Role of root UV-B sensing in Arabidopsis early seedling development

Abstract

All sun-exposed organisms are affected by UV-B [(UVB) 280–320 nm], an integral part of sunlight. UVB can cause stresses or act as a developmental signal depending on its fluence levels. In plants, the mechanism by which high-fluence-rate UVB causes damages and activates DNA-repair systems has been extensively studied. However, little is known about how nondamaging low-fluence-rate UVB is perceived to regulate plant morphogenesis and development. Here, we report the identification of an Arabidopsis mutant, root UVB sensitive 1 (rus1), whose primary root is hypersensitive to very low-fluence-rate (VLF) UVB. Under standard growth-chamber fluorescent white light, rus1 displays stunted root growth and fails to form postembryonic leaves. Experiments with different monochromatic light sources showed that rus1 phenotypes can be rescued if the seedlings are allowed to grow in light conditions with minimum UVB. We determined that roots, not other organs, perceive the UVB signal. Genetic and molecular analyses confirmed that the root light-sensitive phenotypes are independent of all other known plant photoreceptors. Three rus1 alleles have been identified and characterized. A map-based approach was used to identify the RUS1 locus. RUS1 encodes a protein that contains DUF647 (domain of unknown function 647), a domain highly conserved in eukaryotes. Our results demonstrate a root VLF UVB-sensing mechanism that is involved in Arabidopsis early seedling morphogenesis and development.

Plants depend on light as both an energy source and as a signaling cue for morphogenesis and development. In a dynamic light environment, plants must perceive the direction, intensity, quality, and duration of light to fine-tune their metabolism and growth (1–3). Plants have developed sophisticated photoreceptor classes, including the well-characterized phytochromes (PHY) (4), cryptochromes (CRY) (5), and phototropins (PHOT) (6). Whereas PHYs are responsible for sensing red/far-red wavelengths (600–750 nm), CRYs and PHOTs perceive UV-A [(UVA)/blue (320–500 nm)] wavelengths (7). Specific and overlapping roles for the 3 classes of photoreceptors have been documented, and molecular mechanisms of how they perceive light signals have been extensively studied.

UV-B [(UVB) 280–320 nm], despite being only ≈1.5% of the total solar energy, represents an important environmental hazard to living organisms. UVB can adversely react with many biological molecules including amino acids, nucleic acids, proteins, and lipids and elicit stress responses at the molecular, cellular, and whole-organism levels (8–10). Plant responses to UVB damage have been extensively characterized (11–16). High-fluence UVB-elicited responses share gene activation and signal transduction pathways commonly associated with other biotic and abiotic stresses (8, 17, 18). Microarray studies have revealed that a large number of genes are commonly induced by both UVB and other stresses, suggesting that plants may defend themselves against UVB through mechanisms that overlap with general stress-response systems (19, 20). To protect themselves from harmful levels of UVB, plants may thicken their epidermis and accumulate UV-absorbing compounds such as flavonoids and other related phenolic compounds (21, 22).

Depending on fluence-rate levels, UVB can be either toxic or beneficial to plant life (10, 23–25). Whereas high-fluence-rate (HF) UVB (>1 μmol m⁻² s⁻¹) causes cellular damage and triggers stress responses, low-fluence-rate (LF) UVB (0.1–1 μmol m⁻² s⁻¹) can serve as a signal to regulate plant growth and development (10, 26). In Arabidopsis, LF UVB is known to regulate developmental processes including inhibition of hypocotyl and root elongation, enhancement of cotyledon opening/epinasty, and induction of specific sets of genes required for UVB tolerance (27–29). Analyses of genes encoding key enzymes in the biosynthesis of UV-absorbing pigments (e.g., flavonoids) have revealed that UVB stimulates the expression of both the CHALCONE SYNTHASE (CHS) gene and the HY5 gene via a specific signaling pathway (8, 21, 22). Known components in this UVB-mediated signaling pathway include UV RESISTANCE LOCUS8 (UVR8), a chromatin binding protein, and COP1, which regulate promoters of transcription factors such as HY5 and HYH (8, 20). Recent studies have shown that UVB promotes rapid nuclear localization of UVR8 and activates its function in the nucleus (30, 31).

It is widely accepted that there is a specific UVB receptor in plants, but its molecular nature remains unclear. The difficulties arise from the complexity of cellular and physiological effects elicited by various UVB fluence levels. HF UVB causes cellular damage that in turn triggers universal systemic stress responses. LF UVB levels are known to induce expressions of a number of genes including the well-studied CHS gene (20). Although these responses show specificity for UVB radiation, cells may actually be responding to downstream cellular effects of UVB, and not the UVB itself, because of the UVB absorption by many biological molecules. It has been speculated that UVB-specific receptors should respond to very-low-fluence UVB (<0.1 μmol m⁻² s⁻¹) that contains enough quanta to elicit cellular responses through a signaling mechanism involving signal amplification. However, physiological and morphological responses specifically associated with VLF UVB are yet to be identified and characterized (10). Identifying the UVB-specific receptor has been technically difficult because of the lack of unambiguous screenable VLF UVB-specific phenotypes. We report here the identification of an Arabidopsis mutant that is hypersensitive to VLF UVB. Our findings suggest a UVB-sensing mechanism that is closely associated with Arabidopsis early seedling development.

Results

rus1-1 Mutation Causes Developmental Arrest.

We screened a T-DNA-insertion mutagenized Arabidopsis population (ecotype Columbia) for mutants that show root-growth defects. A bleached mutant with extremely stunted growth was identified and named root-UVB-sensitive1-1 (rus1-1, see below). We determined genetically that rus1-1 plants carry a single recessive mutation. When grown vertically on Murashige and Skoog (MS) medium Petri dishes under standard growth-chamber fluorescent light (70–100 μmol m⁻² s⁻¹), rus1-1 seeds germinated, but subsequent development was severely retarded (Fig. 1A). rus1-1 seedlings failed to develop true postembryonic leaves, and their 2 cotyledons remained yellow and had chlorophyll levels <20% that of WT. Primary root elongation was arrested, and no lateral root initiation was observed in rus1-1. Whereas roots of 7-day-old WT seedlings reached ≈2.7 cm in length, those of the rus1-1 seedlings were <0.1 cm, <4% of that of WT. Seven-day-old rus1-1 seedlings recovered almost completely when transferred from Petri dishes to soil. Four days after the seedlings were transferred to soil, rus1-1 cotyledons became green, and postembryonic true leaves appeared (Fig. 1C). Although the sizes of transplanted rus1-1 seedlings were smaller than WT seedlings, they continued to develop normally and were able to set seeds. When seeds were sown directly on soil, rus1-1 was almost indistinguishable from WT at every stage of development (Fig. 1B).

Fig. 1.

rus1-1 seedlings are hypersensitive to ambient growth light and transfer of rus1-1 seedlings from Petri dish to soil rescues the phenotypes. (A) Seven-day-old seedlings for both WT and rus1-1 are shown. Seeds of WT and rus1-1 were germinated vertically on MS plates in a standard growth chamber. An enlarged image of rus1-1 seedling is shown in Inset. (B) rus1-1 is indistinguishable from WT when germinated and grown in soil directly. Fourteen-day-old seedlings are shown. (C) Seven-day-old rus1-1 seedlings are rescued when transferred from plate to soil. Vertically grown 7-day-old seedlings of both rus1-1 and WT were transferred from the Petri dish to soil. Representative seedlings of 0, 4, 7, and 11 days after transfer (DAT) are shown. (Scale bar: 1 cm.)

Exposure of rus1-1 Roots to Light Results in Developmental Arrest.

Dramatic phenotypic differences between rus1-1 seedlings grown on MS medium versus soil led us to suspect that rus1-1 might be hypersensitive to specific nutritional differences between MS medium and soil. rus1-1 seedlings were grown on a series of modified MS media that had specific mineral components (e.g., sugar, nitrogen, potassium, sodium, and vitamins) removed. Under standard growth-chamber light, rus1-1 phenotypes remained strong when grown on the modified MS media (data not shown).

Another difference between soil and Petri dishes is the light environment. We tested rus1-1 growth responses under various light-fluence-rate conditions. Neutral density (ND) filters were used to wrap Petri dishes to achieve a range of light-fluence rates, including 1 that mimicked soil-surface light conditions [4.7 μmol m⁻² s⁻¹ photosynthetically active radiation (PAR)] (32). rus1-1 was sensitive to all tested light irradiances in the standard growth chamber (Fig. 2A). However, all reduced fluence rates progressively alleviated the rus1-1 phenotype. At 47.8 μmol m⁻² s⁻¹, rus1-1 seedlings showed partially green cotyledons but strongly inhibited roots. Under fluence rates <47.8 μmol m⁻² s⁻¹, shoot development in rus1-1 was significantly better. The cotyledons were greener, and postembryonic leaves were formed. rus1-1 root growth, as a percentage of WT, is closely correlated with the measured PAR levels (Fig. 2B). As PAR levels decreased, the root lengths of rus1-1 were significantly longer, reaching as much as 50% of that of WT in the lowest PAR levels tested (4.7 μmol m⁻² s⁻¹ PAR). Seedlings showed partially etiolated morphology in this condition, as indicated by elongated hypocotyls. rus1-1 and WT seedlings germinated in complete darkness showed little differences in their morphology (Fig. 2A), suggesting that the rus1-1 mutation does not affect skotomorphogenesis and that the nutritional composition of MS medium is unlikely to be responsible for the rus1-1 phenotypes observed in light. The 4 ND filters reduced the fluence rates at wavelengths in both the visible and UVB regions in a relatively even manner (Fig. 2C and Table 1).

Fig. 2.

Reduced fluence rates alleviate rus1-1 phenotypes, and limiting root exposure to light rescues rus1-1 phenotypes. (A) Relationship between fluence rates and rus1-1 phenotypes. Seven-day-old WT and rus1-1 seedlings were grown vertically on plates wrapped with neutral density filters to achieve various light fluence rates (4.73, 18.8, 39.2, and 47 μmol m⁻² s⁻¹ PAR). As controls, unwrapped (no filter, 71.2 μmol m⁻² s⁻¹ PAR) and black foil-wrapped (dark, 0) plates are included. (Scale bars: 1 cm.) (B) Correlation between fluence rates and the rus1-1 phenotype severity. Root lengths of both WT and rus1-1 seedlings grown under light conditions shown in A were measured and compared. Average percentages of rus1-1 root lengths against that of the WT were calculated and plotted against light fluence rates. n = 45. Error bars = SD. (C) Emission spectra (220–850 nm) of neutral density (ND) filters. ND filters reduced the fluence rates in both the visible light region (400–800 nm) and the UVB region (UV-B); see also Table 1. (D and E) rus1-1 phenotypes can be rescued by eliminating light exposure to roots. Seeds of WT and rus1-1 were plated on MS growth media with either a clear surface (D) or a surface covered by a layer of black aluminum foil (E). Close-up views for the media surface are shown as insets at the left corners in D and E. Holes (0.4-mm diameter) were generated on black foil to allow seeds to contact growth media directly (arrowheads, E). Images are of 7-day-old seedlings. Representative seedlings from each group are shown on the right. (Scale bars: 1 cm.)

Table 1.

Fluence rates reduced by ND filters

Filter condition	PAR (μmol m−2 s−1)	UVB (μmol m−2 s−1)
No filter	71.2	0.300
ND #298	47.8	0.117
ND #209	39.21	0.077
ND #210	18.8	0.055
ND #299	4.73	0.025

We hypothesized that the root is the site of light perception in rus1 mutants, because rus1-1 seedlings survived after being transferred to soil where the root is covered. To test this hypothesis, we germinated both WT and rus1-1 seeds on horizontal plates on MS growth media that had either a standard translucent surface or a black foil-covered surface with the hydrated seeds placed over pin holes in the foil (Fig. 2E Inset). On the translucent surface, rus1-1 showed growth and developmental arrests as described above (Fig. 2D). Compared with rus1-1 seedlings grown on vertically placed plates, the phenotypes of rus1-1 grown on horizontally placed plates were slightly weaker (Fig. 2D). Whereas limiting light exposure to roots had little effect in WT, the growth and development of rus1-1 seedlings was far better on black foil-covered MS media (Fig. 2E). These results suggest that the root is the location of light perception for the rus1 phenotypes.

UVB Is Responsible for rus1-1 Light Hypersensitivity.

To test whether rus1-1 phenotypes depend on any of the known photoreceptors (crytochromes, phototropins, and phytochromes), we crossed rus1-1 to a number of photoreceptor mutants, including cry1, cry2, cry1 cry2, phyA, phyB, phyA phyB, phot1, phot2, phot1 phot2, nph3. Analyses of crosses homozygous for rus1-1 and the various photoreceptor genes showed that none of these mutations suppressed the rus1-1 phenotype (data not shown). We next tested rus1-1 responses to various visible-light wavelengths. As shown in Fig. 3A, monochromatic blue, red, or far-red light had little effect on rus1-1 root growth.

Fig. 3.

rus1-1 is hypersensitive to UVB. (A) rus1-1 growth responses under blue, red, or far-red light. Representative WT and rus1-1 seedlings grown under the indicated monochromatic lights are shown. Light fluence rates were 15, 15, and 10 μmol m⁻² s⁻¹ for blue, red, and far-red light, respectively. (Scale bars: 1 cm.) (B) rus1-1 hypersensitivity is specifically related to UVB. Hydrated WT and rus1-1 seeds were exposed for 6 h to white light or white light filtered by transmission cutoff filters (WG280 and WG320) before being placed in dark. Root lengths of 7-day-old etiolated rus1-1 seedlings were calculated and compared with that of WT. n = 40. Error bar = SD. The UV transmission cutoff curves for the 2 filters are shown in the Inset. (C) Effect of UVB on WT and rus1-1 seedlings. WT and rus1-1 seedlings grown vertically in a dark growth chamber without (−UVB) or with (+UVB) supplementary UVB (0.8 μmol m⁻² s⁻¹ 1 h daily). (Scale bars: 1 cm.) (D) Reduced UVB fluence alleviates rus1-1 phenotypes. Combinations of various light sources were used to create light conditions with either reduced UVB fluence rate (0.05 μmol m⁻² s⁻¹, 280–320 nm) (−UVB) or unreduced UVB fluence rate (0.8 μmol m⁻² s⁻¹, 280–320 nm) (+UVB). Images of 7-day-old seedlings are shown. (Scale bars: 1 cm.) (E) Semiquantitative RT-PCR analysis of UVB-regulated genes in WT and rus1-2 in response to UVB treatment. Transcript levels for the 5 genes in 6-day-old seedlings treated with or without 2-hr UVB (1.2 μmol m⁻² s⁻¹) are shown. Constitutively expressed EF1α is used as a control.

rus1-1 light hypersensitivity occurs in very early seedling development [supporting information (SI) Fig. S1]. Exposure of the hydrated rus1-1 seeds to light (regular white fluorescence light, 80 μmol m⁻² s⁻¹ PAR) for as little as 6 h before placing them in the dark resulted in >50% root inhibition in etiolated rus1-1 seedlings (Fig. 3B). This observation provided a sensitive and specific way to assay rus1-1 light sensitivity. Experiments in which white light was replaced by various monochromatic light sources (red, green, blue, UVA, and UVB) indicated that UVB was responsible for the observed rus1-1 root inhibition (Fig. S2). Emission spectra for the various light sources are shown in Fig. S3. To confirm the UVB specificity, transmission cutoff filters (WG280 and WG320) (Fig. S4) were used to test rus1-1 responses under conditions with or without UVB filtered out. As shown in Fig. 3B, rus1-1 demonstrated the same hypersensitivity either under white light or under white light plus the WG280 filter. In contrast, rus1-1 hypersensitivity is diminished under white light plus the WG320 filter, a condition where UVB is cut off (Fig. 3B Inset).

UVB hypersensitivity was also tested directly on etiolated or light-grown rus1-1 seedlings. As shown in Fig. 3C, exposure of etiolated seedlings of rus1-1 to UVB (0.8 μmol m⁻² s⁻¹, 1 h daily from germination, see Fig. S3 for emission spectrum of the UVB source) resulted in >80% root growth inhibition as compared with the WT. The UVB fluence rate in standard Arabidopsis growth-chamber light is ≈0.8 μmol m⁻² s⁻¹. We modified the white light source in the chamber to give reduced UVB (0.05 μmol m⁻² s⁻¹). As shown in Fig. 3D, under this light condition, rus1-1 root growth from light-grown seedlings reached as much as 50% that of WT (Fig. 3D Left, −UVB). When LF UVB was added to this light condition (fluence rate = 0.8 μmol m⁻² s⁻¹), rus1-1 root growth was severely suppressed (Fig. 3D, +UVB). rus1-1 growth was affected even under the lowest tested UVB fluence rates (Fig. 2 A–C and Table 1). A strong correlation between UVB fluence rates (calculated in Table 1) and rus1-1 root inhibition was established (Fig. S5). A hypothetical UVB fluence-rate point where there would be zero root inhibition in rus1-1 was determined to be ≈2.27 nmol m⁻² s⁻¹ (Fig. S5). Our analyses demonstrate that the mutant, specifically the mutant root, is hypersensitive to UVB light, hence the name root UVB-sensitive1 (rus1).

In the rus1-2 background, we analyzed the expression of the genes CHS (At5g13930), HY5 (At5g11260), At1g19020, At4g15480, and At5g05410 (19), all of which are known to be transcriptionally up-regulated by UVB light over time. As in WT, all analyzed UVB-regulated marker genes were up-regulated by HF UVB (1.2 μmol m⁻² s⁻¹, 2 h) in whole rus1-2 seedlings (Fig. 3E). Additionally, none of these genes (with the exception of CHS) was up-regulated in rus1-2 under standard growth-chamber light conditions. These results suggest that the expression of these known UVB-induced genes remains similar in WT and rus1-2.

Mapping and Cloning of RUS1.

The rus1-1 allele was created by T-DNA insertional mutagenesis but does not have a T-DNA associated with it. We mapped rus1-1 using simple sequence-length polymorphism (SSLP) and cleaved amplified polymorphic sequence (CAPS) markers (33, 34). No recombinants were observed with the F16L2-HaeIII marker after analyses of 1,000 F2 rus1-1 homozygotes (Fig. 4A). Genomic fragments within this region were used to transform rus1-1 (35). A 9-kb fragment containing At3g45890 complemented rus1-1 completely, and sequencing of this gene in the rus1-1 background revealed that a foreign fragment of 22 bp is inserted at the −31 position and that a 53-bp fragment (from −26 to +27) is deleted (Fig. 4 B and C). The deleted region contains the promoter-proximal region (26 bp), the 5′-UTR region (16 bp), and the first 4 codons (Fig. 4C). To test further whether this mutation is responsible for the rus1-1 phenotypes, we fused the At3g45890 gene with its promoter to the GFP coding sequence and used it to complement rus1-1. No differences were observed for root elongation and seedling development between transgenic rus1-1 plants carrying At3g45890::At3g45890-GFP and WT under both light and UVB-supplemented dark conditions (Fig. 4D). We conclude that RUS1 is At3g45890.

Fig. 4.

Map-based cloning and complementation of rus1-1. (A) The rus1-1 mutation mapped to a 100-kb region on T16L2 on chromosome 3. Values are the number of recombinant chromosomes divided by the total number of chromosomes tested. Markers in the adjacent BACs units are indicated. (B) The gene structure of RUS1 (At3g45890) and the nature of the 3 rus1 alleles. Shaded boxes indicate exons. Position 1 is the transcriptional initiation site. Positions relative to the transcriptional initiation site in genomic and cDNA are 2631 and 2054, respectively. The insertions and deletions are represented as blue and red boxes, respectively, on the genomic fragment. The position of the nonsense rus1-3 mutation is indicated as a hexagon. (C) Molecular nature of the 3 rus1 alleles. Numbers of −45, 2205, and 2178 refer to the genomic DNA positions relative to the transcriptional initiation site. Mutated nucleotide sequences of the 3 rus1 alleles are compared with the corresponding WT sequences. Red bars indicate deleted sequences; black triangles indicate inserted sequences. The rus1-3 G-to-A nonsense mutation is underlined. (D) Complementation of rus1-1 by RUS1::RUS1-GFP. Seven-day-old WT, rus1-1, and transgenic RUS1-GFP gene (Compl) plants grown in normal growth chamber (Light) or in the dark supplemented by UVB (Dark + UVB) are shown. (Scale bars: 1 cm.) (E) RUS1 belongs to a large family of proteins with DUF647 domains commonly found in multicellular organisms. Amino acid sequence alignment of Arabidopsis RUS1 and RUS1-like proteins from various nonplant eukaryotes. Identical and conserved amino acids are highlighted in black and gray, respectively. Dashes indicate gaps in the sequence to optimize the alignment. (F) RUS1 is expressed in roots. Confocal microscopic images of GFP fluorescence of the RUS1-GFP fusion protein in complemented rus1-1 roots from 7-day-old etiolated seedling. (Left) Primary root. (Center) Emerging lateral root. (Right) Lateral root. (Scale bars: 10 μm.)

Two additional rus1 alleles were obtained by screening for the rus1 phenotype. rus1-2, generated by fast-neutron mutagenesis in the Col background, carries a 49-bp insertion at genomic position 2232 followed by 6-bp deletion (Fig. 4 B and C). rus1-3, generated by EMS mutagenesis in the Landsberg erecta (Ler) background, carries a single nucleotide (G-to-A) mutation at genomic position 2192 resulting in a nonsense mutation (TGG-to-TGA) (Fig. 4 B and C). Both rus1-2 and rus1-3 are recessive, and their phenotypes are identical to that of rus1-1 (data not shown).

RUS1 Encodes a Protein Containing DUF647 and Is Expressed in Roots.

The RUS1 gene encodes a 608-aa protein (66.4 kDa). Arabidopsis has 5 additional RUS1-like genes based on their sequence homologies to RUS1. Sequence similarities among the 6 proteins range from 31% to 44%. All 6 proteins contain a Domain of Unknown Function647 (DUF647, IPR006968) found in most eukaryotes that have sequence data available. Only the RUS1-like gene At5g49820 (EMB1879) was previously isolated in a screen for defective embryo development, and no defined biochemical functions are currently known for any of the RUS1-like genes. Fig. 4E shows the partial RUS1 amino acid sequence alignments with DUF-containing proteins from a number of nonplant eukaryotes. Within the highly conserved region (243-aa residues), the identities and the similarities between RUS1 and mouse NP663563 protein, for example, are 36% and 60%, respectively. A mouse full-length cDNA encoding this protein was fused to the RUS1 promoter, but the hybrid construct was not able to complement rus1-1 (data not shown).

Complemented transgenic rus1-1 plants carrying a RUS1::RUS1-GFP gene were used to analyze RUS1 localization in planta. Confocal microscopic analyses localized GFP fluorescence to the roots. GFP signals are found near the root apical meristem and in the cortex region of the root elongation zone and not in extreme root apical meristem or root cap (Fig. 4F). High levels of RUS1-GFP appear in emerging embryonic roots. RUS1-GFP is also highly expressed in lateral roots and emerging lateral roots (Fig. 4F).

Discussion

Throughout their lives, plants remain physiologically flexible and ready to respond and adapt to new light conditions. How plant tissues perceive UVA/blue (340–450 nm) and red/far-red (680–780 nm) light to regulate plant growth and development has been well documented, and numerous receptor proteins have been identified. Physiological data suggest the presence of a UVB-perception pathway in plants, but the molecular nature of the UVB-specific receptor(s) remains elusive. Roots are critically important for the success of land plants, yet little is known about whether and how they sense light. We postulate here the discovery of a UVB-sensing mechanism in roots that is involved in Arabidopsis morphogenesis and early seedling development.

The strong correlation between light irradiance and rus1 root lengths suggests that there is a fluence-dependent mechanism that affects rus1 root growth. We conclude that rus1 is hypersensitive to light and that RUS1 may function in postgermination seedling development. A number of light-sensitive mutants have been identified before, but none of them shows sensitivities to light at such a low fluence rate (24, 36, 37).

How photons are sensed and perceived by roots is currently unclear. Genes encoding photoreceptors such as phytochromes and phototropins are highly expressed in roots, but our genetic crosses showed that mutations of these photoreceptors were unable to suppress the rus1 phenotypes. Light-quality experiments with various combinations of filters and monochromatic LED lights specifically pinpointed UVB as the responsible source for the rus1 light hypersensitivity. White-fluorescence light tubes are considered to be a light source that produces little UVB. Nevertheless, that small amount of UVB (0.3 μmol m⁻² s⁻¹) is enough to cause the severe effects seen in rus1. Our calculations suggest that rus1 is able to respond to extremely low UVB, as low as 2.27 nmol m⁻² s⁻¹.

Many biological molecules can absorb and react with HF UVB, causing damage that cells interpret as signals for cellular damage and stress. Well-documented UVB responses include induced synthesis of pigments that absorb UV photons or free radicals and formation of damaged DNA products [cyclobutane pyrimidine dimers (CPDs); pyrimidine (–4) primidone photoproducts] that trigger DNA repair and apoptosis (11, 13). In animals, the amino acid tryptophan has been shown to react with UVB to form 6-formylindolo[3,2-b]carbazole (FICZ). Upon binding to FICZ, an arylhydrocarbon receptor travels to the nucleus to regulate the expression of genes associated with UVB radiation toxicity responses (9).

Little is known about signaling events that underlie VLF UVB-provoked physiological response. Our studies genetically demonstrated that roots are able to respond to VLF UVB light. The extremely low levels of UVB fluence rates that induce responses in rus1 may suggest a quantum-dependent and receptor-mediated cell signaling process that regulates growth and development. Known photoreceptors (phytochromes, crytochromes, and phototropins) all consist of proteins containing light-absorbing chromophores. It is not clear how UVB is absorbed by the unidentified UVB receptor(s), but the low-quantum UVB sensitivities exemplified in rus1 suggest the presence of specific chromophore(s) for UVB receptor(s). It also remains to be determined whether and how the RUS1-mediated signaling pathway is related to UVR8, a known player in LF UVB signaling (8, 10).

The physiological relationship between root UVB perception and seedling development is currently unknown. Although normally functioning in a dark environment, roots can be exposed to various levels of light for periods of time. Roots emerging from germinating seeds that are rested on the soil surface are usually exposed to light until seedlings are established in soil. Roots can also be exposed to light due to rain washes, geological movements, and herbivore activities. Our findings suggest that VLF UVB can be perceived by roots to regulate early seedling morphogenesis and development. Exactly how VLF UVB influences early seedling development is unclear at this point. Although it is clear that UVB specifically regulates certain elements of morphogenesis such as hypocotyl elongation, root elongation, and cotyledon opening, it appears that UVB is not absolutely required for WT seedling development because WT Arabidopsis seedlings develop normally without UVB. This could be because some of the UVB-regulated physiological processes are also regulated by phytochromes and cryptochromes. Similarly, WT plants appear to grow normally under visible-light spectra that lack UVA, although it is well established that UVA plays a role in photomorphogenesis.

Database searches identified 1 prominent hypothetical domain called DUF647 (IPR006968) in RUS1. DUF647-containing proteins make up a large family with members commonly found in eukaryotic organisms. Our studies provide a clue for functional roles of DUF647-containing proteins. It is yet to be determined whether DUF647-containing proteins in other organisms function in a similar way to RUS1. Further structure–function studies on how RUS1 functions in Arabidopsis UVB responses will provide crucial biochemical insights into the DUF647 domain functions. It has been difficult to identify the UVB photoreceptor mainly because of the lack of genetically screenable UVB-specific phenotypes. The striking phenotypes and extreme UVB sensitivities of rus1 will provide a feasible platform to identify genetic suppressors that could be candidates for UVB-specific receptor(s) and other signaling components in the UVB pathway.

Materials and Methods

Plant Material and Growth Conditions.

Arabidopsis plants were grown as described (38). For Petri dish-grown seedlings, surface-sterilized seeds were either cold-treated at 4 °C for at least 48 h or without any cold treatment before being plated on MS growth medium in square plates that were held vertical in a growth chamber (Model CU36L5; Percival) except where specifically noted. For soil-grown plants, seeds with or without the 4 °C cold treatment were directly sowed in pots containing sterilized soil medium and kept in a growth chamber (Model AR-66L; Percival) with an 16-h/8-h light/dark cycle at a constant temperature (22 °C). Mutant seeds for cry1, cry2, cry1cry2, nph3, and npt2 were obtained from the Arabidopsis Biological Resource Center (ABRC) (Ohio State University, Columbus, OH). Seeds for phyA, phyB, and phyAphyB were generously provided by Peter Quail (University of California, Berkeley, CA).

Mutant Screening and Crosses.

The rus1-1 allele was identified in a SALK T-DNA insertional mutagenesis pool of Columbia glabrous seeds (cat. no. CS63650; ABRC). The rus1-2 allele was obtained from a pool of mutants generated by fast neutron mutagenesis of WT Col-0 seeds (cat. no. M2F-02–01; Lehle Seeds). The rus1-3 allele was obtained from a pool of mutants generated by EMS mutagenesis of Landsberg-0 erecta seeds (cat. no. M2E-04–06; Lehle Seeds). Homozygous double or triple mutants from photoreceptor mutant crosses were identified by analyzing phenotypes in segregating progeny and confirmed by PCR markers.

Light Sources and UVB Treatments.

Various filters and light sources used to generate light of specific qualities and quantities are listed in SI Text. Light-fluence measurements and spectral analyses were carried out by using a Wideband Spectroradiometer (Model RPS900-R) and its software (International Light).

Mapping and Complementation.

A mapping population was created by selecting F2 (Col ×Ler) individuals with rus1-1 phenotypes. SSLP and CAPS markers were developed and used to map rus1-1 to near the CIW4 marker (chr. 3 at 18,901,818 bp). Additional markers were used to fine-map rus1-1 to a 50-kb region. Complementation was carried out by transforming rus1-1 with a Col-0 genomic library constructed in the binary vector pBIC20 (gift from C. Somerville, Carnegie Institution for Science).

Confocal Microscopy and RT-PCR Analyses.

rus1-1 was complemented by the chimeric At3g45890(RUS1)-GFP gene driven by RUS1 promoter. GFP Confocal and RT-PCR analyses were carried out as previously described (38). RT-PCR analyses were carried as previously described (49). Primer sequences are listed in SI Text.