# Identification of Nuclear Localization Sequence (NLS) Sites in R2R3-MYB Transcription Factor Involved in Anther Development

## TL;DR

This study identifies specific nuclear localization sequences in the R2R3-MYB transcription factor MS188, which are essential for its role in plant anther development.

## Contribution

The study identifies and validates specific NLS regions in MS188 that are critical for its nuclear localization and function in plant development.

## Key findings

- Three NLS regions in MS188 were identified at amino acid positions 12–15, 18–22, and 96–107.
- The NLS at amino acids 18–22 is sufficient for nuclear localization, as mutations here result in cytoplasmic signals.
- Conserved NLSs in other R2R3-MYB TFs are also required for nuclear localization and developmental regulation.

## Abstract

The R2R3-MYB family of transcription factors (TFs) plays a crucial role in cell specification and secondary metabolism regulation during plant development. In Arabidopsis, MS188, a typical R2R3-MYB protein, is essential for tapetal development and pollen wall formation. However, the nuclear localization sequence (NLS) responsible for directing MS188 into the nucleus has not been fully elucidated. In this study, the subcellular localization of the NLS-containing proteins was determined by GFP tagging in tobacco leaves, and three NLS regions within MS188 were identified: two located at the N-terminus of R2-MYB and one at the C-terminus of R3-MYB. We further narrowed the NLSs located at amino acids (AAs) 12–15, 18–22, and 96–107 via point mutation analysis. Combined with the cytoplasmic protein FBA6, these NLSs fusion proteins could localize in the nucleus. Importantly, the proteins with mutations in AAs 18–22 exhibited completely cytoplasmic signals, whereas other mutated sites partially abolished the nuclear signals. These findings suggest that the NLS at AAs 18–22 is sufficient for nuclear localization. To confirm the NLS functions in vivo, we constructed the vectors including the MS188 gene without the NLS sites, which failed to complement the male sterile phenotype of ms188. We also searched the highly conserved NLSs in other R2R3-MYB TFs and showed they are required for nuclear localization. Collectively, these findings revealed the specific NLS regions within R2R3-MYB transcription factors and highlighted their critical role for subcellular localization in plant developmental regulation.