Deletion of the RS domain of RRC1 impairs phytochrome B signaling in Arabidopsis

Abstract

Phytochrome B (phyB), a major photoreceptor in plants, interacts with transcription factors to regulate gene expression and induce various light responses. Recently, we identified an SR-like splicing factor, RRC1 (reduced red-light responses in cry1cry2 background 1), as a novel component of phyB signaling in Arabidopsis. RRC1 has a C-terminal arginine/serine-rich (RS) domain that is generally important for the regulation of alternative splicing. Whereas rrc1 hypomorphic mutant alleles produce truncated RRC1 proteins that lack the C-terminal region, including the RS domain, and exhibit splicing defects and reduced phyB signaling, the rrc1-4 null allele additionally displays pleiotropic developmental abnormalities with more severe splicing defects. Here, we show that transgenic Arabidopsis plants that express truncated RRC1 lacking the RS domain in the rrc1-4 null allele background exhibited the same phenotype as the hypomorphic alleles. Hence, we conclude that deletion of the RS domain of RRC1 reduces phyB signaling, probably due to aberrant regulation of alternative splicing of target genes.

Figure 1. RRC1∆RS-FLAG rescues pleiotropic developmental defects in rrc1-4. (A) Schematic diagrams of the RRC1-FLAG and RRC1∆RS-FLAG proteins, which represent full-length RRC1 (946 amino acid) and truncated RRC1 lacking the RS domain (870 amino acid), respectively, with a C-terminal 3 × FLAG tag. Each construct was introduced into rrc1-4 and driven by the native RRC1 promoter. These plants are referred to as RRC1-FLAG and RRC1∆RS-FLAG plants, respectively. Red boxes, RNA recognition motif; green boxes, SWAP/Surp domain; blue boxes, RPR or ENTH/VHS domain; orange boxes, cwf21 domain; yellow boxes, RS domain; and black boxes, 3 × FLAG tag. (B) Immunoblot analysis of RRC1 in wild-type (WT), rrc1–3, RRC1-FLAG, and RRC1∆RS-FLAG plants. Each lane was loaded with 50 µg of total protein that was extracted from 2-week-old plants grown under continuous white light (cW; 40 µmol m⁻² s⁻¹). Anti-RRC1-a, anti-RRC1-b, and anti-FLAG were used as primary antibodies. The regions in the RRC1 polypeptide that are recognized by the anti-RRC1-a and anti-RRC1-b antibodies are underlined and labeled with letters a and b, respectively in panel (A). The Coomassie brilliant blue (CBB)-stained gel image is shown as a loading control. (C) Mature plants grown under cW for 3 weeks. Scale bar: 1 cm. (D) Opened siliques resulting from the self-fertilization of each line specified in the panel. Scale bar: 2 mm. (E) Alternative splicing pattern of SR34b. Total RNA was isolated from 2-week-old plants grown under cW and RT-PCR analysis was performed as previously described.TUB2 was used as an internal control.

Figure 2.RRC1∆RS-FLAG plants show elongated hypocotyls specifically under continuous red light. Wild-type (WT), rrc1-3, RRC1-FLAG, RRC1∆RS-FLAG, and phyB-9 seedlings were grown for 5 d under continuous red light (cR; 7.7 µmol m⁻² s⁻¹, top panel), continuous far-red light (cFR; 31 µmol m⁻² s⁻¹, middle panel), or in darkness (Dark, bottom panel), and hypocotyl lengths were measured. Data represent the mean ± SE (n = 30).