Cloning And Functional Analysis Of OsCYP2(Cyclophilin2) In Regulation Of Lateral Root Development

Lateral roots (LR) are major components of rice root system. The development of LR is a postembryonic event. The morphological and physiological description of LR development in plants have been extensively reported, little is known about molecular mechanisms of lateral root development in rice. Through screening for mutants with defect of LR, we isolated three allelic mutants defective in lateral roots Irl1 from an EMS mutated rice library (Kasalath, indica) and Irl2 and Irl3 from an T-DNA inserted rice library(Zhonghua-ll, japonica). The phenotypic, physiological and genetic characterizations of the three allelic mutants were investigated. The gene for OsCYP2 for the mutation was cloned using map-based cloning strategy. The results are summarized as follows:1. Function-loss of OsCYP2 severely disrupts the lateral root formation and reduces root response to gravity. Significant differences in shoot length, primary root length, adventitious root number, average length of adventitious roots, lateral root number and average length of lateral roots were found between the mutants and wild-type seedlings. Analysis of anther and pollen staining and morphology at mature stage showed that OsCYP2 mutation greatly affects plant reproduction, reduces pollen activity, seed fertility and one thousand grain weight.2. Compared with wild-type plants, the Oscyp2 mutants showed similar response to auxin transport inhibitor NPA. Exogenous application of 0.1μM NAA can not rescue the defect in the lateral root of the mutants, but the average lateral root length of Oscyp2-1 is induced by 0.1μM NAA. IAA concentration analysis showed that the ratio of IAA concentration of leaf and root in Oscyp2-1 and wild-type seedlings 7 days after germination is not significantly different (P>0.05). These results show that OsCYP2 mutation disrupts auxin-mediated signaling pathway required for LR formation.3. Genetic analysis indicated that allelic mutants Oscyp2-1, Oscyp2-2, Oscyp2-3 possess a recessive mutation in a single nuclear locus. The OsCYP2 gene is located on the short arm of chromosome 2, flanked by the dCAPS marker dEcoRI and SNP marker PmaCI, covered by two BAC clones of OSJNBb0088N06 and OJ1020_C02. Sequecing results show that each of the three known alleles contains a unique mutation of substitution G to C, C to A and deletion of 59 bp in the coding sequence of OsCYP1, respectively.4. Alleles OsCYP2-1 and OsCYP2-2 contain single nucleotide point mutations that generate an amino acid change(G₇₂A) and a stop codon (C₂₅Stop), respectively, while OsCYP2-3 has a frame-shift mutation (A₁₅E_Δ15-35. Homology analysis showed that both rice cyclophilins (OsCYP2 and OsCYP1) and those from other plants have an extra four amino acids (position 51-54) that are absent from other eukaryotic cyclophilins. Due to the high degree of conservation of these four amino acids, they probably compose a domain that is responsible for a function unique to plants. 5.Expression in several tissues by real time RT-PCR analysis suggests that OsCYP2 gene is constitutively expressed in all tested tissues, the expression level of OsCYP2 gene in Oscyp2-1 is similar to wild-type Kasalath in root, the junction of root and stem, stem, leaf and flower. RT-PCR and QRT-PCR analysis show that OsCYP2 mutation affects the expression of partial auxin-responsive genes, such as OsPIN10a and OsIAA20, implying that OsCYP2 gene probably regulates lateral root development in an auxin-responsive signaling pathway.