Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Young-Hwan Ju,Tatsuhiko Shiraiwa,Ju-Young Choi,Aritra Roy Choudhury,Swapan Kumar Roy,Tomoyuki Katsube-Tanaka,Md Atikur Rahman,Moon-Soon Lee,Sun-Hee Woo,Soo-Jeong Kwon,Kun Cho

doi:10.3390/ijms22115879

Abstract

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 μM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Highlights

Heavy metal pollution is rapidly increasing, which induce many serious problems to the environment [1]
Using the gel-free quantification method, we found that the abundance of 44 differentially abundant proteins (DAPs) significantly changed in Cu treated seedlings compared with the control
Our study revealed that exogenous citric acid (CA) application alleviated the Cu toxicity by reducing oxidative stress and enhancing antioxidant enzyme activities, and plays a vital role in biochemical regulatory processes, in protein modulation

Summary

Introduction

Heavy metal pollution is rapidly increasing, which induce many serious problems to the environment [1]. Excess Cu induces phytotoxicity in plants that may impair many physiological processes, including photosynthesis and pigment synthesis, oxidative stress, lipid peroxidation in membrane, and other metabolic disturbances [5,6]. CA, as a low molecular weight organic acid, plays an important role as an intermediate of the trichloroacetic acid (TCA) cycle, and improves the bioavailability and removal efficiency of heavy metals from contaminated soil [11]. The protective role of CA towards metal-induced oxidative stress has captured scientists’ attention to increase pivotal enzymes’ activity in the antioxidant defense system [12,13,14]

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