A Lotus basic leucine zipper protein with a RING-finger motif negatively regulates the developmental program of nodulation

Abstract

The developmental program of nodulation is regulated systemically in leguminous host species. A mutant astray (Ljsym77) in Lotus japonicus has lost some sort of its ability to regulate this symtem, and shows enhanced and early nodulation. In the absence of rhizobia, this mutant exhibits characteristics associated with defects in light and gravity responses. These nonsymbiotic phenotypes of astray are very similar to those observed in photomorphogenic Arabidopsis mutant hy5. Based on this evidence, we predicted that astray might contain a mutation in the HY5 homologue of L. japonicus. The homologue, named LjBzf, encodes a basic leucine zipper protein in the C-terminal half that shows the highest level of identity with HY5 of all Arabidopsis proteins. It also encodes legume-characteristic combination of motifs, including a RING-finger motif and an acidic region in the N-terminal half. The astray phenotypes were cosegregated with LjBzf, and the failure to splice the intron was detected. Nonsymbiotic and symbiotic phenotypes of astray were complemented by introduction of CaMV35SLjBzf. It is noteworthy that although Arabidopsis hy5 showed an enhancement of lateral root initiation, Lotus astray showed an enhancement of nodule initiation but not of lateral root initiation. Legume-characteristic combination of motifs of ASTRAY may play specific roles in the regulation of nodule development.

Fig 1.

(A) Sequence of LjBzf cDNA and the predicted protein sequence. Cysteine residues in the RING-finger domain are encircled, and the acidic region is enclosed in a gray box. Serine, the predicted CKII phosphorylation site, is underlined. The basic region and leucine residues in the bZIP domain are indicated in white type and boxed, respectively. (B) Comparison of the primary structures of HY5, LjBZF, STF1, and VFBZIPZF. The RING-finger consensus sequence is highlighted and cysteine repeats are indicated in red type.

Fig 2.

(A) Genomic structure of LjBzf. Exons are enclosed in boxes, and introns are in black lines. LjBzf comprises seven exons and six introns. The nucleotide sequence in the vicinity of the mutation site is presented. Alteration in the astray mutant is indicated in gray type. (B) Segregation analysis of the genomic DNA in the F₂ population. M shows size markers. (C) RT-PCR analysis. Primers expected to amplify an 859-bp fragment within the LjBzf ORF were used.

Fig 3.

(A) Complementation of the astray mutant with 35S:LjBzf. Plants used were the wild type, the astray mutant, and the astray mutant carrying 35S:LjBzf (from left to right). Plants were incubated on agar plates 2 weeks after inoculation. Grids were placed at 1-cm intervals. (B) Comparison of nodule development. Plant roots are the wild type, the astray mutant, and the astray mutant carrying 35S:LjBzf (from left to right). Pictures were taken 5 days after inoculation. Arrows indicate nodule primordia. (Bar = 1 mm.)

Fig 4.

Accumulation of ASTRAY (LjBzf) transcripts. LjUbiquitin was used as internal control. Lanes 1–7: leaves, cotyledons, stems, hypocotyls, uninfected roots, infected roots, and 18-day nodules, respectively.

Fig 5.

A plausible role of ASTRAY in comparison with HAR1 and HY5. HY5 is involved in repression of lateral root development. HAR1 represses nodule and lateral root development. ASTRAY acts as a negative regulator of nodule development.