Conservation of lotus and Arabidopsis basic helix-loop-helix proteins reveals new players in root hair development

Abstract

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmental processes, relatively few have been functionally characterized. Here, we describe the map-based cloning of the Lotus japonicus ROOTHAIRLESS1 (LjRHL1) locus. Deleterious mutations in this locus prevent root hair development, which also aborts root hair-dependent colonization of the host root by nitrogen-fixing bacteria. We show that the LjRHL1 gene encodes a presumed bHLH transcription factor that functions in a nonredundant manner to control root hair development in L. japonicus. Homology search and cross-species complementation experiments defined three members of the Arabidopsis (Arabidopsis thaliana) bHLH protein family, At2g24260, At4g30980, and At5g58010, as functionally equivalent to LjRHL1. Curiously, At2g24260 and At4g30980 mRNA species accumulate independently from the known positive regulators of root hair cell fate, while all three genes act in a partially redundant manner to regulate root hair development in Arabidopsis.

Figure 1.

Root hair phenotypes of L. japonicus wild-type Gifu (left), Ljrhl1-1 mutant (center), and Ljrhl1-2 mutant (right) lines. Six-day-old seedlings are shown. Note the emerging difference in root length between wild-type and mutant plants.

Figure 2.

Map-based cloning of the LjRHL1 locus. A, A schematic of L. japonicus chromosome VI. B, Two overlapping transformation-competent artificial chromosome clones, TM1904 and BM2019, with genetic markers linked to the LjRHL1 locus are indicated. Number of recombinants versus total number of homozygous mutant individuals tested for a given marker is shown in parentheses. C, The region delimited by BK003 and BK001 genetic markers was predicted to contain four genes encoding a presumed zinc finger protein (ZFP), an alcohol dehydrogenase (ADH), unknown protein (UP), and a bHLH domain-containing protein (bHLH). D, Exon-intron structure of the LjRHL1 gene. The positions of genetic lesions in Ljrhl1-1 and Ljrhl1-2 mutant alleles are indicated.

Figure 3.

Alignment of the predicted LjRHL1 bHLH domain with a consensus sequence for plant bHLH (Heim et al., 2003). Amino acid residues that have been predicted to be important for contact with a nucleotide base (stars), DNA backbone (plus signs), and protein-protein interactions (circles) are indicated, as in Heim et al. (2003).

Figure 4.

Phylogenetic analysis of the LjRHL1 protein. Unrooted tree based on an amino acid alignment of full-length sequences of L. japonicus LjRHL1-1 and members of the Arabidopsis bHLH domain protein family belonging to group XI (top) and subgroups IIIf (bottom left) and VIIIc (bottom right). EGL3 and GL3 (subgroup IIIf) and AtRSL1 and AtRHD6 (subgroup VIIIc) were previously shown to be involved in specification of root epidermal cell fate in Arabidopsis (Bernhardt et al., 2003; Heim et al., 2003; Menand et al., 2007).

Figure 5.

Complementation of the Ljrhl1-2 root-hairless phenotype and localization of the LjRHL1 protein. The Ljrhl1-2 mutant was inoculated with the A. rhizogenes strain AR10, and the resulting transgenic hairy roots carrying either a LjRHL1 promoter-GFP control construct (A) or a chimeric GFP-LjRHL1 gene construct driven by the LjRHL1 promoter (B and C) are shown. The equivalent mature root regions, where fully developed root hairs are normally present, are shown in A and B. C shows the meristematic/elongation zone of the transgenic hairy root showing expression of GFP-LjRHL1 in the nuclei of epidermal cells.

Figure 6.

Cross-species complementation experiments. Under the control of the CaMV 35S promoter, the expression of At2g24260, At4g30980, and At5g5810, but not At1g03040 and At4g02590, complements the root-hairless phenotype of Ljrhl1-1 (top row) and Ljrhl1-2 (bottom row) allelic mutant lines in transgenic hairy roots.

Figure 7.

AtLRL1, AtLRL2, and AtLRL3 act redundantly to positively regulate root hair development in Arabidopsis. A, Roots of 5-d-old homozygous single, double, and triple mutant lines are shown in comparison with the wild type (Col-0). The corresponding genotypes are indicated. Note that for Atlrl1-1 × Atlrl2-1 and Atlrl1-2 × Atlrl2-1 crosses, the phenotype of only one representative progeny, AtLRL1 Atlrl1-2/Atlrl2-1 Atlrl2-1, is shown. Bars = 500 μm.

Figure 8.

RT-PCR expression analysis of At2g24260, At4g30980, At5g58010, and RHD6 in roots of Arabidopsis mutant and wild-type (Col-0) lines (see text for further details). Actin serves as a control. Note that lack of the RHD6 cDNA product in rhd6-3 rsl1-1 is caused by the T-DNA insertion in the RHD6 locus.