 6

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The presented scheme represents a working integrative model based on the experimental data obtained in this study and supported by previously published literature. The components directly supported by our experimental results are distinguished from those reflecting interpretation within the established UV-B signalling framework, with relevant references provided. UV-B–induced signalling and downstream physiological responses in the wild type (WT) and spa1, hy5, and bic1,2 mutants are shown, integrating three regulatory layers: UV-B perception, signal transduction, and execution of photoprotective responses. At the perception level, UV-B radiation induces monomerization of the UVR8 photoreceptor to its active form (UVR8*), initiating UV-B signalling [11]. UVR8 is a homodimeric, chromophore-less protein receptor that senses UV-B via intrinsic tryptophan residues [3]. Signal attenuation is mediated by RUP proteins, whose expression is positively regulated by HY5, forming a negative feedback loop that promotes UVR8 redimerization and signal termination [31]. At the signal transduction level, activated UVR8 associates with the COP1–SPA complex, causing its dissociation from the CUL4–DDB1 E3 ubiquitin ligase and functional inactivation of COP1-mediated repression [15]. BIC proteins restrict the duration and intensity of UVR8 signalling by limiting the UVR8–COP1 interaction and promoting signal attenuation; loss of BIC function in bic1,2 results in prolonged UVR8 signalling [10,11]. In the spa1 mutant, reduced SPA function weakens COP1–SPA E3 ligase activity, leading to increased stabilization and accumulation of HY5 [8]. DET1 acts as a chromatin-associated negative regulator that constrains the transcriptional output downstream of HY5 and limits light-induced gene activation [36]. At the execution level, HY5 and its paralogue HYH regulate the expression of UV-B-responsive genes, including CHS, PAL, and APX, thereby controlling the accumulation of anthocyanins, carotenoids, and UV-absorbing pigments (UAPs) and shaping physiological responses such as transpiration, Y(II), and NPQ. These regulatory interactions give rise to genotype-specific differences in photoprotective capacity and photosynthetic performance under repeated daytime UV-B exposure. The dashed outlines indicate gene mutations or strong reductions in regulatory activity. The size of individual components reflects their relative contribution within the signalling network, whereas arrow thickness indicates the strength of regulatory interactions or signal flow. The hourglass recognizes that the regulation process is time-dependent and can behave differently under long-term and short-term influence. The symbols “+”, “++”, “−” and “− −” denote relative increases, strong increases, or decreases, respectively, compared with the WT.

4. Materials and Methods

4.1. Growing Conditions

Seeds of the mutant lines A. thaliana hy5 (elongated hypocotyl 5, HY5 allele used in this study corresponds to the T-DNA insertion line SALK_096651C (NASC stock number N663420, (AT5G11260)), bic1,2 (blue light inhibitor of cryptochromes, CS71577) and spa1 (SUPPRESSOR OF PHYA-105 1, AT2G46340, CS87912), as well as wild-type (WT, Col-0) plants, were obtained from the Arabidopsis Biological Resource Center (ABRC). All the plants were derived from F1 seeds obtained directly from the seed bank, and each seed batch underwent routine quality control and germination testing in our laboratory. These mutants have been widely used in studies of light signalling. The hy5 mutant line has been employed in research on growth regulation and photomorphogenesis [37]. The bic1,2 double mutant (CS71577) has been widely used to analyse cryptochrome inhibition and blue-light signalling [10,38,39]. Since their identification, spa1 mutants (AT2G46340) have provided critical insights into PHYA signalling and the regulatory functions of COP1–SPA complexes in light-controlled development [40,41,42]. Mutant lines were additionally verified at the transcript level by RT–PCR using gene-specific primers designed to amplify regions corresponding to full-length exons (Table S1). The absence of amplification products corresponding to the full-length transcripts confirmed the disruption of gene expression in the mutant lines.

After a three-day vernalization period, the seeds were sown onto a perlite substrate and grown in a climate-controlled chamber using Hoagland nutrient solution. The growth conditions included an 8 h photoperiod, air temperatures of 23/18 °C (day/night), and 75% relative humidity. Illumination was provided by Philips TD-L 58 W/33-640 (Pila, Poland) fluorescent lamps at an intensity of 100 µmol (photons) m−2 s−1. The seedlings were cultivated for approximately 25 days until they reached the developmental stage appropriate for the experimental procedures.

The experimental group, comprising all the investigated genotypes, was exposed to supplemental narrowband UV-B radiation (311 ± 3 nm) during the photoperiod. Under WFL + UV-B conditions, the plants received 43.7 mmol photons m−2 of UV-B per 8 h photoperiod, corresponding to a daily UV-B dose of 16.8 kJ m−2. The irradiation was applied for 8 h per day over three consecutive days, resulting in cumulative UV-B doses of 16.8, 33.6, and 50.4 kJ m−2 after 1, 2, and 3 days of treatment, respectively, whereas the 0 h time point served as the non-UV-B control (Figure 7).

Figure 7.

Figure 7

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Spectral composition of the irradiation conditions used in the experiment. (A) Emission spectrum of white fluorescent lamps (WFL) used as background illumination, showing the presence of a UV-A component and a low-intensity UV-B fraction. Under WFL conditions, plants received 1.50 mmol photons m−2 of UV-B and 7.46 mmol photons m−2 of UV-A per 8 h photoperiod, corresponding to 0.58 and 2.44 kJ m−2, respectively (total UV dose: 3.02 kJ m−2; UV-B/UV-A ratio = 0.20). (B) Emission spectrum of a WFL supplemented with a narrowband UV-B source (Philips TL/PL-S 311). Under WFL + UV-B conditions, the plants received 43.7 mmol photons m−2 of UV-B and 9.9 mmol photons m−2 of UV-A per 8 h photoperiod, corresponding to 16.8 and 3.24 kJ m−2, respectively (total UV dose: ~20.0 kJ m−2; UV-B/UV-A ratio = 4.42). The vertical line in the UV-B region indicates the peak emission at 311 nm. Because both the background fluorescent lamps and the UV-B source contributed to the UV-A spectral range, the treatment represents UV-B supplementation with a small fraction of UV-A.

The applied daily UV-B dose (16.8 kJ m−2) falls within the upper range of natural solar UV-B exposure recorded while taking into account natural fluctuations in solar radiation during clear summer days at temperate latitudes and therefore represents physiologically relevant, yet elevated, daytime irradiation rather than an extreme artificial stress treatment [43,44]. The daily dose of UVB is approximately equal to the maximum dose of UVB under natural conditions, but after 2 and 3 days of UVB exposure, it cumulatively exceeds this dose, which can lead to the development of moderate oxidative stress.

4.2. Pigments

The contents of chlorophyll a, chlorophyll b, and total carotenoids were determined spectrophotometrically (Genesys 10 UV–Vis, Thermo Fisher Scientific, Waltham, MA, USA). Leaf material (approximately 100 mg fresh mass) was homogenized in cold 80% (v/v) acetone in the presence of a small amount of calcium carbonate to prevent pheophytinization. All extraction procedures were performed under dim light conditions, avoiding direct sunlight, and the extracts were immediately covered with dark cloth to minimize pigment degradation. The homogenates were subsequently centrifuged at 10,000× g for 10 min at 4 °C. The pellet was repeatedly washed with cold 80% acetone until complete discolouration occurred. The supernatants were combined and brought to a defined final volume with cold 80% acetone. The absorbance was recorded at 470, 646, and 663 nm, and the pigment concentrations were calculated according to the standard equations described previously [45]. The results are expressed on a fresh mass basis. Spectrophotometric analysis was used to assess relative differences in total pigment pools among genotypes.

Anthocyanins and UV-absorbing pigments (UAPs; methanol-soluble phenylpropanoid derivatives, including flavonol glycosides such as kaempferol and quercetin derivatives, as well as hydroxycinnamic acids and sinapate esters) were extracted from 15 to 20 leaf sections in acidic methanol (methanol:water:HCl = 78:20:2, v/v/v) for 24 h at 4 °C in darkness. After incubation, the extracts were clarified by centrifugation at 10,000× g for 10 min at 4 °C. The anthocyanin content was determined spectrophotometrically at 530 nm with correction at 657 nm to minimize chlorophyll interference, according to [46]. The content of soluble UV-absorbing phenolic compounds was estimated from the absorbance at 327 nm, corresponding to the absorption maximum of methanol-soluble flavonols and hydroxycinnamic acid derivatives. This wavelength reflects the absorbance characteristics of these compounds and is not related to the spectral range of the applied UV-B radiation (280–320 nm). UAP values were normalized to 100 mg fresh mass (FM) and used for comparative analysis among genotypes.

4.3. Fluorescence and Gas Exchange Parameters

Fluorescence induction curves were measured using a mini-PAM fluorometer II (Walz, Effeltrich, Germany) on dark-adapted plants (30 min). Data calculations and analysis were performed using WinControl-3 v3.32 software (Walz, Effeltrich, Germany). Here, Fm and Fm’ are the dark-adapted and light-adapted maximum fluorescence at illumination with a pulse of saturating light (7000 μmol (photon) m−2 s−1), respectively. Fv is the photoinduced change in fluorescence, Fs is the steady-state fluorescence level of Chl a, and F0 is the initial fluorescence level of Chl. The effective quantum yield of PSII (Y(II)) was determined under actinic light of 100 μmol photons m−2 s−1 and calculated as Y(II) = (Fm′ − Fs)/Fm′. Nonphotochemical quenching (NPQ) was calculated according to the equation NPQ = (Fm − Fm′)/Fm′. The fluorescence parameters characterizing the PSII state (such as Fv/Fm and Y(II) = (Fm’ − FS)/Fm) were determined according to [47,48].

Net photosynthetic (Pn) and transpiration (Tr) rates, as well as stomatal conductance (GS), were measured using a CIRAS-3 infrared gas analyser (PP Systems, Amesbury, MA, USA) in a 2.5 cm2 chamber at an irradiance of 120 μmol m−2 s−1 and a CO2 concentration of 400 ppm.

4.4. RNA Extraction, RT–PCR and Rationale for the Selection of Target Genes

RNA was isolated from A. thaliana leaves as described previously [49]. Gene expression analysis was carried out using a CFX96 Touch™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Gene-specific primers (Table S1) were designed for the following genes: elongated hypocotyl 5 (HY5, NM_001343175.1), E3 ubiquitin-protein ligase (COP1, NM_001336418.1), deetiolated 1 (DET1, NM_117086.4), phenylalanine ammonia-lyase 1 (PAL1, NM_129260.3), chalcone synthase (CHS, NM_121396.4), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL, NM_105379.4), chlorophyll a/b binding protein 1 (CAB1, NM_102733.3), glutathione peroxidase 6 (GPX6, NM_117229.4), and ascorbate peroxidase 3 (APX3, NM_119666.4) (Table S1).

Primer sequences were designed using nucleotide sequences obtained from the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov), UniProt (www.uniprot.org), and Phytozome (https://phytozome-next.jgi.doe.gov/), accessed on 1 February 2025, with Vector NTI Suite 9 software (Invitrogen, Carlsbad, CA, USA). The transcript levels were normalized to those of Actin 1 (NM_001336664.1) and Tubulin alpha-3 (TUA3) (NM_121982.4), and the gene expression levels in the WT plants were set to 1.

The selected genes represent key components of UV-B signalling, photoprotection, photosynthetic regulation, and oxidative stress responses. HY5 functions as a central nuclear transcription factor in UVR8-dependent signalling, whereas DET1 and COP1 regulate HY5 stability and modulate downstream light responses. PAL1 and CHS encode cytosolic enzymes of the phenylpropanoid pathway, and their expression reflects the activation of UV-screening compound biosynthesis. CAB1 and RBCL serve as markers of photosynthetic apparatus status, representing light harvesting (thylakoid membranes) and carbon fixation (chloroplast stroma), respectively. APX3 encodes a peroxisomal ascorbate peroxidase involved in H2O2 detoxification, and GPX6 represents the glutathione-dependent redox system contributing to peroxide scavenging. Together, this set of genes enables the assessment of UV-B perception, transcriptional regulation, photoprotective adjustment, and ROS detoxification.

4.5. Statistics

Physiological and biochemical parameters were assessed in fully expanded middle-tier leaves. Photosynthetic fluorescence parameters, net photosynthesis, transpiration rates and stomatal conductance were measured in 4–5 biological replicates per treatment, as indicated in the corresponding figure legends. Pigment content, anthocyanin content, UV-absorbing compounds and gene expression were analysed using four independent biological replicates, each consisting of individually flash-frozen plants with at least eight leaves pooled per replicate.

Statistical analyses were performed using one-way analysis of variance (ANOVA). Differences among genotypes at each time point were evaluated using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Data were tested for normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s test) prior to ANOVA. All the datasets met the assumptions required for parametric testing. These differences are indicated in the figures by different lowercase letters. The data are presented as mean values ± SD (SigmaPlot 12.3, Systat Software).

5. Conclusions

These results demonstrate that UV-B tolerance in A. thaliana depends on the specific regulatory configuration of the UVR8 signalling cascade. Although both bic1,2 and spa1 mutants show tolerance close to that of the wild type, their resistance is achieved through distinct mechanisms.

In bic1,2, prolonged UVR8 activation sustains the transcription of UV-responsive genes and maintains the expression of photosynthesis-related genes during repeated UV-B exposure. However, the relatively weak induction of NPQ and moderate accumulation of UV-screening phenolic compounds indicate that this tolerance is supported mainly by sustained transcriptional activation rather than reinforced energy dissipation or pigment-based shielding.

In contrast, spa1 exhibits a coordinated protective phenotype characterized by increased accumulation of anthocyanins and other UV-absorbing phenylpropanoids, increased expression of antioxidant-related genes, and pronounced NPQ induction, resulting in greater stability of PSII performance. This phenotype is consistent with the enhanced expression of HY5 and more efficient activation of downstream protective pathways.

The hy5 mutant shows pronounced UV-B hypersensitivity, including reduced induction of phenylpropanoid biosynthesis genes, limited accumulation of UV-screening compounds, insufficient NPQ activation, and repression of photosynthetic activity and photosynthesis-related genes, underscoring the central role of HY5 in coordinating UV-B acclimation.

Overall, our study demonstrates that enhanced or weakened UV-B tolerance can arise from distinct regulatory configurations within the UVR8 pathway and that sustained UVR8 activation alone does not necessarily ensure optimal photoprotective adjustment. By analysing repeated daytime UV-B exposure, we show that efficient acclimation requires coordinated activation of many different processes, including phenylpropanoid biosynthesis, photosynthesis and nonphotochemical energy dissipation. These findings refine the current understanding of how UVR8-dependent signalling translates into functional stabilization of photosynthesis and antioxidant systems under recurrent UV-B conditions.

Acknowledgments

Figure 7 was made using the software biorender.com.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms27052436/s1.

ijms-27-02436-s001.zip (61.8KB, zip)

Author Contributions

Conceptualization, P.P. and A.A.; methodology, P.P. and V.D.K.; software, P.P.; validation, A.A., P.P. and V.D.K.; formal analysis, P.P. and A.A.; investigation, A.A. and M.V.; resources, P.P.; data curation, P.P.; writing—original draft preparation, P.P.; writing—review and editing, A.A. and P.P.; visualization, P.P.; supervision, V.V.K.; project administration, V.V.K.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Funding Statement

This work was funded by the Russian Science Foundation (no. 25-14-00037). The data in Figure 1 and Table 1 were obtained within the state assignment of the Ministry of Science and Higher Education of the Russian Federation (No. 126012615950-2).

Footnotes

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Associated Data

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

Supplementary Materials

ijms-27-02436-s001.zip (61.8KB, zip)

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Articles from International Journal of Molecular Sciences are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

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