# Overexpression of milk thistle SOD gene enhances drought tolerance in tobacco by improving photosynthesis and photoprotection

## TL;DR

A gene from milk thistle improves tobacco plants' ability to withstand drought by boosting photosynthesis and protecting against stress.

## Contribution

Transferring the SmSOD gene from milk thistle to tobacco enhances drought tolerance through improved photosynthesis and photoprotection.

## Key findings

- Transgenic tobacco plants with the SmSOD gene showed 5 times higher SOD gene expression and 1.8 times higher enzyme activity under drought stress.
- Transgenic plants had higher photosystem II efficiency and non-photochemical quenching compared to wild-type plants under drought.
- Stomatal width in transgenic plants was smaller under drought stress compared to wild-type plants.

## Abstract

Superoxide dismutase (SOD) is involved in the plant’s primary defense mechanism against the adverse effects of reactive oxygen species (ROS), maintaining ROS homeostasis within cells. Therefore, it is essential to investigate and compare the stress tolerance mechanism in both tolerant and sensitive plants. This study investigates the drought stress tolerance of Tobacco following transferring of a SOD gene obtained from a drought-tolerant plant. The SOD gene was isolated from the Milk thistle (Silybum marianum L.) plant (SmSOD), cloned into the pBI121 (Binary Agrobacterium plasmid) expression vector, and subsequently transformed into Agrobacterium tumefaciens. Pre-cultivated Tobacco (Nicotiana tabacum L.) plants were inoculated with the recombinant bacteria. Following validation of transgene integration via PCR, plants subjected to drought stress corresponding to ~ 50% field capacity for 5 days. Results demonstrated that the expression of SOD and its enzyme activity in drought-imposed transgenic plants were respectively 5 and 1.8 times higher than its expression and activity in wild-type plants. Imaging of chlorophyll fluorescence showed that drought-imposed transgenic plants had a higher maximum quantum yield of photosystem II (Fv/Fm) and Non-photochemical quenching (NPQ) compared with wild-type plants. Stomatal density in drought-imposed plants was lower than in control plants, consistent across both transgenic and wild-type groups. Stomatal width decreased under stress in both plant types, with transgenic plants showing smaller width than wild-type plants. Stomatal length showed no significant differences between transgenic and wild-type plants or between drought-stressed and control conditions. In conclusion, SmSOD gene transfer from the Milk thistle to Tobacco plants increased the drought tolerance of the transgenic plants; this shed light on the path toward reaching drought-tolerant crops. These findings highlight the potential of SmSOD as a key gene for improving drought resistance in crops, offering promising applications in developing drought-tolerant crops for agriculture in water-scarce regions.

The online version contains supplementary material available at 10.1038/s41598-025-31510-3.