# Knockout of a single aquaporin, OsPIP2;4, decreases root water permeability in rice

## TL;DR

Knocking out the OsPIP2;4 aquaporin in rice reduces root water permeability, showing its important role in water transport.

## Contribution

This study identifies OsPIP2;4 as a key aquaporin influencing root hydraulic conductivity in rice through knockout experiments.

## Key findings

- OsPIP2;4 knockout rice plants had significantly lower root hydraulic conductivity compared to wild-type plants.
- Reduced OsPIP2;4 transcript and protein levels were observed in knockout lines.
- Overexpression of OsPIP2;4 did not significantly increase root hydraulic conductivity.

## Abstract

Aquaporins (AQPs) are membrane proteins that facilitate water transport and are present in nearly all bacterial, animal, and plant cells. In plants, AQPs are classified into four or more subfamilies, with plasma membrane intrinsic proteins (PIPs) playing a key role in root water uptake and cellular water regulation. Previous studies have demonstrated that PIPs contribute to root hydraulic conductivity (Lpr) in various plant species. In this study, we examined the specific role of OsPIP2;4, one of the PIP-type aquaporins among 11 rice (Oryza sativa) PIP2s, in regulating Lpr. Transgenic rice plants, including OsPIP2;4-knockout (KO) and overexpressing (Ox) lines, were used for this investigation. Two independent KO lines, generated via the CRISPR-Cas9 system and T-DNA insertion mutagenesis, respectively, showed significantly lower Lpr compared to wild-type rice plants. The decrease in Lpr in the T-DNA KO line was associated with reduced OsPIP2;4 transcript levels, measured by real-time PCR, and lower OsPIP2;4 protein levels, as shown by immunohistochemical analysis. Conversely, no notable increase in Lpr was observed in the Ox lines. These results suggest that OsPIP2;4 is expressed in appropriate tissues in rice roots and is a key factor influencing Lpr. This research represents a significant step toward further understanding the physiological functions of OsPIP2;4 in rice.

The online version contains supplementary material available at 10.1007/s10265-025-01691-z.