# P-Type Pentatricopeptide Repeat Proteins YS1 and YS2 Function in Splicing of petB Intron to Maintain Chloroplast Homeostasis During Rice Seedling Development

## TL;DR

This study identifies two PPR proteins, YS1 and YS2, that are essential for splicing a chloroplast gene in rice, which is crucial for seedling development and chloroplast function.

## Contribution

The novel contribution is the discovery of YS1 and YS2's role in petB intron splicing, revealing their functional redundancy in chloroplast homeostasis during rice development.

## Key findings

- YS1 and YS2 jointly regulate rice seedling development by facilitating petB intron splicing.
- Loss of YS1 and YS2 leads to chloroplast dysfunction, reduced photosynthesis, and seedling death.
- YS1 and YS2 are vital for chloroplast RNA processing and carbohydrate metabolism in rice.

## Abstract

Regulating chloroplast gene expression is crucial for maintaining chloroplast function and plant development. Pentatricopeptide repeat (PPR) proteins form a vast protein family that regulates organelle genes and has multiple functions during plant development. Here, we found that two P-type PPR proteins, YS1 (yellow-green seedling 1) and YS2, jointly regulated seedling development in rice. The loss of YS1 and YS2 exhibited the collapsed chloroplast thylakoids and decreased photosynthetic activity, leading to the yellowing and death of rice seedlings. YS1 and YS2 could directly bind to the transcript of the psbH-petB intergenic region to facilitate the splicing of petB intron, thereby affecting the splicing efficiency of petD, which is located downstream of petB in the five-cistronic transcription unit psbB-psbT-psbH-petB-petD. The mutations in YS1 and YS2 led to decreased mature transcripts of petB and petD after splicing, significantly reducing the protein levels of PetB and PetD. This further led to deficiencies in the cytochrome b6/f and photosystem I complexes of the electron transport chain (ETC), ultimately resulting in decreased ETC-produced NADPH and reduced contents of carbohydrates in ys mutants. Moreover, transcriptome sequencing analysis revealed that YS1 and YS2 were vital for chloroplast organization and carbohydrate metabolism, as well as chloroplast RNA processing. In previous studies, the mechanism of petB intron splicing in the five-cistronic transcription unit psbB-psbT-psbH-petB-petD of rice is unclear. Our study revealed that the two highly conserved proteins YS1 and YS2 were functionally redundant and played critical roles in photosynthesis and seedling development through their involvement in petB intron splicing to maintain chloroplast homeostasis in rice. This work broadened the perspective on PPR-mediated chloroplast development and laid a foundation for exploring the biofunctions of duplicated genes in higher plants.