Introduction

Sunlight is an environmental factor regulating plant growth and development. Plants have evolved a battery of photoreceptors to sense and respond to different wavelengths of light (Jenkins, 2017; Liu et al., 2020a). UV-B (280–315 nm) comprises the shortest wavelength of sunlight on the earth surface. In general, low fluence rate UV-B does not generate damage effects on plants, rather it induces plant photomorphogenic responses (Yin and Ulm, 2017). High fluence rate UV-B may damage macromolecules, including nucleic acids, protein and membranes (Hideg et al., 2013). The UV-B photoreceptor UVR8 protein ((UV RESISTANCE LOCUS 8) perceives the low dose UV-B and triggers UV-B specific signal transduction. The photomorphogenic responses mediated by UVR8 play a key role in UV-B acclimation and plant survival in nature (Favory et al., 2009). UVR8 exists as homodimer in its ground state and UV-B perception leads to dimer dissociation into monomer, which is the active form for signaling (Rizzini et al., 2011). In Arabidopsis, RUP1 (REPRESSORS OF UV PHOTOMORPHOGENESIS 1) and RUP2 are two key negative regulators of UV-B signaling (Gruber et al., 2010). RUP1 and RUP2 promote the reversion of UVR8 monomer to homodimer (Heijde and Ulm, 2013).

Tomato (Solanum lycopersicum) is an agriculturally and economically important vegetable crop. In agriculture, tomato seedlings may face sudden UV-B exposure when transferred from seedling bed in green house, where UV-B is blocked by plastic cover or glass, to open field. Therefore, it is important to investigate how tomato seedlings respond to UV-B. Previously, we demonstrated that Tomato UVR8 (SlUVR8) regulates seedling photomorphogenesis and UV-B stress tolerance (Liu et al., 2020b). However, the negative regulation of UV-B signaling is not known.

Results

In Arabidopsis, the two highly related WD40 proteins RUP1 and RUP2 (AtRUP1 and AtRUP2) were identified to be repressors of UVR8-mediated UV-B signaling pathway (Gruber et al., 2010). We found that AtRUP1 and AtRUP2 show high identity to only one putative protein encoded by Solyc11g005190 in Tomato genome (55.5 and 56.1%, respectively). The Solyc11g005190 was therefore called tomato RUP (SlRUP). Previously, SlRUP was shown to be a negative regulator of photomorphogenesis in white light, where it was named as LeCOP1Like (Liu et al., 2004). For clarity we renamed LeCOP1Like as SlRUP herein. Like AtRUP1 and AtRUP2, SlRUP mainly consists of WD40-repeats (Trp-Asp) domain with apparently no additional domains (Fig. S1A&B). Transcript of SlRUP was induced by UV-B in a SlUVR8- and SlHY5-dependent manner (Fig. 1A).

Fig. 1
figure 1

SlRUP negatively regulates the UVR8-mediated UV-B photomorphogenesis. (A) Transcript analysis of SlRUP in response to UV-B in Sluvr8, Slhy5 mutants compared with wildtype (AC, Ailsa Craig) by qRT-PCR. 6d old seedlings were irradiated with UV-B for different lengths. Mean and SE of three biological replicates are presented. (B) Representative seedlings of wildtype and Slrup mutants (CR8 and CR22), and SlRUP-GFP lines grown in white light or white light supplemented with UV-B. Bar = 1 cm. (C) and (D) Hypocotyl length of 6 d old seedlings grown under white light (WL) or white light supplemented with UV-B (UV-B), Mean and SE are presented (n = 30). (E) The hypocotyl length ratio of UV-B/WL. Asterisks indicate significant differences between transgenic lines and wildtype, differences with a P < 0.05 (*) and P < 0.01 (**) resulting from a one-way analysis of variance test. (F) UV-B induced anthocyanin accumulation. Mean and SE are presented (n = 3). (G) and (H) qRT-PCR analysis of SlHY5 and SlCHS1 in response to UV-B in wildtype, Slrup mutant, and SlRUP-GFP line for different UV-B radiation time. Mean and SE of three biological samples are presented. (I) SlRUP promotes redimeriation of SlUVR8 post UV-B treatment. 6 d old seedlings were irradiated with broadband UV-B for 0.5 h, followed by recovery in white light (WL) in the absence of UV-B for different time. Anti-SlUVR8 was used as the primary antibody for immunoblot analysis. Ponceau staining serves as loading control

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To analyze the physiological functions of SlRUP, we generated SlRUP overexpression lines with C-terminal GFP, and Slrup mutants by CRISPR/CAS9 approach (Fig. S2). Three independent SlRUP-GFP overexpression lines were selected by immunoblot analysis (Fig.S2A). Two homozygous Slrup mutant lines were identified by PCR-resequencing, with 111 bp and 113 bp deletion, respectively (Fig.S2B-E). Under white light, Slrup mutants developed shorter hypocotyls than wildtype (AC, Ailsa Craig) (Fig. 1B&C). UV-B can inhibit hypocotyl elongation of seedlings, the hypocotyl length of wildtype seedling under UV-B was only about 56% of that under white light (Fig. 1D&E). Slrup mutants and RUP-GFP overexpression seedlings were hypersensitive and hyposensitive to UV-B, respectively (Fig. 1B, D, E). Consistent with the key function of SlUVR8 in UV-B signaling, hypocotyl length of Sluvr8 mutant was only slightly shortened under UV-B in comparison to that under white light. Relative to white light control, UV-B induced anthocyanin content to about 3 folds in wildtype, 6–7 folds in Slrup mutants, and only 1.5–2 folds in SlRUP-GFP overexpression lines (Fig. 1F). HY5 and CHS1 play key roles in anthocyanin biosynthesis. Transcription of both SlHY5 and SlCHS1 was induced by UV-B much more pronounced in Slrup than in wildtype and SlRUP-GFP lines (Fig. 1G & H, Fig. S3). Intriguingly, the UV-B-induced expression of SlHY5 and SlCHS was similar in wildtype and SlRUP-GFP overexpression lines. Based on those observations, we conclude that SlRUP is a negative regulator of UVR8-mediated UV-B photomorphogenesis.

We tested possible mechanism for SlRUP in UV-B signaling. SlUVR8 protein levels were similar in Slrup mutant and wildtype (Fig. S4), suggesting that SlRUP does not regulate SlUVR8 protein levels. In response to UV-B, UVR8 homodimers dissociate rapidly to monomers, which trigger UV-B signaling in plants (Rizzini et al., 2011). We tested whether SlRUP regulates the dimer/monomer conformational change of SlUVR8. Under white light, SlUVR8 was detected as homodimer and UV-B induced the dissociation of homodimer into monomer in wildtype, the Slrup mutant and the SlRUP-GFP overexpression line. In wildtype seedling, SlUVR8 showed dimer recovery initiated within 15 min, and completed within 2 h post UV-B exposure. The rate of SlUVR8 redimerization in the Slrup mutant was slow and monomer was detectable even 4 h post UV-B (Fig. 1I, Fig. S5A). SlUVR8 redimeration appears to be slightly faster in SlRUP-GFP overexpression line than wildtype post UV-B (Fig. 1I, Fig. S5B). Thus, we conclude that SlRUP accelerates redimeration of UV-B-activated SlUVR8 monomer.

To survive in nature, plants need to acquire mechanisms to cope with high dose UV-B stress. We previously showed that the acclimation to high dose UV-B stress was dependent on photoreceptor SlUVR8 (Liu et al., 2020b). We tested whether SlRUP participates in UV-B stress tolerance. Exposure of seedlings to stress UV-B (broadband) caused seriously stress response in wildtype, SlRUP-GFP, and Sluvr8 mutant seedlings, whereas, Slrup mutant was obviously more resistant (Fig. S6). Consistent with the previous report, a beforehand exposure of seedlings to photomorphogenic UV-B (acclimated) for 2 d enhanced tolerance to broadband UV-B stress in wildtype, but not in Sluvr8 mutant (Fig. S6) (Liu et al., 2020b). Interestingly, this acclimation effect was attenuated in SlRUP-GFP lines and enhanced in Slrup mutants (Fig. S6). This result demonstrated that SlRUP could negatively regulate UV-B acclimation and tolerance to UV-B stress of tomato seedlings.

Discussion

In agricultural, tomato seedlings may face sudden UV-B exposure at seedling stage when transferred from UV-B-shielded green house to UV-B-rich open field. Thus, the understanding of UV-B responses of tomato seedlings is important for better agriculture practice. In this work, we characterized a key negative regulator of UV-B signaling pathway in tomato. Slrup mutants were hypersensitive to low fluence rate UV-B in the assays tested including hypocotyl elongation, UV-B target gene expression and anthocyanin accumulation. Through comparison of SlUVR8 photocycle in wildtype, Slrup mutant and SlRUP overexpression line, it is evident that SlRUP inhibits UV-B signaling via promoting the reversion of active SlUVR8 monomer to inactive homodimer. This mechanism is well conserved in Arabidopsis (Heijde and Ulm, 2013). Previous study showed that pigments and carotenoids are altered in SlRUP-RNAi tomato fruits (Liu et al., 2004). Further work is needed to investigate how SlRUP regulates tomato fruit metabolisms.

In a previous report, the SlRUP-RNAi line developed shorter hypocotyl and dark green leaves than wildtype in white light and dark (Liu et al., 2004). Thus, SlRUP is a negative regulator of photomorphogenesis not only in UV-B but also in white light. Intriguingly, overexpression of SlRUP also leads to shorter hypocotyls in white light. Thus, balanced expression of RUP is required for proper seedling photomorphogenesis. The mechanism for the function of SlRUP in white light awaits further investigation.

In summary, this study revealed that SlRUP is a key repressor of SlUVR8-mediated UV-B photomorphogenesis in tomato. SlRUP inhibits UV-B signaling via accelerating the conversion of active SlUVR8 monomer to inactive homodimer. In addition, SlRUP negatively regulates UV-B stress tolerance of tomato seedlings, which is important for agriculture practice.