Nitrogen response and growth trajectory of sorghum CRISPR-Cas9 mutants using high-throughput phenotyping

Inorganic nitrogen (N) fertilizer has emerged as one of the key factors driving increased crop yields in the past several decades. However, the overuse of chemical N fertilizer has led to severe ecological and environmental burdens. Understanding how crops respond to N fertilizer has become a central topic in plant science and plant genetics, with the ultimate goal of enhancing N use efficiency (NUE) in crop production. As one of the most essential macronutrients, N significantly influences crop performance across different developmental stages of plants, and phenotypic traits result from the cumulative effects of genetic factors, prevailing environmental conditions (specifically N availability), and their complex interactions. Previous studies have selected a set of genes potentially affecting Sorghum nitrogen responsiveness to be characterized. The knockout mutants of these genes are generated using the CRISPR-Cas9 technique. Using a LemnaTec plant imaging system, this study obtained time series imagery data from 29 to 130 d after sowing (DAS) for these CRISPR-edited mutants under high N and low N greenhouse conditions. After imagery data analysis, temporal pixel count and greenness index traits were extracted as a proxy of plant growth and N responses, which, subsequently, were modeled by mathematical functions, allowing us to estimate seven key parameters from the growth curves. Our findings revealed that the wildtype and the edited sorghum lines exhibited differences in N responses for several of the key growth-related parameters, with the Edit 1 showing especially reduced sensitiveness to use the available N resources. This high-throughput N phenotyping pipeline paves the way for a better understanding of the N responses of edited lines in a dynamic manner and sheds light on further improvements in crop NUE.