Transgenic Medicago truncatula Plants That Accumulate Proline Display Enhanced Tolerance to Cadmium Stress

Vanesa S García De La Torre,José J Pueyo,M Mercedes Lucas,Teodoro Coba De La Peña

doi:10.3389/fpls.2022.829069

Abstract

Cadmium (Cd) accumulation in agricultural soils constitutes a serious problem for crop yields and food safety. It is known that proline (Pro) can rapidly accumulate in plant tissues in response to abiotic stress. To analyze the potential protective effect of Pro accumulation against Cd toxicity, we compared the response to Cd stress of wild-type (WT) Medicago truncatula and a transgenic line that we had previously obtained and characterized (p18), which expressed the Δ1-pyrroline-5-carboxylate synthetase gene from Vigna aconitifolia (VaP5CS), and accumulated high Pro levels. Cadmium significantly reduced germination of WT seeds compared to p18 seeds, and seedling relative root growth, a valid indicator of metal tolerance, was significantly higher for p18 than WT seedlings. We analyzed the relative expression of genes related to Pro metabolism, phytochelatin biosynthesis. antioxidant machinery, and NADPH recycling, which are relevant mechanisms in the response to Cd stress. They presented differential expression in the seedlings of both genotypes both under control conditions and under Cd stress, suggesting that the Cd response mechanisms might be constitutively activated in the transgenic line. Pro accumulation promoted higher survival, enhanced growth performance, and minor nutrient imbalance in transgenic p18 plants compared to WT plants. These facts, together with the recorded gluthatione levels, lipid peroxidation and antioxidant enzyme activities strongly suggested that VaP5CS expression and Pro accumulation conferred enhanced Cd tolerance to M. truncatula p18 plants, which was likely mediated by changes in Pro metabolism, increased phytochelatin biosynthesis and a more efficient antioxidant response. Moreover, p18 roots accumulated significantly higher Cd amounts than WT roots, while Cd translocation to the aerial part was similar to WT plants, thus suggesting that high Pro levels increased not only Cd tolerance, but also Cd phytostabilization by rhizosequestration.

Highlights

The occurrence of polluted soils with trace metal elements, known as heavy metals (Pourret et al, 2020), is an increasing agricultural problem due to industry-derived metal contamination and to the use of soil amendments and agrochemicals
NAPDH is considered a limiting metabolite in the plant antioxidant capacity because many reactive oxygen species (ROS)-detoxifying reactions are NADPH-dependent (Foyer and Noctor, 2011). This enzyme cofactor and reducing agent can be produced in different pathways, including enzymatic reactions catalyzed by isocitrate dehydrogenase (ICDH), glucose 6-phosphate dehydrogenase (G6PDH) or 6-phosphogluconate dehydrogenase (6PGDH), which are induced in response to oxidative stress caused by different factors, including Cd stress (Marino et al, 2007, 2013; Pérez-Chaca et al, 2014)
Our results indicated that the expression of Vigna aconitifolia ∆1-pyrroline-5-carboxylate synthetase gene (VaP5CS), and the consequent Pro accumulation, remarkably enhanced the capacity of M. truncatula transgenic plants to cope with Cd stress

Summary

Introduction

The occurrence of polluted soils with trace metal elements, known as heavy metals (Pourret et al, 2020), is an increasing agricultural problem due to industry-derived metal contamination and to the use of soil amendments and agrochemicals. NAPDH is considered a limiting metabolite in the plant antioxidant capacity because many ROS-detoxifying reactions are NADPH-dependent (Foyer and Noctor, 2011). This enzyme cofactor and reducing agent can be produced in different pathways, including enzymatic reactions catalyzed by isocitrate dehydrogenase (ICDH), glucose 6-phosphate dehydrogenase (G6PDH) or 6-phosphogluconate dehydrogenase (6PGDH), which are induced in response to oxidative stress caused by different factors, including Cd stress (Marino et al, 2007, 2013; Pérez-Chaca et al, 2014). The Cd-induced gene expression of these enzymes might be key for Cd tolerance in some M. truncatula cultivars (García de la Torre et al, 2021) Another plant response to Cd stress is the synthesis of phytochelatins (PCs) by phytochelatinsynthases (PCS), using glutathione as a substrate. It has been suggested that PCs are important for Cd chelation, detoxification and tolerance (Schat et al, 2002; Sobrino-Plata et al, 2009; Ahmad et al, 2019)

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