CRISPR/Cas9-mediated targeted mutation of the E1 decreases photoperiod sensitivity, alters stem growth habits, and decreases branch number in soybean

Abstract

The distribution of elite soybean (<i>Glycine max</i>) cultivars is limited due to their highly sensitive to photoperiod, which affects the flowering time and plant architecture. The recent emergence of CRISPR/Cas9 technology has uncovered new opportunities for genetic manipulation of soybean. The major maturity gene <i>E1</i> of soybean plays a critical role in soybean photoperiod response. Here, we performed CRISPR/Cas9-mediated targeted mutation of <i>E1</i> gene in soybean cultivar Tianlong1 carrying the dominant <i>E1</i> to investigate its precise function in photoperiod regulation, especially in plant architecture regulation. Four types of mutations in the <i>E1</i> coding region were generated. No off-target effects were observed, and homozygous trans-clean mutants without T-DNA were obtained. The photoperiod sensitivity of <i>e1</i> mutants decreased relative to the wild type plants; however, <i>e1</i> mutants still responded to photoperiod. Further analysis revealed that the homologs of <i>E1</i>, <i>E1</i>-<i>La</i>, and <i>E1</i>-<i>Lb</i>, were up-regulated in the <i>e1</i> mutants, indicating a genetic compensation response of <i>E1</i> and its homologs. The <i>e1</i> mutants exhibited significant changes in the architecture, including initiation of terminal flowering, formation of determinate stems, and decreased branch numbers. To identify <i>E1</i>-regulated genes related to plant architecture, transcriptome deep sequencing (RNA-seq) was used to compare the gene expression profiles in the stem tip of the wild-type soybean cultivar and the <i>e1</i> mutants. The expression of shoot identity gene <i>Dt1</i> was significantly decreased, while <i>Dt2</i> was significantly upregulated. Also, a set of MADS-box genes was up-regulated in the stem tip of <i>e1</i> mutants which might contribute to the determinate stem growth habit.