# Overexpression of RpKTI2 from Robinia pseudoacacia Affects the Photosynthetic Physiology and Endogenous Hormones of Tobacco

## TL;DR

A gene from Robinia pseudoacacia, when overexpressed in tobacco, affects photosynthesis and plant hormones, with notable impacts on stomata and antioxidant capacity.

## Contribution

This study reveals the effects of RpKTI2 overexpression in tobacco, highlighting its influence on photosynthetic physiology and endogenous hormones.

## Key findings

- RpKTI2 overexpression increased maximum photochemical efficiency (Fv/Fm) and decreased intercellular CO2 concentration (Ci) in transgenic tobacco.
- Stomatal size and specific hormone levels were reduced, while brassinosteroid content increased in transgenic plants.
- Random forest regression identified RpKTI2's strongest impact on carotenoid content, initial fluorescence, and stomatal area.

## Abstract

Kunitz trypsin inhibitor genes play important roles in stress resistance. In this study, we investigated RpKTI2 cloned from Robinia pseudoacacia and its effect on tobacco. RpKTI2 was introduced into the tobacco cultivar NC89 using Agrobacterium-mediated transformation. Six RpKTI2-overexpressing lines were obtained. Transgenic and wild-type tobacco plants were then compared for photosynthetic characteristics and endogenous hormone levels. Transgenic tobacco showed minor changes in chlorophyll content, fluorescence, and photosynthetic functions. However, the maximum photochemical efficiency (Fv/Fm) increased significantly while intercellular CO2 concentration (Ci) decreased significantly. Stomatal size and hormone content (indole-3-acetic acid, zeatin riboside, gibberellin, and indole-3-propionic acid) were reduced, while brassinosteroid content increased. Random forest regression revealed that RpKTI2 overexpression had the biggest impact on carotenoid content, initial fluorescence, Ci, stomatal area, and indole-3-acetic acid. Overall, RpKTI2 overexpression minimally affected chlorophyll synthesis and photosynthetic system characteristics but influenced stomatal development and likely enhanced the antioxidant capacity of tobacco. These findings provide a basis for future in-depth research on RpKTI2.