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bioRxiv · 10.1101/2024.05.06.592765

The major nucleoid-associated protein WHIRLY1 promotes chloroplast development in barley

Krupinska, K.·Eirich, J.·Repnik, U.·Desel, C.·Saeid Nia, M.·Schmidt, A.·Voigt, U.·Bennewitz, B.·Bilger, W.·Finkemeier, I.·Hensel, G.
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Abstract

WHIRLY1 is a DNA-binding protein of high abundance in chloroplast nucleoids, which have a complex proteome consisting of proteins involved in gene expression and unexpected proteins indicating links to energy production and biosynthetic activities of chloroplasts. In addition, WHIRLY1 has a second localization in the nucleus making it an excellent candidate for chloroplast-to-nucleus communication. To unravel the role of WHIRLY1 for structure and protein composition of nucleoids and its potential involvement in retrograde signaling during chloroplast development, knockout mutants of HvWHIRLY1 were prepared by site-directed mutagenesis using Cas9 endonuclease. In contrast to mutants of rice and maize, which die after the seedling stage, the barley why1 mutants survive and produce grains. Leaves of the mutants are initially pale and get green with time (xantha-to-green phenotype). However, the chlorophyll content of primary leaves stayed distinctly lower than that of the wild-type leaves, coinciding with a rather heterogeneous plastid population, whereby only 50% developed a rather normal thylakoid membrane system. For comparison, mature foliage leaves had almost normal levels of chlorophyll but a severely reduced photosynthetic capacity. A proteome analysis of chloroplasts isolated from mature foliage leaves revealed that in the absence of WHIRLY1, the abundances of a considerable fraction of proteins were downregulated. The fraction included multiple nucleoid-associated proteins including components of the transcriptional apparatus. Furthermore, ribosomal proteins, subunits of pyruvate dehydrogenase, CLP protease, ATP synthase, Rubisco and chaperons/chaperonins were found to be downregulated. In conclusion, the characterization of the barley why1 mutant plants revealed that WHIRLY1 is not essential for chloroplast development. Rather, it ensures a fast and failure-free progression of chloroplast development by remodeling nucleoids, which serve as assembly platforms for a concerted workflow of the numerous processes required for chloroplast development. Gene expression analyses revealed that the disturbance of chloroplast development is signaled to the nucleus, indicating that WHIRLY1 is not part of the biogenic retrograde signaling of plastids.

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Krupinska, K., Eirich, J., Repnik, U., Desel, C., Saeid Nia, M., Schmidt, A., Voigt, U., Bennewitz, B., Bilger, W., Finkemeier, I., Hensel, G.. 2024-05-07. The major nucleoid-associated protein WHIRLY1 promotes chloroplast development in barley. https://doi.org/10.1101/2024.05.06.592765

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