Functional analysis of heavy metal transporter HMA5 gene in symbiotic nodulation of <italic>Medicago truncatula</italic>

Abstract

<p indent=0mm>Leguminous plants are able to grow in a nitrogen-deprived environment because they have evolved a symbiotic relationship with rhizobia in the soil, leading to the formation of new organs, namely nodules. In root nodules, rhizobia reduce nitrogen to ammonia through biological nitrogen fixation and provide ammonia to the host legumes in exchange for carbon source and energy they need. In the symbiosis, legumes also provide the metal trace elements to rhizobia, including copper (Cu). As one of the essential elements for plant growth, Cu participates in multiple biological processes. However, excess Cu induces oxidative stress to the detriment of many physiological processes including symbiotic nitrogen fixation. Therefore, the copper content in plants must be maintained within a certain physiological range. The higher plants have evolved complex physiological mechanisms to maintain the copper homeostasis in cells. The heavy metal transporter, P-type ATPase (HMA), is mainly involved in the transport of heavy metals including copper in plants. All members of the HMA family in the dicot model plant <italic>Arabidopsis</italic> have been identified and classified. Preliminary research in the laboratory found that HMA5 gene is related to copper stresses and the symbiotic nodulation of <italic>Medicago</italic>. In this study, the function of HMA5 homologous gene was investigated during symbiotic nodulation in <italic>Medicago truncatula</italic> (Mt), a model leguminous plant. A phylogenetic tree was constructed based on the currently classified HMA family genes in Medicago truncatula and the expression patterns of the genes were analyzed. A homologous gene <italic>MTR</italic>_<italic>8g079250</italic> of HMA5 was selected for further study. Subcellular localization assays showed that<italic> MTR_8g079250 </italic>was localized to the cell membrane and nucleus. The fusion expression analysis of GFP::MTR_8g079250 showed that MTR_8g079250 was mainly expressed in the meristem, infection zone and cortex of the nodules, as well as in the stele and cap of the roots. Knockdown of <italic>MTR_8g079250</italic> by RNA interference (RNAi) technology had no significant effect on the symbiotic nitrogen fixation phenotype. However, overexpression of <italic>MTR_8g079250 </italic>impaired the infection process and nodule development. Compared with the control, the fresh weight and nodule number per plant were dramatically reduced in<italic> MTR_8g079250-</italic>overexpression plants. The numbers of the infection threads and nodule primordia were also significantly decreased. Ultrastructure analyses showed that the nodule cells contained fewer bacteria in the overexpression plants. In particular, the remarkable deformation of bacteroids and enlargement of peribacteroid space occurred in the infected cells, suggesting premature aging of the nodules. qRT-PCR analyses showed that the expression levels of the nodulation marker genes, <italic>NIN</italic>, <italic>DMI1</italic>, <italic>ENOD11</italic> and leghemoglobin gene were down-regulated to different extent. These results indicate that the overexpression of<italic> MTR_8g079250</italic> inhibited the symbiotic nodulation. In summary, this study preliminarily assessed the function of HMA5 in symbiotic nodulation, laying a foundation for the subsequent exploration of the absorption, transport and equilibrium mechanisms of copper ions during root nodule symbiosis.