Regulation of the Later Stages of Nodulation Stimulated by IPD3/CYCLOPS Transcription Factor and Cytokinin in Pea Pisum sativum L

Abstract

The IPD3/CYCLOPS transcription factor was shown to be involved in the regulation of nodule primordia development and subsequent stages of nodule differentiation. In contrast to early stages, the stages related to nodule differentiation remain less studied. Recently, we have shown that the accumulation of cytokinin at later stages may significantly impact nodule development. This conclusion was based on a comparative analysis of cytokinin localization between pea wild type and ipd3/cyclops mutants. However, the role of cytokinin at these later stages of nodulation is still far from understood. To determine a set of genes involved in the regulation of later stages of nodule development connected with infection progress, intracellular accommodation, as well as plant tissue and bacteroid differentiation, the RNA-seq analysis of pea mutant SGEFix^--2 (sym33) nodules impaired in these processes compared to wild type SGE nodules was performed. To verify cytokinin's influence on late nodule development stages, the comparative RNA-seq analysis of SGEFix^--2 (sym33) mutant plants treated with cytokinin was also conducted. Findings suggest a significant role of cytokinin in the regulation of later stages of nodule development.

Keywords: IPD3/CYCLOPS transcription factor; Pisum sativum; cytokinin; gene expression; transcriptomic data.

Figure 1

Analysis of root length (A) and the number of nodules in SGEFix^--2 (sym33) mutant (B) and wild-type cv. SGE (C) plants 2 weeks after inoculation with Rhizobium leguminosarum bv. viciae CIAM1026 and treatment with 10 μM 6-BAP (cytokinin, CK). The graphs show the results of three independent experiments (4–5 plants per one variant were used). The error bars represent the mean ± SEM of three repeats. The asterisks indicate statistically significant differences based on Student’s t-test (** p < 0.01).

Figure 2

Light microscopy images of 2-week-old nontreated nodules of wild-type (A,B) and sym33 mutant (C,D) and treated with 10 μM 6-BAP (cytokinin) wild-type (E,F) and sym33 mutant (G,H). IC—infected cells, NIC—noninfected cells, IT—infection threads. (A,C,E,G) images have 40× magnification. (B,D,F,H) images have 100× magnification. Arrowheads indicate infection threads. Scale bars are 20 μm (A) and 10 μm (B).

Figure 3

Transmission electron microscopy images of 2-week-old wild-type nodules nontreated (A,B) or treated (C,D) with 10 μM 6-BAP (cytokinin) plants. IC—infected cells, NIC—noninfected cells, IT—infection thread. Arrowheads indicate infection threads. Scale bars are 5 μm (A,C) and 1 μm (B,D).

Figure 4

Transmission electron microscopy images of 2-week-old nodules of sym33 mutant nontreated (A,B) or treated (C,D) with 10 μM 6-BAP (cytokinin). IT—infection thread. Scale bars are 2 μm (C), 1 μm (A,B), and 500 nm (D).

Figure 5

Differential gene expression in pea nodules of cv. SGE wild type and SGEFix^--2 (sym33) mutant. Heatmap represents top differentially expressed transcripts in wild-type nodules to sym33 mutant nodules as normalized counts after variance stabilizing transformation of raw read counts per transcript. The data of three independent biological repeats were combined for each variant—sym33 and wild type (wt) nodules.

Figure 6

Differential gene expression in pea nodules of SGEFix^--2 (sym33) mutant plants untreated or treated with 10 µM BAP (cytokinin). Heatmap represents top differentially expressed transcripts in nodules of sym33 mutant plants non-treated and treated with cytokinin (CK) as normalized counts after variance stabilizing transformation of raw read counts per transcript. The data of three independent biological repeats were combined for each variant—sym33 nodules without treatment and sym33 after cytokinin (CK) treatment.

Figure 7

Gene ontology pathways that are overrepresented in differentially expressed genes in wild-type nodules compared to sym33 mutant nodules (up-and down-regulated separately).

Figure 8

Gene ontology pathways that are overrepresented in differentially expressed genes in nodules of untreated sym33 mutant plants compared to cytokinin treated (up-and down-regulated separately).