Unable to move, plants must adapt to their surroundings. An important and unavoidable component of a plant’s environment is solar radiation, which includes both beneficial visible light and damaging ultraviolet (UV) rays. UV radiation harms a variety of cellular components including DNA. UV damaged DNA, primarily pyrimidine photodimers, is repaired by photolyases, using the energy from visible light (light repair), and by nucleotide excision repair (NER) (dark repair) (Pang and Hays, 1991; Molinier, 2017).

Nucleotide excision repair is a conserved multistep pathway involving damage recognition, strand unwinding, excision, repair synthesis, and ligation. Damage recognition is via one of two NER sub-pathways. Global genomic repair (GGR) identifies UV damage in DNA throughout the genome, while transcription coupled repair (TCR) initiates repair of transcribed strands. TCR has been well studied in humans, where deficiencies in this process can result in Cockayne Syndrome and UV-sensitive syndrome (Gregersen and Svejstrup, 2018). UV damaged DNA arrests progression of RNA polymerase II (RNAP), resulting in stabilization of RNAP – Cockayne Syndrome B (CSB) interaction. CSB then recruits the Cockayne Syndrome A (CSA)-DDB1-cullin 4 complex, which ubiquitinates CSB, followed by UV Stimulated Scaffold protein A (UVSSA) and Ubiquitin Specific Peptidase 7 (USP7), which stabilize CSB. Subsequently, core NER components, such as TFIIH and the XPG and XPF endonucleases, are recruited, and resulting in damage excision and repair. Re-initiation of transcription following repair is thought to involve the TFIIS elongation factor (Geijer and Marteijn, 2018).

In plants, UV damage in transcribed strands is preferentially repaired, and this process is regulated by the circadian clock (Fidantsef and Britt, 2012; Oztas et al., 2018). The Arabidopsis homologs of CSA, CSB, USP7, and TFIIS have previously been identified and described. Arabidopsis thaliana has two CSA homologs, AtCSA-1/ CSAat1A (At1g27840) and AtCSA-2/ CSAat1B (At1g19750) (Kunz et al., 2005). Despite the fact that these two proteins are 92% identical, they are both required for tolerance to UV and MMS and repair of transcribed strands. The CSA homologs interact with DDB1A, localize to the nucleus, and form heterotetramers (Biedermann and Hellmann, 2010; Zhang et al., 2010). The Arabidopsis CSB homolog is SWI2/SNF2 protein Chromatin Remodeling 8 (CHR8, At2g18760) (Kunz et al., 2005; Singh et al., 2010). CHR8 RNAi lines result in UV sensitivity, but do not exhibit ionizing radiation or intrachromosomal recombination rate phenotypes, consistent with a role in NER (Shaked et al., 2006). UBP12 (At5g06600) and UBP13 (At3g11910) are the Arabidopsis USP7 homologs and have been implicated in plant immunity, flowering, seed, and root development, as well as jasmonate signaling (Ewan et al., 2011; Cui et al., 2013; Derkacheva et al., 2016; Jeong et al., 2017; An et al., 2018). The Arabidopsis TFIIS homolog is Reduced Dormancy 2 (RDO2, At2g38560), which is required for regulation of seed dormancy by Delay of Germination 1 (DOG1) (Léon-Kloosterziel et al., 1996; Grasser et al., 2009; Liu et al., 2011; Mortensen and Grasser, 2014). RDO2/TFIIS has also been implicated in mRNA processing in plants, including in response to light (Dolata et al., 2015; Antosz et al., 2017; Godoy Herz et al., 2019). In this study we identify the Arabidopsis UVSSA homolog and examine the roles of UVSSA, UBP12/13, and RDO2 in UV tolerance.

In this study we identify the Arabidopsis UVSSA homolog. Arabidopsis UVSSA (encoded by At3g61800) is 39% and 28% identical to rice and human UVSSA, respectively. Clear UVSSA homologs are found throughout the animal and plant kingdoms including angiosperms, gymnosperms, ferns, and moss. One million maximum parsimony phylogenetic search replicates for UVSSA homolog amino acid sequences recovered a single most parsimonious tree (score 2561) (Figure 1) that is topologically congruent with well supported hypotheses of plant evolutionary history (Morris et al., 2018). Conserved domains in UVSSA proteins include ENTH/VHS in the N terminus and DUF2043 in the C terminus (Figure 2). ENTH/VHS domains are multi-helical with an alpha-alpha 2-layered structural fold, while DUF2043 is an approximately 100 amino acid long UVSSA-specific domain, which includes three conserved cysteines and a CP(y/l)HG motif (Marchler-Bauer et al., 2017). AtUVSSA has a potential bipartite NLS in the C terminus and SUBAcon predicts nuclear localization (score 0.994) (De Castro et al., 2006; Hooper et al., 2014), consistent with a role in DNA repair.

Public gene expression data was examined for Arabidopsis UVSSA and the other TCR gene homologs: AtCSA-1, CHR8 (CSB homolog), UBP12 and UBP13 (USP7 homologs), and RDO2 (TFIIS homolog). With respect to absolute levels of expression (Supplementary Figure S1A), UBP12, UBP13, and RDO2 are expressed throughout the plant, consistent with the broad role of these genes in development (Grasser et al., 2009; Cui et al., 2013; Derkacheva et al., 2016), while AtCSA-1, CHR8, and UVSSA are expressed at lower levels (Schmid et al., 2005). With respect to relative levels of expression (Supplementary Figure S1B), CHR8 and UVSSA are enriched in mature pollen, while RDO2, AtCSA-1, CHR8, and UVSSA are up-regulated more than two-fold in dry seed, perhaps contributing to maintenance of seed genome integrity (Waterworth et al., 2015).

Public expression data was also examined to determine the effect of potentially mutagenic stress on expression of these genes. CHR8 was found to be upregulated by genotoxic stress induced by bleomycin and mitomycin C treatment, consistent with previous reports (Molinier et al., 2005), in both the shoot and root, but the other genes were not, while UV-B treatment did not result in major changes to the levels of any of the genes (Supplementary Figure S2; Kilian et al., 2007).

In order to examine the role of these genes in Arabidopsis UV tolerance, T-DNA insertion mutants were obtained. Previously described alleles of AtCSA-1 (SALK_030558) (Lee et al., 2010) and RDO2 (SALK_027259) (Grasser et al., 2009; Liu et al., 2011) were utilized. UBP12 allele GABI_742C10 (ubp12-2) has previously been shown to result in reduced levels of both UBP12 and UBP13, thus acts as a weak double mutant (Cui et al., 2013). In previous studies RNAi lines of CHR8 were shown to exhibit UV sensitivity (Shaked et al., 2006). Here we examine two T-DNA alleles of CHR8, chr8-1 (SALK_000799) and chr8-2 (SAIL_273_G11) (Figure 3A). For UVSSA, two T-DNA alleles were examined, uvssa-1 (SAIL_58_C12), located 38 bp upstream of the start codon, and uvssa-2 (SALK_061538), located in the first intron past the start codon.

We examined the effect of these alleles on gene expression using semi-quantitative RT-PCR. Primers flanking the chr8-1 and chr8-2 insertion sites detected no CHR8 transcript, indicating these are null alleles (Figure 3B). Semi-quantitative RT-PCR with T-DNA insertion flanking primers also confirmed loss of transcript in the atcsa-1, ubp12, and rdo2 lines (Supplementary Figure S3). For UVSSA, we utilized primers in the first and second coding exons, since the effect of T-DNA insertion on coding sequences was our primary concern. uvssa-2 results in a null allele, but in uvssa-1 both the predicted band and a larger band were detected (Figure 3C). The size of the larger band was consistent with that of the unspliced transcript, so we hypothesized that uvssa-1 insertion affected intron splicing [note the uvssa-1 samples did not result in larger gDNA-size bands of CHR8, thus were not gDNA contaminated (data not shown)]. Real-time qPCR with an intron-specific primer was used to quantify the effect of the uvssa-1 allele on splicing, and large amounts of the unspliced product were detected (Figure 3D). Due to the presence of an in frame stop codon in the intron, this transcript results in a truncated 77 amino acid product. uvssa-1 also resulted in increased levels of correctly spliced UVSSA. Thus uvssa-1 would be predicted to result in increased levels of both full length and truncated UVSSA.

Mutant alleles of the TCR genes were grown in long day conditions with their respective controls and their developmental phenotypes examined. ubp12-2 mutants exhibited decreased rosette size, early flowering (days), and decreased apical dominance (increased number of shoots) (Supplementary Figure S4), consistent with previously described phenotypes (Cui et al., 2013; Derkacheva et al., 2016). The other mutant alleles did not exhibit any developmental phenotypes with the exception of a slight increase in apical dominance in chr8-2. RDO2 mutants have been described as early flowering (Grasser et al., 2009), however, additional analysis indicates this phenotype is observed with respect to number of leaves, rather than number of days, at flowering (Mortensen and Grasser, 2014), consistent with our results.

The UV tolerance of the mutant alleles of the TCR genes was then assessed. Since TCR is a sub-pathway of NER, or dark repair, we assessed UV tolerance in seedlings following dark incubation after UV treatment. As previously described (Shaked et al., 2006; Biedermann and Hellmann, 2010; Zhang et al., 2010), AtCSA-1 and CHR8 (CSB) loss of function resulted in increased UV sensitivity in the dark (Figure 4A–C and Supplementary Figures S5A,B). The UVSSA loss of function allele, uvssa-2, also resulted in increased UV sensitivity in the dark (Figure 4E). The uvssa-1 allele, which results in increased levels of both truncated and full length UVSSA, did not exhibit either increased or decreased UV tolerance following 2 or 3 days of dark incubation (Figure 4D and Supplementary Figure S5C). ubp12-2 also exhibited increased UV sensitivity in the dark (Figure 4F and Supplementary Figure S5D). rdo2 exhibited increased dark UV sensitivity in hypocotyls (but not roots) after 2 days of incubation, but not after 3 days (Figure 4G and Supplementary Figure S5E). We also examined UV sensitivity in adult plants following dark incubation and found that, as in seedlings, atcsa-1, chr8, uvssa-2, ubp12, and rdo2 mutants exhibit UV sensitivity, while uvssa-1 does not (Figure 5).

To examine the specificity of the UV sensitivity of these alleles, they were also incubated in light (long day) following UV treatment. atcsa-1, uvssa-2, and ubp12 were not UV sensitive in the light (Figure 4A,E,F), consistent with the dark specific role of NER. Surprisingly, both chr8 alleles displayed UV sensitivity following light incubation (Figure 4B,C), exhibiting the expected dose dependence, with the more severely truncated chr8-1 allele demonstrating a stronger root phenotype in both light and dark. This result suggests that CHR8 plays a role in light repair, distinct from the other components of the TCR pathway.

In this study, we examined the UV sensitivity of mutant alleles of Arabidopsis homologs of genes implicated in mammalian TCR. As previously reported, we find atcsa-1 mutants exhibit increased dark specific UV sensitivity (Biedermann and Hellmann, 2010). Our atcsa-1 dark root phenotype is not as strong as that of mutants in other TCR components such as CSB/CHR8 and UVSSA, this may be due to redundancy with AtCSA-2/CSAat1B.

The Arabidopsis homolog of mammalian CSB [also known as Excision Repair Cross-Complementing 6 (ERCC6)] and yeast Rad26 is CHR8 (Kunz et al., 2005; Singh et al., 2010). In this study, we utilized CHR8 T-DNA lines and observed increased UV sensitivity following dark incubation, consistent with previous studies using CHR8 RNAi lines (Shaked et al., 2006). Also, unique among the TCR mutants we examined, chr8 alleles exhibited increased UV sensitivity following light incubation. Mammalian CSB has been implicated in regulation of transcription and base excision repair in addition to TCR (Stevnsner et al., 2008; Boetefuer et al., 2018), so one of these roles may contribute to the chr8 light UV sensitivity phenotype.

In humans, mutation of UVSSA results in defective TCR and UV sensitive syndrome (Cleaver, 2012). Loss of the C. elegans UVSSA homolog also results in increased UV sensitivity (Babu and Schumacher, 2016). While UVSSA is conserved throughout the animal kingdom (Nakazawa et al., 2012), it is absent from Drosophila. However, Drosophila also lack CSA and CSB homologs, and do not appear to perform TCR (Sekelsky, 2017). Yeast also lack UVSSA, although both S. cerevisiae and S. pombe have CSB homologs and perform TCR (Li and Li, 2017; Xu et al., 2017). Here we show that UVSSA is found throughout the plant kingdom, with conserved ENTH/VHS and DUF2043 domains. Recently, the region corresponding to amino acid 400–415 of human UVSSA was found to be well conserved in animals and required for TFIIH interaction (Okuda et al., 2017). Although this region is still acidic in plants, it not well conserved with human UVSSA and plants lack F408 and V411, which are required for TFIIH interaction and TCR in humans (Okuda et al., 2017), as well as K414, which is mono-ubiquitinated (Higa et al., 2018). In addition, residues 251–254 of human UVSSA have been shown to be required for USP7 interaction, CSB stability, and TCR (Higa et al., 2016), yet this sequence is also not conserved in plants. Nonetheless our data show that lack of UVSSA results in dark specific UV sensitivity in Arabidopsis, consistent with a role in NER.

Arabidopsis USP7 homologs UBP12 and UBP13, like other ubiquitin specific proteases, play important roles in plant development and environmental response (Zhou et al., 2017). UBP12/13 interact with LHP1 and deubiquitinate RGFR1 and MYC2 (Derkacheva et al., 2016; Jeong et al., 2017; An et al., 2018). Our results here indicate that UBP12 (and UBP13) are involved in UV tolerance, suggesting they may also deubiquitinate UVSSA and CSB, as has been proposed for mammalian USP7 (Geijer and Marteijn, 2018). UBP12 and UBP13 act redundantly, and double null alleles are inviable due to pollen defects (Ewan et al., 2011; Derkacheva et al., 2016). Here we use an allele of UBP12, ubp12-2, which also results in a partial decrease in UBP13 level, and resulting in a weak double mutant (Cui et al., 2013). However, because this is a weak (non-null) double mutant, we may be underestimating the role of UBP12/13 in UV tolerance.

In mammals, in addition to acting during transcript elongation, TFIIS has been shown to facilitate transcription re-initiation following RNAP arrest, and is recruited to the stalled polymerase in a CSB and CSA dependent manner (Donahue et al., 1994; Kalogeraki et al., 2005; Fousteri et al., 2006; Dutta et al., 2015). In yeast, loss of TFIIS only results in increased UV sensitivity in a GGR-deficient background, however, the same is true of CSB homolog Rad26 (Wong and Ingles, 2001). In mammals, reduction of TFIIS resulted in reduced RNA synthesis recovery, but had no effect on UV sensitivity (Jensen and Mullenders, 2010). In this study we detected a UV sensitive phenotype in TFIIS deficient Arabidopsis (rdo2), however, it was milder than observed for the other TCR mutants and not detectable 3 days after seedling UV treatment. Interestingly, the UV sensitive phenotype of both rdo2 and atcsa-1 was stronger in hypocotyls than in roots, at 2 days than at 3 days, and in adults than in seedlings, suggesting the role of these genes in UV tolerance may vary with tissue, time, and phenotype assessed (growth versus tissue death).

In this study, we have identified the Arabidopsis UVSSA homolog and shown that Arabidopsis UVSSA, USP7 (UBP12/13), and TFIIS (RDO2) homologs contribute to UV tolerance, along with CSA and CSB (CHR8) homologs, suggesting conservation in the mechanisms of TCR.

All datasets for this study are included in the manuscript and the Supplementary Files.

WAK, AS, and DS performed the experiments. JM conducted the phylogenetic analysis. DS wrote the first draft of the manuscript. All authors contributed to revised manuscript and approved the final version, designed the experiments and analyzed the data.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.