Expression of γ-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stresses by reducing reactive oxygen species

The -tocopherol methyltransferase (-TMT) is an important enzyme regulating synthesis of four tocopherols (, , and ). In this report, we investigated the role of -TMT in regulating abiotic stress within chloroplasts. The At -tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high-level expression of -TMT converted most of -tocopherol to -tocopherol in transplastomic seeds (similar to 10-fold higher) in the absence of abiotic stress. When grown in 400mm NaCl, -tocopherol content in transplastomic TMT leaves increased up to 8.2-fold and 2.4-fold higher than wild-type leaves. Likewise, under heavy metal stress, -tocopherol content in the TMT leaves increased up to 7.5-fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3-fold within 48h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild-type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that -tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.