A variable gene family encoding nodule-specific cysteine-rich peptides in pea (Pisum sativum L.)

Abstract

Various legume plants form root nodules in which symbiotic bacteria (rhizobia) fix atmospheric nitrogen after differentiation into a symbiotic form named bacteroids. In some legume species, bacteroid differentiation is promoted by defensin-like nodule-specific cysteine-rich (NCR) peptides. NCR peptides have best been studied in the model legume Medicago truncatula Gaertn., while in many other legumes relevant information is still fragmentary. Here, we characterize the NCR gene family in pea (Pisum sativum L.) using genomic and transcriptomic data. We found 360 genes encoding NCR peptides that are expressed in nodules. The sequences of pea NCR genes and putative peptides are highly variable and differ significantly from NCR sequences of M. truncatula. Indeed, only one pair of orthologs (PsNCR47–MtNCR312) has been identified. The NCR genes in the pea genome are located in clusters, and the expression patterns of NCR genes from one cluster tend to be similar. These data support the idea of independent evolution of NCR genes by duplication and diversification in related legume species. We also described spatiotemporal expression profiles of NCRs and identified specific transcription factor (TF) binding sites in promoters of “early” and “late” NCR genes. Further, we studied the expression of NCR genes in nodules of Fix– mutants and predicted potential regulators of NCR gene expression, one among them being the TF ERN1 involved in the early steps of nodule organogenesis. In general, this study contributes to understanding the functions of NCRs in legume nodules and contributes to understanding the diversity and potential antibiotic properties of pea nodule-specific antimicrobial molecules.

Identification and Expression Analysis of the C-TERMINALLY ENCODED PEPTIDE Family in Pisum sativum L.

“…To study the expression levels of the PsCEPs genes in symbiotic nodules in more detail, we analyzed transcriptomic data by Zorin et al [ 29 ] on the transcript abundance in the root tips and symbiotic nodules at different stages of nodulation: 12 days, three weeks, and four weeks after rhizobial inoculation (dpi) ( Figure 10 ). The transcripts of six PsCEP genes were found in this set of data: PsCEP1 , PsCEP2 , PsCEP7 , PsCEP13 , PsCEP16 , and PsCEP17 , where PsCEP1 , PsCEP2 , and PsCEP7 were abundant at earlier stages of nodulation (12 dpi), and PsCEP7 , PsCEP13 , PsCEP16 , and PsCEP17 demonstrate high expression levels in mature nodules after four weeks of rhizobial inoculation.…”

“…These mutant lines carry mutations in transcription factor genes PsEFD and PsIPD3 , respectively [ 30 , 31 ], which result in Fix - -phenotype (i.e., the mutant lines form white round-shaped ineffective nodules, where nitrogen fixation does not occur). Recently, the analysis of transcription changes in three-week-old nodules of SGEFix – -1 ( Pssym40-1 ), SGEFix – -2 ( Pssym33-3 ), and SGE (wild type) was performed [ 29 ]. Here, we analyzed the expression of PsCEP genes in mutant nodules with use of the available data ( Figure 11 ).…”

“…Transcriptomic analysis was performed using the reads obtained by Zorin et al [ 29 ]. All raw reads were processed to filter out adaptor sequences and low-quality sequences using BBDuk from BBMap toolkit ( , accessed on 11 July 2022).…”

“… Heatmap illustrating the expression levels of the PsCEP genes in symbiotic nodules. Only those PsCEP genes which have transcript abundance ≥10 according to the data by Zorin et al [ 29 ] are shown. …”

TheMedicago truncatulanodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules

“…The irreversible transition of rhizobia in M. truncatula nodules is mediated by host-produced defensin-related nodule-specific cysteine-rich (NCR) peptides (Van De Velde et al ., 2010). The genome of M. truncatula codes for about 700 NCR peptides and similar genes in different numbers have been identified in other IRLC legume species (Mergaert et al ., 2003; Montiel et al ., 2017; Zorin et al ., 2022; Wei et al ., 2022). Almost all NCR genes are specifically expressed in infected cells of M. truncatula root nodules and they are activated in successive waves during nodule organogenesis and bacteroid differentiation (Nallu et al ., 2013; Guefrachi et al ., 2014).…”