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. 2020 Jan 31;9:e45310. doi: 10.7554/eLife.45310

Figure 5. det1 restores PLB formation, plastid development and cotyledon greening in ccr2.

(A) Schematic structure of the wild type DET1 gDNA, DET1 protein and alternative spliced DET1-154 protein. A G->A mutation at the end of exon 4 (1449 bp) of AT4G10180 (6347991 bp) was confirmed by Sanger sequencing that leads to the skipping of exon 4 (69 bp). The DET1-154 splice variant produces a shorter protein (521 aa). Exon 4 comprises 23 amino acids in-frame, having homology to the six-hairpin glycosidase-like (IPR008928) domain. (B) Rosette images of WT, ccr2, ccr2 det1-154, and ccr2 det1-154::DET1-OE showing leaf pigmentations in newly emerged leaves from plants shifted from a 16 hr photoperiod (2 weeks old) to an 8 hr photoperiod for 1 week. Images are representative of 122/149 T1 generation ccr2 det1-154 plants from 12 independent lines surviving Basta herbicide selection after being transformed with pEARLEY::DET1-OE. (C) Carotenoid profiles of 7-d-old dark grown cotyledons from WT, ccr2, ccr2 det1-154 and det1-1 etiolated seedlings. Wavelengths close to the absorption maxima of A440 (major carotenoids and ζ-carotene isomers) show neoxanthin (N); violaxanthin (V); lutein (L), β-carotene (β-C) in WT and neurosporene isomers (1 and 2) tetra-cis-lycopene (3); pro-neurosporene (4), and pro-ζ-carotene (5) in ccr2 and to a less extent in ccr2 det1-154. (D) Etiolated seedling morphology of WT, ccr2, ccr2 det1-154 and det1-154. Seedlings were grown in the dark for 7 d on MS media without sucrose. Representative images (>100 seedlings from independent experiments) depict a typical apical hook for WT and ccr2, and shorter hypocotyl with open cotyledons for ccr2 det1-154 and det1-154. (E) Chlorophyll levels in cotyledons following de-etiolation. ccr2, ccr2 det1-154 and WT were etiolated for 4 d in darkness and thereafter exposed to continuous white light. Chlorophyll measurements were taken at 0, 24, 48 and 72 hr after de-etiolation. Letters within a time point denote statistical analysis by one-way ANOVA with a post-hoc Tukey test (n > 20 seedlings). Error bars denote standard error of means.

Figure 5.

Figure 5—figure supplement 1. det1-154 has alternative splicing and reduced pigments, cis-carotenes and restored PLB formation in ccr2.

Figure 5—figure supplement 1.

(A) qRT-PCR confirms alternative splicing of exon four in ccr2 det1-154 leaf tissues. Primers were designed to quantify the full length (+ Exon 4; spanning exons 3–4 and 4–5 junctions) and the spliced (- Exon 4: spanning exon 3–5 and 6–7 junctions) DET1-154 mRNA transcript levels in WT and ccr2 det1-154 leaf tissues, respectively. Standard error bars are shown (n = 4). (B) ccr2 det1-154 displays phenotypes resembling det1-1, including a small rosette, shorter floral architecture and partial sterility in comparison to WT and ccr2. (C) ccr2 det1-154 shows reduced pigment levels compared to ccr2. Neoxanthin (N); violaxanthin (V); antheraxanthin (A), lutein (L), β-carotene (β-C), total carotenoids (T) and total chlorophylls (Chl) were quantified at a 440 nm. Mean values are displayed and error bars denote standard error (n = 3). Star denotes significance (ANOVA, p<0.05). Data is representative of multiple experiments. (D) det1-154 reduces cis-carotene content in ccr2. phytoene (phyt), phytofluene (pflu), tri-cis-ζ-carotene (3ζ-C), di-cis-ζ-carotene (2ζ-C), pro-neurosporene (p-N), tetra-cis-lycopene (p-lyc) and total cis-carotenes were quantified at absorption wavelengths providing maximum detection. Star denotes significance (ANOVA, p<0.05). Data is representative of two independent experiments and error bars show standard error (n = 4). (E) Transmission electron micrographs of a representative etioplast from 5-d-old dark grown cotyledons showing a well-developed PLB in ccr2 det1-154.