Search for Nodulation and Nodule Development-Related Cystatin Genes in the Genome of Soybean (Glycine max)

Abstract

Nodulation, nodule development and senescence directly affects nitrogen fixation efficiency, and previous studies have shown that inhibition of some cysteine proteases delay nodule senescence, so their nature inhibitors, cystatin genes, are very important in nodulation, nodule development, and senescence. Although several cystatins are actively transcribed in soybean nodules, their exact roles and functional diversities in legume have not been well explored in genome-wide survey studies. In this report, we performed a genome-wide survey of cystatin family genes to explore their relationship to nodulation and nodule development in soybean and identified 20 cystatin genes that encode peptides with 97–245 amino acid residues, different isoelectric points (pI) and structure characteristics, and various putative plant regulatory elements in 3000 bp putative promoter fragments upstream of the 20 soybean cystatins in response to different abiotic/biotic stresses, hormone signals, and symbiosis signals. The expression profiles of these cystatin genes in soybean symbiosis with rhizobium strain Bradyrhizobium japonicum strain 113-2 revealed that 7 cystatin family genes play different roles in nodulation as well as nodule development and senescence. However, these genes were not root nodule symbiosis (RNS)—specific and did not encode special clade cystatin protein with structures related to nodulation and nodule development. Besides, only two of these soybean cystatins were not upregulated in symbiosis after ABA treatment. The functional analysis showed that a candidate gene Glyma.15G227500 (GmCYS16) was likely to play a positive role in soybean nodulation. Besides, evolutionary relationships analysis divided the cystatin genes from Arabidopsis thaliana, Nicotiana tabacum, rice, barley and four legume plants into three groups. Interestingly, Group A cystatins are special in legume plants, but only include one of the above-mentioned 7 cystatin genes related to nodulation and nodule development. Overall, our results provide useful information or clues for our understanding of the functional diversity of legume cystatin family proteins in soybean nodulation and nodule development and for finding nodule-specific cysteine proteases in soybean.

RNA-Seq analysis of nodule development at five different developmental stages of soybean (Glycine max) inoculated with Bradyrhizobium japonicum strain 113-2

“…In this report, 6 cystatins were identified from our RNA-Seq ( Table S5 ), among them, two cystatins (Glyma18g12240 and Glyma18g00690) were not in the seven cystatins 11 . Glyma18g12240 was significantly increased in roots at 0.5 h of post inoculation and during nodule developments 58 and different expressed in 6 Groups ( Table S5 ), indicating that Glyma18g12240 play roles in nodulation and nodule development. The symbiotic function of Glyma18g12240 was studied and the results showed that it really promotes nodulation in L. japonicus and performs an essential role in delaying nodule senescence ( Fig.…”

“…Previous studies have shown that cysteine proteases and cystatins are actively transcribed during nodule development and senescence 11 , and inhibition of some cysteine proteases delay nodule senescence 56 57 . Cystatin genes, nature inhibitors of cysteine proteases, are very important in nodule symbiosis and 7 soybean cystatins play different roles in nodulation, nodule development and senescence 58 . In this report, 6 cystatins were identified from our RNA-Seq ( Table S5 ), among them, two cystatins (Glyma18g12240 and Glyma18g00690) were not in the seven cystatins 11 .…”

Genome-Wide Identification and Classification of Soybean C2H2 Zinc Finger Proteins and Their Expression Analysis in Legume-Rhizobium Symbiosis

“…The symbiosis-related soybean C2H2s selected from our previous RNA-seq data (Yuan et al, 2016a , 2017 ) were further evaluated using qPCR, and the methods refer to our previous studies (Yuan et al, 2016b ). Briefly, purified RNA samples were treated with DNase I (Takara) and reversely transcribed into cDNA using a Prime Script RT reagent Kit (Perfect Real Time) with gDNA Eraser (Takara Bio, Inc) and oligo (dT) as the primer.…”

Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.)

“…Based on the molecular mass (11.4 kDa) and other molecular characteristics of BnCPI (described above), we concluded that it should be clustered into group I. Subgroups (Group I, Group II and Group III) of the phytocystatin family were generated on the basis of phylogenetic analysis [ 31 ]. Introns are common in the coding regions of cystatins from plants and animals, and some phytocystatins contain more than one intron [ 32 , 33 , 34 , 35 ]. Intron–exon structure is also often used to characterize cystatin genes; those lacking introns in their ORF are categorized as MCOG C, whereas those containing three introns are classed into subgroup B1, and those with only one intron in the DNA sequence—located between the coding sequences for the conserved motifs LARFAV and QxVxG—are placed in either MCOG A or subgroup B2 [ 36 ].…”