LATERAL ROOTLESS2, a Cyclophilin Protein, Regulates Lateral Root Initiation and Auxin Signaling Pathway in Rice

Dear Editor, Cyclophilins (CYP) are a class of highly conserved peptidyl–prolyl cis–trans isomerases (PPIases) that play important roles in various biological processes in eukaryotes (reviewed in Romano et al., 2004Romano P.G. Horton P. Gray J.E The Arabidopsis cyclophilin gene family.Plant Physiol. 2004; 134: 1268-1282Crossref PubMed Scopus (189) Google Scholar). In higher plants, a conserved single domain cyclophilin has been identified as a novel component of the auxin signaling pathway by analyzing the tomato diageotropica (dgt) mutant (Ivanchenko et al., 2006Ivanchenko M.G. Coffeen W.C. Lomax T.L. Dubrovsky J.G Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle.Plant J. 2006; 46: 436-447Crossref PubMed Scopus (61) Google Scholar; Oh et al., 2006Oh K. Ivanchenko M.G. White T.J. Lomax T.L The diageotropica gene of tomato encodes a cyclophilin: a novel player in auxin signaling.Planta. 2006; 224: 133-144Crossref PubMed Scopus (89) Google Scholar). The dgt mutant displays a lateral-rootless and auxin-resistant phenotype (Ivanchenko et al., 2006Ivanchenko M.G. Coffeen W.C. Lomax T.L. Dubrovsky J.G Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle.Plant J. 2006; 46: 436-447Crossref PubMed Scopus (61) Google Scholar). Further studies revealed that mutations in the DGT-like genes of Physcomitrella patens also exhibited an auxin-resistant phenotype, suggesting a conserved role of DGT-like proteins in auxin signaling. Moreover, mutation in PpDGT gene suppresses the auxin hypersensitive phenotype conferred by the overexpression of the auxin receptor gene PpAFB2, indicating that PpDGT acts downstream of PpAFB2 (Lavy et al., 2012Lavy M. Prigge M.J. Tigyi K. Estelle M The cyclophilin DIAGEOTROPICA has a conserved role in auxin signaling.Development. 2012; 139: 1115-1124Crossref PubMed Scopus (40) Google Scholar). Mutations in the rice OsCYP2 gene cause a similar auxin-resistant phenotype and defects in lateral root development (Kang et al., 2013Kang B. Zhang Z. Wang L. Zheng L. Mao W. Li M. Wu Y. Wu P. Mo X OsCYP2, a chaperone involved in AUX/IAA degradation, plays crucial roles in rice lateral root initiation.Plant J. 2013; https://doi.org/10.1111/tpj.12106Crossref Scopus (62) Google Scholar). Moreover, OsCYP2 protein physically interacts with OsSGT1 and the degradation of AUX/IAA protein with auxin treatment is reduced in oscyp2, suggestive of a role of OsCYP2 in the degradation of AUX/IAA proteins (Kang et al., 2013Kang B. Zhang Z. Wang L. Zheng L. Mao W. Li M. Wu Y. Wu P. Mo X OsCYP2, a chaperone involved in AUX/IAA degradation, plays crucial roles in rice lateral root initiation.Plant J. 2013; https://doi.org/10.1111/tpj.12106Crossref Scopus (62) Google Scholar). We have previously identified a rice mutant with defective lateral development, designated as lrt2 (for lateral rootless) in a genetic screen for auxin-resistant mutants (Wang et al., 2006Wang H. Taketa S. Miyao A. Hirochika H. Ichii M Isolation of a novel lateral-rootless mutant in rice (Oryza sativa L.) with reduced sensitivity to auxin.Plant Sci. 2006; 170: 70-77Crossref Scopus (36) Google Scholar) (see also Supplemental Figures 1 and 2). To characterize at which stage the lateral root development was impaired, we performed a histological analysis of the wild-type (Nipponbare; NPB) and lrt2 mutant roots. Through longitudinal section, a series of lateral root primordia (LRPs), including the stage I LRP, was observed in wild-type roots (Supplemental Figure 3A–3E). However, we found neither LRP nor LRP-like cells present in lrt2 roots (Supplemental Figure 3F). The cross-sections also showed that no LRPs were found in lrt2 roots (Supplemental Figure 3I and 3J). These results indicate that there is no patterned cell division for lateral root initiation in lrt2. Moreover, the radial cell arrangement in lrt2 roots is disrupted. Generally, in wild-type, there would be a large vessel surrounded by seven little vessels at the center of basal elongation zone and two to three large vessels at the upper elongation zone (Supplemental Figure 3G and 3H). However, cells in lrt2 stele were disorganized. Instead of the regularly arranged vessel pattern, the vessel cells in lrt2 are abutted in the central cylinder (Supplemental Figure 3I). In addition, the number of cortex cell layers in lrt2 was less than that in wild-type, which might lead to the slender-root phenotype in the mutant (Supplemental Figure 3G–3J). Map-based cloning revealed a 50-bp deletion in a candidate gene OsCYP2 (LOC_Os02g02890, Supplemental Figure 4A), which encodes a protein with 172 amino acid residues and contains a single cyclophilin-like domain (Figure 1A). To further confirm the identity of LRT2, a genetic complementation was performed. An LRT2–His–FLAG fusion gene under the control of the LRT2 native promoter was transformed into the lrt2 mutant (Supplemental Figure 4B). In all the analyzed six transgenic lines, the LRT2–His–FLAG transgene fully rescued the lrt2 mutant phenotype (Figure 1, Figure 1). We also generated an anti-LRT2 polyclonal antibody in mouse. The anti-LRT2 antibody specifically recognized both the endogenous LRT2 and the LRT2–His–FLAG fusion proteins, but detected no signal in the lrt2 mutant (Figure 1D). Together, these data suggest that the lrt2 phenotype was caused by a mutation in the OsCYP2 gene. A protein sequence alignment revealed that LRT2/OsCYP2 shared considerable similarity with the cyclophilin proteins of other plant species (Supplemental Figure 5A and 5B), suggesting that LRT2/OsCYP2 may play conserved roles in plant growth and development. In the rice genome, 27 cyclophilin-like genes were annotated (Ahn et al., 2010Ahn J.C. Kim D.W. You Y.N. Seok M.S. Park J.M. Hwang H. Kim B.G. Luan S. Park H.S. Cho H.S Classification of rice (Oryza sativa L. Japonica nipponbare) immunophilins (FKBPs, CYPs) and expression patterns under water stress.BMC Plant Biol. 2010; 10: 253Crossref PubMed Scopus (69) Google Scholar), and the other cyclophilin-like proteins share relatively lower sequence similarities with LRT2. Cyclophilins have been reported to be localized in the cytosol (Ahn et al., 2010Ahn J.C. Kim D.W. You Y.N. Seok M.S. Park J.M. Hwang H. Kim B.G. Luan S. Park H.S. Cho H.S Classification of rice (Oryza sativa L. Japonica nipponbare) immunophilins (FKBPs, CYPs) and expression patterns under water stress.BMC Plant Biol. 2010; 10: 253Crossref PubMed Scopus (69) Google Scholar). LRT2 protein has no distinctive signal peptide for organelle localization when predicted using the SignalP 4.0 Server (Petersen et al., 2011Petersen T.N. Brunak S. von Heijne G. Nielsen H SignalP 4.0: discriminating signal peptides from transmembrane regions.Nat. Methods. 2011; 8: 785-786Crossref PubMed Scopus (7092) Google Scholar). To characterize the subcellular localization of LRT2, we expressed the LRT2–GFP fusion protein in tobacco leaves driven by the LRT2 promoter. The GFP fluorescence signals were likely accumulated in cytosol and nucleus (Figure 1E). To further characterize the subcellular localization of LRT2, we also detected the LRT2 accumulation in various subcellular fractions. Immunoblot with anti-LRT2 antibody showed that LRT2 protein was accumulated in all fractions investigated, including the cytosol (membrane-depleted and nuclei-depleted fractions), total membrane fraction, and the nucleus (Figure 1F and 1G). These results suggest that LRT2/OsCYP2 is localized at the cytosol and the nuclei as previously reported (Ahn et al., 2010Ahn J.C. Kim D.W. You Y.N. Seok M.S. Park J.M. Hwang H. Kim B.G. Luan S. Park H.S. Cho H.S Classification of rice (Oryza sativa L. Japonica nipponbare) immunophilins (FKBPs, CYPs) and expression patterns under water stress.BMC Plant Biol. 2010; 10: 253Crossref PubMed Scopus (69) Google Scholar; Kang et al., 2013Kang B. Zhang Z. Wang L. Zheng L. Mao W. Li M. Wu Y. Wu P. Mo X OsCYP2, a chaperone involved in AUX/IAA degradation, plays crucial roles in rice lateral root initiation.Plant J. 2013; https://doi.org/10.1111/tpj.12106Crossref Scopus (62) Google Scholar). A qRT–PCR (quantitative reverse transcription PCR) analysis showed that LRT2 was expressed in all examined tissues/organs, including roots, coleoptiles, and leaves. In particular, a substantially higher expression level of LRT2 was observed in the basal internodes (Supplemental Figure 6A). To further characterize its expression pattern, both the in situ RNA hybridization and staining of the transgenic plants carrying the GUS reporter gene driven by the putative LRT2 promoter were performed. The in situ hybridization results revealed that the LRT2 gene was mainly expressed in LRPs throughout its initiation and development (Figure 1H). Similarly, the GUS signals were predominantly detected in LRPs (Supplemental Figure 6E, 6F, and 6H). Moreover, the GUS signals were also observed intensively in stele and basal meristem but rarely near the quiescent center (QC) at the root tip (Supplemental Figure 6C, 6D, and 6G). The expression pattern of LRT2/OsCYP2 is consistent with its regulatory role in lateral root initiation and radial cell arrangement. The lrt2 mutant showed reduced sensitivity to auxin in a root elongation assay, and the lateral-rootless phenotype of lrt2 could not be rescued by auxin (Supplemental Figure 2). To further analyze the impaired auxin response in lrt2, DR5::GUS, a marker for monitoring the auxin level (Ulmasov et al., 1997Ulmasov T. Murfett J. Hagen G. Guilfoyle T.J Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.Plant Cell. 1997; 9: 1963-1971Crossref PubMed Scopus (1589) Google Scholar), was introduced into the lrt2 mutant. The expression of DR5::GUS was examined in the crown roots of 7-day-old seedlings of wild-type and lrt2 mutant. The expression of DR5::GUS was reduced more in lrt2 than that in the wild-type (Supplemental Figure 7). Moreover, we also investigated the expression of several auxin-responsive genes by qRT–PCR, including OsIAA20, OsIAA23, OsIAA31, and OsARF16. Compared with that in wild-type, the auxin-induced expression level of these genes was reduced in lrt2 roots (Figure 1I), suggesting that the auxin signaling pathway is impaired in lrt2. Previous studies have revealed that the auxin influx carrier mediated auxin transport and the NPA-dependent auxin transport in lrt2 remained largely unaltered (Wang et al., 2006Wang H. Taketa S. Miyao A. Hirochika H. Ichii M Isolation of a novel lateral-rootless mutant in rice (Oryza sativa L.) with reduced sensitivity to auxin.Plant Sci. 2006; 170: 70-77Crossref Scopus (36) Google Scholar). To further assess whether the reduced auxin sensitivity in lrt2 is caused by defects in the auxin efflux carrier-dependent polar auxin transport, we analyzed PIN1-like proteins by immunostaining using an anti-AtPIN1 antibody. In wild-type, the signals of PIN1-like proteins were basally localized and mainly accumulated in the steles. Unexpectedly, the polar localization and distribution of PIN1-like protein in lrt2 was similar to that in wild-type (Supplemental Figure 8A). Moreover, the transcriptional level of OsPIN1a and OsPIN1b in lrt2 was unaltered compared with that in wild-type (Supplemental Figure 8B). These results suggest that the PIN1-like protein-dependent polar auxin transport is not altered in lrt2. Our results provide evidence for an important role of LRT2/OsCYP2 in auxin signaling. During the course of this study, Kang et al., 2013Kang B. Zhang Z. Wang L. Zheng L. Mao W. Li M. Wu Y. Wu P. Mo X OsCYP2, a chaperone involved in AUX/IAA degradation, plays crucial roles in rice lateral root initiation.Plant J. 2013; https://doi.org/10.1111/tpj.12106Crossref Scopus (62) Google Scholar independently characterized two additional lrt2/oscyp2 mutant alleles. Along with previous studies on cyclophilins in tomato and Physcomitrella patens (Ivanchenko et al., 2006Ivanchenko M.G. Coffeen W.C. Lomax T.L. Dubrovsky J.G Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle.Plant J. 2006; 46: 436-447Crossref PubMed Scopus (61) Google Scholar; Lavy et al., 2012Lavy M. Prigge M.J. Tigyi K. Estelle M The cyclophilin DIAGEOTROPICA has a conserved role in auxin signaling.Development. 2012; 139: 1115-1124Crossref PubMed Scopus (40) Google Scholar), these studies reveal a highly conserved regulatory mechanism of auxin signaling mediated by cyclophilins in dicots and monocots as well as in lower plants. It will be of great interest to determine the precise biochemical nature and the direct targets of the highly conserved peptidyl–prolyl cis–trans isomerases in auxin signaling. Supplementary Data are available at Molecular Plant Online. This work was supported by grants from the National Natural Science Foundation of China (No. 31071385), the Joint Research Program between the Zhejiang Academy of Agricultural Sciences and the Chinese Academy of Sciences, the Fundamental Program of Zhejiang Academy of Agricultural Sciences, the Zhejiang Provincial Natural Science Foundation of China (No. Z3110509), and the grant from the Ministry of Agriculture of China (No. 2011ZX08010002). No conflict of interest declared.