A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development
- Western Sydney University, Australia
- The Australian National University, Australia
Figures
Figure 1 with 2 supplements
A shorter photoperiod alters plastid development and pigmentation in ccr2.
(A) Three-week-old wild type (WT) and ccr2 plants growing under a 16 hr light photoperiod. (B) Two-week-old plants were shifted from a 16 hr to 8 hr photoperiod for one week and newly emerged or …
Figure 1—figure supplement 1
cis-carotene biosynthesis and regulation of PLB formation during skotomorphogenesis.
(A) A pathway for cis-carotene and xanthophyll synthesis. Tri-cis-ζ-carotene and tetra-cis-lycopene are isomerised by ZISO and CRTISO to form di-cis-ζ-carotene and lycopene, respectively. ziso and cc…
Figure 1—figure supplement 2
A shorter photoperiod promotes yellow leaf virescence affecting chlorophyll levels and carotenoid composition in ccr2.
(A) WT and ccr2 plants were grown under a lower intensity of light (50 µmol m−2 s−1) and representative images taken 14 DAG. (B) and (C) WT and ccr2 plants were grown under a very short 8 hr …
Figure 2
Altered plastid development in ccr2 is linked with cis-carotene accumulation and not to a perturbation in ABA or SL.
(A) Mutants that perturb the levels of lutein, ABA, SL and accumulate cis-carotenes (ccr2, ccr1 and ziso) were grown for two weeks under a 16 hr photoperiod and then shifted to a shorter 8 hr …
Figure 3
A forward genetics screen identified revertant lines of ccr2 having reduced lutein and normal chlorophyll accumulation when grown under a shorter photoperiod.
(A) Representative images of rccr2−155 and rccr2−154 rosettes one week after shifting two-week old plants from a 16 hr to 8 hr photoperiod. (B) Percentage lutein relative to total carotenoids in …
Figure 4
ziso alters cis-carotene profile to restore PLB formation, plastid development and cotyledon greening in ccr2.
(A) Schematic structure of the wild type ZISO gDNA, ZISO protein and the truncated version of the ZISO-155 genomic sequence. ccr2 ziso-155 contains a G->A mutation in AT1G10830 (3606630 bp) as …
Figure 5 with 1 supplement
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 …
Figure 5—figure supplement 1
det1-154 has alternative splicing and reduced pigments, cis-carotenes and restored PLB formation in ccr2.
(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 …
Figure 6 with 1 supplement
The carotenoid cleavage dioxygenase (CCD) inhibitor, D15, restores PLB formation in etiolated ccr2 seedlings, cotyledon greening following de-etiolation and alters cis-carotene accumulation.
(A) Transmission electron micrographs of a representative etioplast from 5-d-old dark grown cotyledons reveal a well-developed PLB in ccr2 treated with the D15, but not in ccr2 treated with ethanol …
Figure 6—figure supplement 1
The loss-of-function in individual members of the carotenoid cleavage dioxygenase gene family cannot restore plastid development in ccr2 rosettes.
Two-week-old WT, ccr2, ccr2 ccd1, ccr2 ccd4, ccr2 ccd7, and ccr2 ccd8 (F3 homozygous double mutant lines) plants were shifted from a 16 hr to 8 hr photoperiod until newly formed leaves in the ccr2 …
Figure 7 with 1 supplement
Chemical inhibition of CCD activity revealed how a ccr2 generated apocarotenoid signal transcriptionally up-regulates POR and PIF3 in parallel to det1-154 during skotomorphogenesis.
(A) Transcript levels of PORA, PIF3 and HY5 in WT, ccr2, ccr2 det1-154 and det1-154 etiolated seedlings growing on MS media (+ /- D15). Statistical analysis denoted as a star was performed by a …
Figure 7—figure supplement 1
The DET1-154 peptide is smaller in det1-154 mutant genotypes.
(A) Representative western blot image showing the reduced DET1 peptide size in ccr2 det1-154 and det1-154 (59 kDa) compared to WT and ccr2 (62 kDa). Gel electrophoresis of the gel membrane from Figur…
Figure 8
Chemical inhibition of CCD activity revealed how a ccr2 generated apocarotenoid signal transcriptionally represses HY5 and LHCB2 expression during photomorphogenesis.
(A) Transcript levels of PIF3 and HY5 in WT, ccr2, ccr2 det1-154 and det1-154 de-etiolated seedlings growing on MS media + /- D15. (B) Representative western blot images showing PIF3 and HY5 protein …
Figure 9
Model showing how a cis-carotene derived apocarotenoid cleavage product controls plastid development in leaves from plants growing under a shorter photoperiod.
Shorter photoperiods that have an extended period of darkness, cause cis-carotenes to accumulate in leaf tissues from plants having impaired or lacking carotenoid isomerase activity. Plants growing …
Tables
Table 1
A cis-carotene derived ACS acts in parallel to DET1 to control PLB formation.
| Germplasm | Hypocotyl Length (mm) | Apical hook | Cotyledon | % PLB (-D15) | % PLB (+D15) | cis-carotenes | |
|---|---|---|---|---|---|---|---|
| WT | Normal | 13.4 ± 0.2 | Yes | Closed | 100 | 100 | None detected |
| ccr2 | normal | 13.8 ± 0.2 | yes | closed | 0 | 85 | phyt, pflu, ζ-C, p-N, p-Lyc |
| ccr2 det1-154 | shorter | *8.3 ± 0.2 | no | open | 69 | 0 | reduced cis-carotenes |
| det1-154 | shorter | *9.9 ± 0.1 | no | open | ND | ND | phyt, pflu and ζ-C |
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ND; not determined; p-N; pro-neurosporene, p-Lyc; pro-lycopene (tetra-cis-lycopene), phyt; phytoene, pflu; phytoflurene, ζ-c; ζ-carotene, *; denotes statistical significance (ANOVA, p<0.05).
Table 2
Contra-regulated differential gene expression in etiolated seedlings and young leaves of ccr2 ziso-155.
| Gene id | GENE | PhANG | Protein encoding description | Etiolated seedlings | Young leaves | det1-1 | NF-1 | NF-2 | ||
|---|---|---|---|---|---|---|---|---|---|---|
| ccr2 | ccr2 ziso-155 | ccr2 | ccr2 ziso-155 | |||||||
| At1g09530 | PIF3 | Transcription factor interacts with photoreceptors and negatively regulates signalling | 30 | 0.1 | 220 | 0.1 | ↓ | −5.0 | NS | |
| At4g10180 | DET1/FUS2 | Encodes a nuclear-localized protein repressor of photomorphogenesis | 5.1 | 0.1 | 5.9 | 0.2 | NS | NS | NS | |
| At3g19390 | Granulin repeat cysteine protease family protein | 4.4 | NS | 6.8 | NS | NS | NS | NS | ||
| At5g13210 | Unknown conserved expressed protein | 3.8 | NS | 0.4 | NS | ↑ | NS | NS | ||
| At3g45730 | Unknown expressed protein | 2.8 | NS | 2.4 | NS | NS | NS | 10.6 | ||
| At5g43500 | ATARP9 | Encodes an expressed protein similar to actin-related proteins | 2.4 | NS | 2.2 | NS | NS | NS | NS | |
| At5g48240 | Unknown expressed protein | 2.1 | NS | 2.2 | NS | NS | NS | NS | ||
| At2g32950 | COP1/FUS3 | Repressor of photomorphogenesis and induces skotomorphogenesis | 2.0 | 0.0 | 8.9 | 0.1 | ↑ | NS | NS | |
| At5g11260 | HY5 | Transcription factor negatively regulated by COP1, promotes light responsive gene expression | 0.5 | 8.1 | 0.3 | 8.4 | NS | NS | 2.8 | |
| At4g02770 | PSAD1 | Expressed protein with similarity to photosystem I subunit II | 0.5 | NS | 0.5 | NS | ↑ | −12.3 | 0.15 | |
| At3g17070 | Peroxidase family expressed protein | 0.5 | NS | 0.5 | NS | NS | NS | NS | ||
| At2g31751 | Potential natural antisense gene, expressed protein | 0.4 | NS | 0.5 | NS | NS | NS | NS | ||
| At4g15560 | DXS/CLA1 | yes | 1-deoxyxylulose 5-phosphate synthase activity in MEP pathway | 0.3 | 4.2 | 0.1 | 16.2 | NS | NS | 0.42 |
| At4g34350 | ISPH/CLB6 | yes | 4-hydroxy-3-methylbut-2-enyl diphosphate reductase in MEP pathway | 0.3 | 9.4 | 0.2 | 11 | ↑ | NS | NS |
| At1g24510 | TCP-1 | T-complex expressed protein one epsilon subunit | 0.3 | 12.0 | 0.1 | 7.9 | NS | NS | NS | |
| At3g59010 | PME35 | Pectin methylesterase that regulates the cell wall mechanical strength | 0.2 | NS | 0.4 | NS | ↓ | NS | NS | |
| At1g29930 | CAB1/LHCB1.3 | yes | Subunit of light-harvesting complex II (LHCII), which absorbs light | 0.2 | 13 | 0.2 | 11 | NS | NS | NS |
| At2g05070 | LHCB2.2 | yes | Light-harvesting chlorophyll a/b-binding (LHC) protein that constitute the antenna system | 0.2 | NS | 0.2 | NS | ↑ | −3.6 | NS |
| At5g13630 | GUN5/CHLH | yes | Magnesium chelatase involved in plastid-to-nucleus signalling | 0.2 | 17 | 0.2 | 20 | ↑ | −3.3 | 0.33 |
| At1g67090 | RBCS1a | yes | Member of the Rubisco small subunit (RBCS) multigene family functions in photosynthesis | 0.1 | 67 | 0.1 | 61 | NS | NS | NS |
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Notes: NS; not significant. Transcriptomic data; det1-1 (Schroeder et al., 2002), norflurazon (NF-1; Page et al., 2017), norflurazon (NF-2; Koussevitzky et al., 2007), PhANG; Photosynthesis associated nuclear gene. Numbers refer to fold change relative to WT = 0 (except for NF-1 where positive and negative numbers indicate up and down-regulation, respectively relative to WT = 1.
Additional files
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Supplementary file 1
Immature ccr2 tissues have an altered cis-carotene and xanthophyll composition.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp1-v1.docx
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Supplementary file 2
D15 and ziso restore PLB formation in ccr2 etiolated cotyledons.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp2-v1.docx
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Supplementary file 3
Transcriptomic analysis of WT, ccr2 and ccr2 ziso-155 etiolated tissues.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp3-v1.xlsx
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Supplementary file 4
Transcriptome analysis of WT, ccr2 and ccr2 ziso-155 immature leaf tissues.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp4-v1.xlsx
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Supplementary file 5
Significantly expressed genes regulated in ccr2 and contra-regulated ccr2 ziso-155 that are common to both etiolated and immature leaf tissues.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp5-v1.xlsx
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Supplementary file 6
det1 reduced carotenoids and caused cis-carotenes to accumulate in leaves and etiolated tissues.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp6-v1.docx
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Supplementary file 7
Primer sequences used for qRT-PCR and ccr2 det154 characterisation.
- https://cdn.elifesciences.org/articles/45310/elife-45310-supp7-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/45310/elife-45310-transrepform-v1.docx
