Large and stable genome edits at the sorghum alpha kafirin locus result in changes in chromatin accessibility and globally increased expression of genes encoding lysine enrichment

Abstract

Introduction: Sorghum is a resilient and widely cultivated grain crop used for feed and food. However, it's grain is deficient in lysine, an essential amino acid. This is due to the primary seed storage proteins, the alpha-kafirins, lacking lysine. It has been observed that reductions in alpha-kafirin protein results in rebalancing of the seed proteome and a corresponding increase in non-kafirin proteins which leads to an increased lysine content. However, the mechanisms underlying proteome rebalancing are unclear. This study characterizes a previously developed gene edited sorghum line, with deletions at the alpha kafirin locus.

Methods: A single consensus guide RNA leads to tandem deletion of multiple members of the gene family in addition to the small target site mutations in remaining genes. RNA-seq and ATAC-seq were utilized to identify changes in gene expression and chromatin accessibility in developing kernels in the absence of most alpha-kafirin expression.

Results: Several differentially accessible chromatin regions and differentially expressed genes were identified. Additionally, several genes upregulated in the edited sorghum line were common with their syntenic orthologues differentially expressed in maize prolamin mutants. ATAC-seq showed enrichment of the binding motif for ZmOPAQUE 11, perhaps indicating the transcription factor's involvement in the kernel response to reduced prolamins.

Discussion: Overall, this study provides a resource of genes and chromosomal regions which may be involved in sorghum's response to reduced seed storage proteins and the process of proteome rebalancing.

Keywords: ATACseq; CRISPR/Cas9; RNAseq; grain quality; lysine; seed protein; sorghum.

Figure 1

Protein phenotype of 19Q4-13. (A) 19Q4-13 displayed a 43% increase in protein-bound lysine content over wild-type Tx430 (p<0.05). (B) 19Q4-13 displayed a 161% increase in free lysine content over wild-type Tx430 (p<0.05). (C) The red arrow points to the 22kDa band, showing a reduction in the 22kDa alpha kafirin. (D) Edited line 19Q4-13 showed a similar level of kernel vitreousness to wild-type Tx430. An example of an edited sorghum line with a comparably less vitreous texture is shown below.

Figure 2

Edit characterization of 19Q4-13. (A) Genomic location of alpha kafirin gene copies on chromosome 5. X-axis values are chromosome 5 basepair 1x10e-7. (B) 8 out of 16 gene copies had an edited allele, ranging from 1bp deletions to large structural variants.

Figure 3

Next generation sequencing data analysis. (A) ATAC-seq alignment displayed an expected enrichment in reads around transcription start sites. (B–E) Blue represents DACR and DEGs upregulated in 19Q4-13, while pink represents those upregulated in Tx430. (B) ATAC-seq read counts in peaks did not appear to show clustering between Tx430 and 19Q4-13. (C) Volcano plot of ATAC-seq peaks. (D) RNA-seq expression data showed clustering between 19Q4-13 and Tx430 using the first 2 principal components. (E) Volcano plot of RNA-seq data.

Figure 4

Expression differences among alpha kafirin gene copies indicates knockdown, but not knockout, of kafirin gene expression. Some kafirin gene copies which were unedited displayed reduced expression. Given the considerable structural variation, disruption in regulatory sequences may be preventing normal transcription of the unedited gene copies. TPM in the y-axis represents transcripts-per-million.

Figure 5

Relationship between log10 fold change and predicted lysine residues among DEGs. Genes upregulated in 19Q4-13 appear to have a higher number of predicted lysine residues than Tx430. Among DEGs, the mean number of lysine residues was 15.03 in 19Q4-13 up regulated genes versus 7.94 in genes upregulated in wild type (p=0.003). Blue indicates DEGs upregulated in 19Q4-13, while pink indicates DEGs upregulated in Tx430.