# Iron allocation to chloroplast proteins depends on the DNA-binding protein WHIRLY1

## TL;DR

The protein WHIRLY1 helps allocate iron to chloroplast proteins, and its absence leads to chlorotic leaves and disrupted photosynthesis.

## Contribution

WHIRLY1's role in iron allocation and chloroplast protein assembly is identified as a novel function.

## Key findings

- WHIRLY1-deficient plants show chlorotic leaves and impaired photosynthesis under high light.
- Levels of chloroplast iron cofactor proteins are reduced in WHIRLY1-deficient plants.
- Ferritin levels increase in WHIRLY1-deficient plants, possibly compensating for its absence.

## Abstract

The DNA-binding protein WHIRLY1, sharing structural similarities with ferritin, plays a role in the formation of iron cofactor proteins within chloroplasts.

Previous studies indicated that barley plants with a knockdown of HvWHIRLY1 containing a minimal amount of the protein are compromised in chloroplast development and photosynthesis, and get chlorotic leaves when grown at high irradiance. Thereby, the leaves display signs of iron deficiency. Metal determination revealed, however, that leaves of WHIRLY1-deficient plants had a regular iron content. Nevertheless, WHIRLY1-deficiency affected the functionality of photosystem II less than that of photosystem I, which has a higher demand for iron. Immunological analyses revealed that components of both photosystems had reduced levels. Additionally, the levels of other chloroplast proteins containing different classes of iron cofactors were lower in the WHIRLY1-deficient plants compared to the wild type. In contrast, the level of the iron sequestering protein ferritin increased in WHIRLY1-deficient lines, whereby high irradiance intensified this effect. RNA analyses showed that the upregulation of ferritin coincided with an enhanced expression of the corresponding gene, reflecting an apparent overload of chloroplasts with free iron. Ferritin and WHIRLY proteins are known to share the same oligomeric structure. Therefore, the high abundance of ferritin in WHIRLY1-deficient plants might be a compensation for the reduced abundance of WHIRLY1. Enhanced expression levels of genes encoding photosynthesis proteins and iron cofactor proteins indicate a demand for protein formation or assembly of protein complexes. The results support a general role of WHIRLY1 in assembly and/or stabilization of chloroplast proteins and, moreover, suggest a specific function in sequestering and supply of iron in chloroplasts.

The online version contains supplementary material available at 10.1007/s00425-025-04736-8.