Effect of Partial Knockout of the Plastid Starch Phosphorylase Gene NtPHO1-L1 on the Metabolism of Carbohydrates and Carotenoids in Nicotiana tabacum L. Leaves

Abstract Starch metabolism is regulated by a complex catalytic network, one of the key enzymes of which is the plastid starch phosphorylase PHO1. In this study, using the CRISPR-Cas9 system, we obtained tobacco ( Nicotiana tabacum L.) plants with a partial knockout of the NtPHO1-L1 gene due to deletion variants of the catalytic domain of the NtPHO1-L1 protein, leading to the formation of nonfunctional forms of the enzyme. The edited lines differed from wild-type plants by increased starch accumulation and decreased content of sugars, chlorophylls, and carotenoids in the leaves. It was shown that, compared to the control, the edited plants were characterized by differential expression of starch ( NtPHO1-L1 , NtGWD , NtBAM1 , NtBAM9 , NtAI ) and carotenoid ( NtPSY2 , NtPDS , NtZDS , NtCRTISO , NtVDE ) metabolism genes, as well as genes encoding MADS-domain transcription factors ( NtFUL1 , NtSEP1 , NtSEP2 , NtSEP3 ), which are presumably involved in the regulation of transcription of the studied metabolic genes. These data suggest that partial knockout of NtPHO1-L1 alters the functional activity of tobacco starch phosphorylase. This, in turn, may influence the coordinated activity of starch catabolism enzymes, as well as chlorophyll and carotenoid synthesis enzymes, possibly through differential expression of MADS-box genes. The results highlight the critical regulatory role of plastid starch phosphorylase in transient starch metabolism and in stimulating plant photosynthesis.