Genome-wide identification and characterization of legume T2 Ribonuclease gene family and analysis of GmaRNS9, a soybean T2 Ribonuclease gene, function in nodulation

Abstract

T2 ribonuclease family (RNaseT2) proteins are secretory and nonspecific endoribonucleases that have a large conserved biological role. Family members of RNaseT2 are found in every organism and carry out important biological functions. However, little is known about the functions of these proteins in legumes, including potential roles in symbiotic nodulation. This study aimed to characterize and perform bioinformatic analysis of RNaseT2 genes in four legume species that their genome was sequenced. In total, 60 RNaseT2 genes were identified and characterized. By analyzing their phylogeny, we divided these RNaseT2 into five clades. Expression analysis of RNaseT2 genes indicated that these genes are expressed in various tissues, and the most expression level was related to the pod, flower, and root. Moreover, GmaRNS9 expression analysis in soybean was consistent with in silico studies and demonstrated that this gene usually has high root tip expression. GmaRNS9 expression was reduced by Bradyrhizobium japonicum inoculation and nodule formation. Reduced expression of this gene was possibly controlled by the GmNARK gene either directly or pleiotropically through increased phosphorus requirements during increased nodulation. However, the nutrient stress (phosphate and nitrate starvation) led to an increase in the expression level of GmRNS9. In silico and quantitative gene expression analyses showed that RNaseT2 genes could play important roles in the growth and development of legumes as well as nodulation.

Keywords: Abiotic stresses; Biochemical properties; Gene expression; In silico; Legume; Phylogeny; RNaseT2.

Fig. 1

Protein sequence alignment of all the RNaseT2 proteins. Residues conserved are shaded. The two conserved active site (CAS) regions and mutations in these sites are indicated, and the two catalytic histidines (No. 354 and 431) are marked with red arrows

Fig. 2

Mutations in conserved active site residues in plant RNasesT2. The catalytic histidines are demonstrated with red arrows. PhvuRNS13, MedtrRNS23, MedtrRNS25, MedtrRNS27, LjRNS5, LjRNS6 and LjRNS7 proteins have lost their CAS I and showed using a yellow box. Mutation in either one or the two histidines leads to loss of their RNases activity. RNS1, RNS5, GmaRNS1, and GmaRNS2 are active RNases that only are shown for comparison

Fig. 3

Phylogenetic tree of the RNaseT2 proteins from G. max, M. truncatula, P. vulgaris, L. japonicus and A. thaliana species based on protein sequences of RNaseT2. Clades I, II, III, IV and V are indicated. Phylogenetic tree made with maximum-likelihood with UGENE program, using PhyML substitution model with 1000 bootstraps and default parameters

Fig. 4

Depicted protein structure of RNaseT2 proteins. Signal peptide (red box), RNaseT2 domain (purple box), GTP-EFTU domain (green box), transmembrane helix regions (yellow box) and thioredoxin domain (blue box). GmaRNS9, GmaRNS10, MedtrRNS11, MedtrRNS12, MedtrRNS14, MedtrRNS16, MedtrRNS22, MedtrRNS23, MedtrRNS24, MedtrRNS25, MedtrRNS26, MedtrRNS27, PhvuRNS2, PhvuRNS6, PhvuRNS7, PhvuRNS11, PhvuRNS12, PhvuRNS13, LjRNS3, LjRNS5, LjRNS6 and LjRNS7 proteins lack a signal peptide. In addition to, MedtrRNS13 and MedtrRNS11 have unusual domains

Fig. 5

Gene expression profiles of RNaseT2 in four legumes and A. thaliana. Transcriptom data of RNaseT2 genes were obtain from the BAR resource for A. thaliana (a), G. max (b) and M. thruncatula (e); Lotus Base for L. japonicus (c); PvGEA for P. vulgaris (d). Heat map of RNaseT2 genes expression profiles obtain for each plant separately. RPKM values are represented by color key bottom of each heat map. (d: day, w: week, HAI: hours after inoculation, WT: wild type)

Fig. 6

Root phenotype and nodule formation of the soybean wild type, Bragg, and nts382 mutant in presence and absence of B. japonicum (CB 1809)

Fig. 7

Expression analysis of GmaRNS9 gene at the root tip of soybean. a Expression of GmaRNS9 gene at the root tip of soybean Bragg and nts382 affected by nodulation. b Expression of GmaRNS9 gene at root tip of soybean Bragg in nitrate and phosphate stresses. Error bars indicate SE