Efficacy of a Plant-Microbe System: Pisum sativum (L.) Cadmium-Tolerant Mutant and Rhizobium leguminosarum Strains, Expressing Pea Metallothionein Genes PsMT1 and PsMT2, for Cadmium Phytoremediation.

Viktor E Tsyganov,Olga A Kulaeva,Viktoria E Kim,Andrey A Belimov,Igor A Tikhonovich,Elena P Chizhevskaya,Anna B Kitaeva,Pyotr G Kusakin,Anna V Tsyganova,Kira A Ivanova,Tatiana A Serova,Elena V Seliverstova,Artemii P Gorshkov

doi:10.3389/fmicb.2020.00015

Abstract

Two transgenic strains of Rhizobium leguminosarum bv. viciae, 3841-PsMT1 and 3841-PsMT2, were obtained. These strains contain the genetic constructions nifH-PsMT1 and nifH-PsMT2 coding for two pea (Pisum sativum L.) metallothionein genes, PsMT1 and PsMT2, fused with the promoter region of the nifH gene. The ability of both transgenic strains to form nodules on roots of the pea wild-type SGE and the mutant SGECdt, which is characterized by increased tolerance to and accumulation of cadmium (Cd) in plants, was analyzed. Without Cd treatment, the wild type and mutant SGECdt inoculated with R. leguminosarum strains 3841, 3841-PsMT1, or 3841-PsMT2 were similar histologically and in their ultrastructural organization of nodules. Nodules of wild-type SGE inoculated with strain 3841 and exposed to 0.5 μM CdCl2 were characterized by an enlarged senescence zone. It was in stark contrast to Cd-treated nodules of the mutant SGECdt that maintained their proper organization. Cadmium treatment of either wild-type SGE or mutant SGECdt did not cause significant alterations in histological organization of nodules formed by strains 3841-PsMT1 and 3841-PsMT2. Although some abnormalities were observed at the ultrastructural level, they were less pronounced in the nodules of strain 3841-PsMT1 than in those formed by 3841-PsMT2. Both transgenic strains also differed in their effects on pea plant growth and the Cd and nutrient contents in shoots. In our opinion, combination of Cd-tolerant mutant SGECdt and the strains 3841-PsMT1 or 3841-PsMT2 may be used as an original model for study of Cd tolerance mechanisms in legume-rhizobial symbiosis and possibilities for its application in phytoremediation or phytostabilization technologies.

Highlights

Plant-microbial systems formed by legumes and soil bacteria, called rhizobia, are widely used to enrich soils with nitrogen (Stagnari et al, 2017)
Negative effects of Cd on nitrogen fixation were demonstrated for nodules of Pisum sativum L. (Hernandez et al, 1995), Glycine max (L.) Merr. (Balestrasse et al, 2004), L. albus (Sánchez-Pardo et al, 2013), M. sativa L. (Shvaleva et al, 2010), M. truncatula Gaertn. (Marino et al, 2013), and in some other species too
The pea (Pisum sativum L.) laboratory line SGE (Kosterin and Rozov, 1993), and its corresponding mutant line SGECdt characterized by an increased tolerance to Cd and higher levels of Cd accumulation (Tsyganov et al, 2007), were both used in this study

Summary

Introduction

Plant-microbial systems formed by legumes and soil bacteria, called rhizobia, are widely used to enrich soils with nitrogen (Stagnari et al, 2017). Nitrogen fixation takes place in symbiotic nodules formed on plant roots (and in some cases on shoots), and this nodule formation creates a new ecological niche for rhizobia. Rhizobia use the infection thread to penetrate inside their roots (Tsyganova and Tsyganov, 2018). From these infection threads, rhizobia are released into host cells’ cytoplasm where they differentiate into bacteroids and form symbiosomes, the main units fixing nitrogen. In P. sativum nodules exposed to Cd, expansion of the peribacteroid space, destruction of the symbiosome membrane, fusion of symbiosomes, and the formation of symbiosomes containing several bacteroids were all observed (Tsyganova et al, 2019). Similar features of Cd’s adverse influence on nodule ultrastructure were reported for nodules of M. sativa (Shvaleva et al, 2010)

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