Reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in cytoplasmic male-sterile Brassica juncea

Abstract

The signal from organelle to nucleus, namely retrograde regulation of nuclear gene expression, was largely unknown. Due to the nuclear-cytoplasmic incompatibility in cytoplasmic male-sterile (CMS) plants, we employed CMS Brassica juncea to investigate the retrograde regulation of nuclear gene expression in this study. We studied how reduced BjRCE1 gene expression caused by the nuclear-cytoplasmic incompatibility altered the auxin response in CMS of B. juncea. We isolated the BjRCE1 gene that was located in the nucleus from B. juncea. Over-expression of BjRCE1 enhanced auxin response in transgenic Arabidopsis. The expression of BjRCE1 was significantly reduced in CMS compared with its maintainer fertile (MF) line of B. juncea. There were fewer lateral roots in CMS than MF under normal and treatment of indole-3-acetic acid (IAA) conditions. Expression patterns of several auxin-related genes together with their phenotypes indicated a reduced auxin response in CMS compared to MF. The phenotypes of auxin response and auxin-related gene expression pattern could be mimicked by inhibiting mitochondrial function in MF. Taken together, we proposed reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in CMS B. juncea. This may be an important mechanism of retrograde regulation of nuclear gene expression in plants.

Figure 1. The characterization of RCE1 gene from Brassica juncea.

A, Genomic structure of RCE1 gene from Brassica juncea. B, Conserved domain and ubiquitin interaction sites of RCE1 gene from Brassica juncea. C, Alignment of RCE1 gene from Brassica juncea and its orthologous from Arabidopsis. D, Sub-cellular localization of RCE1 gene from Brassica juncea. Scale bar = 10 µm. E, Phylogenetic tree of RCE1, RCE1 amino acid sequences are from NCBI database.

Figure 2. The characterization of over-expression of BjRCE1 gene in Arabidopsis.

A, Phenotype of oe-RCE1 of Arabidopsis. B, Expression level of BjRCE1 in oe-RCE1 Arabidopsis. C, Statistic analysis of length of primary root. D, Statistic analysis of number of lateral root. E, Expression of AtPIN2 gene. F, Expression of AtARF1 gene. G, Expression of AtCullin gene. For genes expression, actin gene was used as an internal control. Error bars, mean±SD (three independent biological replications).

Figure 3. The transcriptional expression of BjRCE1 gene in MF, CMS of Brassica juncea.

For BjRCE1 gene expression, 25S gene was used as an internal control. Error bars, mean±SD (three independent biological replications).

Figure 4. The phenotypic analysis of root from MF and CMS of Brassica juncea.

A, Root phenotype of MF, CMS and treated with 0.1 mmol/L and 0.5 mmol/L IAA. B, Statistic analysis of lateral root number. C, Statistic analysis of primary root length. Mean±SD values from 20 seedlings.

Figure 5. Transcriptional expression patterns of auxin-related genes in MF, CMS and MF/CMS treated with IAA in Brassica juncea.

For genes expression, 25 S gene was used as an internal control. Error bars, mean±SD (three independent biological replications).

Figure 6. A proposed model of mitochondrial modulation of auxin response that regulates root development via BjREC1 gene.