SvSTL1 in the large subunit family of ribonucleotide reductases plays a major role in chloroplast development of Setaria viridis.

SUMMARY: Ribonucleotide reductases (RNRs) are essential enzymes in DNA synthesis. However, little is known about the RNRs in plants. Here, we identified a svstl1 mutant from the self‐created ethyl methanesulfonate (EMS) mutant library of Setaria viridis. The mutant leaves exhibited a bleaching phenotype at the heading stage. Paraffin section analysis showed the destruction of the C4 Kranz anatomy. Transmission electron microscopy results further demonstrated the severely disturbed development of some chloroplasts. MutMap analysis revealed that the SvSTL1 gene is the primary candidate, encoding a large subunit of RNRs. Complementation experiments confirmed that SvSTL1 is responsible for the phenotype of svstl1. There are two additional RNR large subunit homologs in S. viridis, SvSTL2 and SvSTL3. To further understand the functions of these three RNR large subunit genes, a series of mutants were generated via CRISPR/Cas9 technology. In striking contrast to the finding that all three SvSTLs interact with the RNR small subunit, the phenotype varied along with the copies of chloroplast genome among different svstl single mutants: the svstl1 mutant exhibited pronounced chloroplast development and significantly fewer copies of the chloroplast genome than the svstl2 or svstl3 single mutants. These results suggested that SvSTL1 plays a major role in the optimal function of RNRs and is essential for chloroplast development. Furthermore, through the analysis of double and triple mutants, the study provides new insights into the finely tuned coordination among SvSTLs to maintain normal chloroplast development in the emerging C4 model plant S. viridis. Significance Statement: Understanding the chloroplast development of C4 plants is very important. Setaria viridis was used as a model system to study the functions of the three RNR large subunit genes in chloroplast development. SvSTL1 was identified by forward genetics, and SvSTL2 and SvSTL3 were identified by homolog analysis. All three genes were knocked out by CRISPR/Cas9. SvSTL1 plays a major role in chloroplast development. This study provides new insights into the fine control of RNRs on chloroplast development in an emerging C4 model. [ABSTRACT FROM AUTHOR]