Fine-tuning sugar content in strawberry

Abstract

Fine-tuning quantitative traits for continuous subtle phenotypes is highly advantageous. We engineer the highly conserved upstream open reading frame (uORF) of FvebZIPs1.1 in strawberry (Fragaria vesca), using base editor A3A-PBE. Seven novel alleles are generated. Sugar content of the homozygous T1 mutant lines is 33.9-83.6% higher than that of the wild-type. We also recover a series of transgene-free mutants with 35 novel genotypes containing a continuum of sugar content. All the novel genotypes could be immediately fixed in subsequent generations by asexual reproduction. Genome editing coupled with asexual reproduction offers tremendous opportunities for quantitative trait improvement.

Keywords: Asexually reproducing crops; Basic leucine zipper; Fine-tuning; Quantitative trait variation; Strawberry; Sugar content; Upstream open reading frame.

Fig. 1

Conserved uORFs of strawberry bZIP genes. a Diagram depicting the sucrose-dependent post-transcriptional control of bZIP genes and the strategy for increasing sugar content by engineering the conserved sucrose control uORF (SC-uORF). The mutant sc-uorf reduced inhibition of translation of the bZIP gene, leading to increased sugar accumulation. uORF, upstream open reading frame. pORF, primary open reading frame. b Phylogenetic tree of the S1 group bZIP genes in Arabidopsis and strawberry. The strawberry S1 group bZIP homologs are shown in red. c Schematic illustration of the organization of the uORFs in the mRNA of the four strawberry bZIP genes. The uORFs are shown by the yellow lines with arrowheads upstream of the blue line with arrowhead representing the pORF. The longest uORF of each gene is the SC-uORF. d Alignment of the conserved SC-uORF amino acid sequences in Arabidopsis, strawberry, and other dicotyledonous and monocotyledonous plants. Black box with white letter, 100% identity; dark gray box with white letter, 80% identity; gray box with black letter, 60% identity

Fig. 2

Engineering the SC-uORF of FvebZIPs1.1 using A3A-PBE. a Diagram of the uORFs in FvebZIPs1.1 and the target of A3A-PBE. Part of the amino acid sequence of the SC-uORF and the corresponding codons in the DNA sequence are shown, and the targeted sequence is underlined. The two ATGs and two codons that encode conserved amino acids (RR) of SC-uORF are marked in red. b Schematic of the A3A-PBE vector. GFP fluorescence was used to identify transgenic plants. pUbi, maize Ubiquitin-1 (Ubi-1) gene promoter. pID, root loci promoter. c Frequencies of mutations induced by A3A-PBE in T0 strawberry plants. GFP⁺, GFP fluorescence positive. d Proportions of the different types of mutation in T0 strawberry plants. e Proportions of C-to-T changes, C-to-G changes, and indels induced by A3A-PBE at the target site. f The four different alleles (single and multiple C-to-T conversions) of the uORF among the T0 mutant plants, and their frequencies. Nucleotide substitutions are indicated in red. The letter subscripts on the cytosines in the WT sequence indicate the positions of these bases in the protospacer, counting from the distal end to the protospacer-adjacent motif. Frequency: number of a given allele /total numbers of alleles

Fig. 3

Effect of the novel alleles on translation of the downstream primary open reading frame. a The novel alleles generated in the T0 generation. Nucleotide substitutions and small deletions are indicated in red. The amino acid residues of SC-uORF corresponding to the codons in the DNA sequence are indicated by yellow filled boxes. Changed amino acids are indicated by red filled boxes. b Schematic of the dual-luciferase reporter vector. 35s pro, cauliflower mosaic virus 35s promoter. REN, Renilla reniformis luciferase. LUC, luciferase. c Effect of the novel alleles on translation of the pORF in the dual-luciferase reporter system. d Effect of the novel alleles on transcription of the pORF in the dual-luciferase reporter system. In c and d, mean values (±SD) are compared to those for wild-type plants using Student’s t tests, **P < 0.01. AL#, allele #

Fig. 4

Sugar content of the novel genotypes. a Model showing the strategy for enriching genotype diversity, and the advantage of asexual reproduction for fixing novel genotypes. b Fructose, glucose, and sucrose contents of the seven T1 homozygous mutants and WT. c Total sugar contents of the seven T1 homozygous mutants and WT. d Transcription of FvebZIPs1.1 in the seven homozygous strains and WT. e Total sugar contents of the 35 novel genotypes  and WT in the T1 generation (means ± SD). In b to d, mean values (±SD) are compared to those for wild-type plants using Student’s t tests, **P < 0.01, *P < 0.05. AL#/AL#, combination of allele# and allele#. Hete-, heterozygote. Homo-, homozygote. Bial-, biallelic

Fig. 5

Growth parameters of homozygotes. a Appearance of the seven homozygous mutant plants and WT. b Leaves of the seven homozygous genotypes and WT. c Leaf lengths, widths, and weights. d Fruits of the eight strains. e Fruit lengths, widths, and weights. In c and e, mean values (±SD) are compared to those of wild-type plants using Student’s t tests. AL#/AL#, homozygote for allele #