Expression Of Soybean GmPM1 And GmPM9 Proteins Enhances Resistance To Copper Stress In Plant

Copper is an essential micronutrient for all living microorganisms, as they are cofactors of many enzymes and play an important role in electron transport, redox reactions, and in a variety of metabolic pathways. Copper contaminated soil from agricultural and industrial activities including mining, use of copper fungicides and fertilizer, as well as in sewage sludge has become an important environmental concern. Excess copper in plants causes physiological alterations, particularly oxidative stress.Research into late embryogenesis abundant（LEA） proteins has been ongo ing for more than 30 years. However, their function has been entirely obscure despite there a strong association of LEA proteins with abiotic stress tolerance, particularly dehydration and cold stress. Since the initial discovery in cotton seeds, LEA proteins have been described in a range of different plants and plant tissues. They belong to the family of intrinsically disordered proteins. It is still unclear about how these flexible LEA proteins recognize different ligands and exercise multiple protection under abiotic stress in plant.Soybean seedlings were cultured in Hoagland solutions. To induce abiotic stresses, the seedlings were transfered to Hoagland solutions containing 150μM Cu SO4. Real time quantitative PC R analyses showed the expression levels of the Gm PM1 and Gm PM9 genes were up-regulated under copper stress at 3h and 24 h, thereby suggesting that these two genes play a role in responses to copper stress.The recombinant yeast plasmids of p YES2/Gm PM1, p YES2/Gm PM1 and empty vector p YES2/C T were transformed into the copper-sensitive yeast mutant Δcup2 to create recombinants of ΔCUP2/Gm PM1, ΔCUP2/Gm PM9, ΔCUP2/p YES2. The growth of recombinants was monitored under SC-Gal medium supplemented with 0μM or 150μM Cu Cl2. The results indicated that the growth rate of ΔCUP2/Gm PM1 and ΔCUP2/Gm PM9 was faster than control ΔCUP2/p YES2 at 24h³6h, suggesting the expression of Gm PM1 and Gm PM9 could confer Cu²⁺ tolerance in yeast.Different truncated polypeptides of Gm PM1 protein were constructed according to the characteristics of helix structure a nd the distribution of alkaline amino acids. Based on these residues, Gm PM1 and its related domains can reduce the formation of hydroxyl radicals induced by Cu. They inhibited generation of hydroxyl radicals in the Cu–ascorbate system. The reduction of hydroxyl radical generation by Gm PM1 and Gm PM9 was dose dependent, and the 50% inhibitory dose（IC50） were 0.66mM and 0.81mM, respectively. High percent of histidine of Gm PM1 C-terminal（Gm PM1-C） play an important role on Cu²⁺ binding and hydroxyl radicals reducing activities.Isothermal titration calorimetry showed Gm PM1, Gm PM9, Gm PM1-C, Gm PM1-C1, Gm PM1-C2 and Gm PM1-C3 could interact with Cu²⁺ and the strength of binding affinity is Gm PM1-C2> Gm PM9> Gm PM1> Gm PM1-C1> Gm PM1-C> Gm PM1-C3. The secondary structure of Gm PM1-C and Gm PM1-C2 have changed obviously with the addition of Cu²⁺ by CD spectra analysis.In this study, it is proposed that C- terminal contained histidine of Gm PM1 protein plays an important role to confer copper stress tolerance.