Accumulation of ENOD2-like transcripts in non-nodulating woody papilionoid legumes

Abstract

Japanese pagodatree (Styphnolobium japonicum [L.] Schott) and American yellowwood (Cladrastis kentukea Dum.-Cours.) Rudd are the first woody, non-nodulating papilionoid legumes shown to possess putative early nodulin 2 (ENOD2) genes. ENOD2 cDNAs from Japanese pagodatree (807 bp) and American yellowwood (735 bp) have 75% to 79% sequence identity to ENOD2 sequences and encode deduced proteins that possess conserved ENOD2 pentapeptides (PPHEK and PPEYQ). Lower percentages of glucose and higher percentages of histidine and valine suggest that SjENOD2 and CkENOD2 are different from other ENOD2s. Hybridization analyses indicate the clones represent ENOD2 gene families of two to four genes in Japanese pagodatree and American yellowwood genomes, and ENOD2-like transcripts were detected in stems and flowers, as well as roots. Only roots of control species that nodulate, Maackia amurensis Rupr. & Maxim. and alfalfa (Medicago sativa), produced pseudonodules after treatment with zeatin or 2,3,5-triiodobenzoic acid, an auxin transport inhibitor. Accumulation of MaENOD2 transcripts was enhanced during the first 10 d of treatment, but 2,3,5-triiodobenzoic acid and zeatin enhanced transcript accumulation after 30 d in roots of Japanese pagodatree and American yellowwood. Characteristics that distinguish ENOD2 gene families in basal, non-nodulating woody legumes from other ENOD2 genes may provide new information about the function of these genes during symbiotic and non-symbiotic organ development.

Figure 1

Comparison of deduced ENOD2 amino acid sequences from Japanese pagodatree (SjENOD2), American yellowwood (CkENOD2), M. amurensis (MaENOD2), soybean (GmENOD2), and Sesbania rostrata (SrENOD2). Alignments were obtained with GCG computer programs Lineup and Pileup. Pentapeptide repeats conserved in ENOD2 proteins are shaded in black, whereas repeats common to ENOD2 proteins are shaded in dark gray. Light gray shading indicates conserved regions in the N-terminal domain. Conserved sequences for C-terminal domains that are not aligned because of differences in sequence length are underlined with a solid line. The consensus line identifies conserved amino acids in four and five presented sequences (bold) or in two and three of the sequences. The putative signal peptide sequence is underlined with a dotted line in the consensus sequence. Asterisks represent presence of stop codons. Dashes (approximately) indicate no sequence data available. Dots represent gaps in the sequence.

Figure 2

Southern hybridizations for Japanese pagodatree (Sj) and American yellowwood (Ck). Genomic DNA was digested with EcoRI (lane 1), BamHI (lane 2), and EcoRI/XhoI (lane 3). Genomic DNA from soybean and maize, digested with EcoRI, was used as positive (lane 4) and negative (lane 5) controls, respectively. Ten micrograms of DNA was loaded in each lane. ³²P-labeled SjENOD2 and CkENOD2 PCR fragments were hybridized to blots for Japanese pagodatree and American yellowwood, respectively.

Figure 3

Northern blots of poly(A⁺) transcripts from leaves (L), stems (S), roots (R), and flowers (F) of Japanese pagodatree (Sj) and American yellowwood (Ck). Each lane contained 2.5 μg of mRNA. ³²P-labeled SjENOD2 and CkENOD2 PCR fragments were hybridized to blots of Japanese pagodatree and American yellowwood RNA, respectively.

Figure 4

TIBA-treated roots of M. amurensis, alfalfa, Japanese pagodatree, and American yellowwood. Plants were grown in nutrient solution with 50 μm TIBA for 40 d. Presumed pseudonodules are indicated by arrows in the positive controls, M. amurensis (A) and alfalfa (B). Root tips of Japanese pagodatree (C) and American yellowwood (D) swelled, but no pseudonodules were observed on root systems. Bar = 2 mm.

Figure 5

Temporal analysis of putative ENOD2 transcripts from TIBA- and zeatin-treated roots of Japanese pagodatree (Sj), American yellowwood (Ck), and M. amurensis (Ma). Plants were grown in nutrient solution with 50 μm TIBA or 100 nm zeatin for 0 to 40 d. Control plants were grown in nutrient solution without TIBA or zeatin. Total RNA was hybridized to ³²P-labeled SjENOD2 (Sj), CkENOD2 (Ck), and MaENOD2 (Ma) PCR fragments. Ten micrograms of RNA was loaded in each lane. Hybridization with a 18S rDNA probe served as a control for loading.