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

Figure 1. 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 expanded leaves appeared yellow in ccr2 (YL; yellow outline), while WT displayed green leaves (GL; green outline). (C) Chlorophyll levels (µg/gfw) and pigment ratios in green (WT and ccr2) and yellow (ccr2) leaves formed one week after a photoperiod shift from 16 hr to 8 hr. Standard error is shown for TChl (n = 5, single leaf from five plants). Star denotes significant differences (ANOVA; p<0.05). (D) Absolute carotenoid levels (μg/gfw) in green (WT and ccr2) and yellow (ccr2) leaves formed one week after a photoperiod light shift from 16 hr to 8 hr. Values represent average and standard error bars are displayed (n = 5, single leaf from five plants). Lettering denotes significance (ANOVA; p<0.05). Neoxanthin (neo), violaxanthin (viol), antheraxanthin (anth), lutein (lut), zeaxanthin (zea), β-carotene (β-car), Total Chlorophyll (TChl), Chlorophyll a/b ratio (Chl a/b), Total carotenoids (TCar). (E)Transmission electron micrograph images showing representative chloroplasts from WT and ccr2 green leaf sectors as well as yellow leaf sectors of ccr2.

Figure 1.

Figure 1—figure supplement 1. cis-carotene biosynthesis and regulation of PLB formation during skotomorphogenesis.

Figure 1—figure supplement 1.

(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 ccr2 mutants accumulate cis-carotenes (Park et al., 2002; Chen et al., 2010). In the light, photoisomerisation facilitates cis-carotene isomerisation. Norflurazon (NF) inhibits PDS activity. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) activity may cleave cis-carotenes to generate an apocarotenoid signal (ACS) (Kachanovsky et al., 2012; Fantini et al., 2013; Avendaño-Vázquez et al., 2014; Álvarez et al., 2016). Chemical treatment of seedlings with D15 can inhibit CCD activity and enhance carotenoid accumulation (Van Norman et al., 2014). Mutants that block the production of lutein (lut2; lutein-deficient 2), strigolactone (max4-3; more axillary branching 4) and abscisic acid (aba1-3; aba deficient 1) were utilised to interrogate the cause of the ccr2 leaf virescence phenotype. (B) Control of prolamellar body (PLB) formation and protein levels during skotomorphogenesis. DET1 acts as a repressor of photomorphogenesis in etiolated tissues to maintain high PIF3 and low HY5 protein levels, which reduce PHOTOSYNTHESIS ASSOCIATED NUCLEAR GENE (PhANG) expression. det1 mutants do not accumulate PORA and do not form a PLB within the etioplast. Upon de-etiolation, the protein levels DET1 and PIF3 decline and HY5 increases, which induces PhANG expression. Grey insert boxes digitally represent published western protein blots for PORA (Lebedev et al., 1995), PIF3 (Dong et al., 2014) and HY5 (Osterlund et al., 2000) in WT and det1 mutant genotypes. Solid black and grey fills represents high and low protein expression, respectively. Green arrows and red lines represent positive and negative regulation, respectively. Abbreviations: GGPP, geranylgeranyl pyrophosphate; PSY, PHYTOENE SYNTHASE; PDS, PHYTOENE DESATURASE, ZDS, ζ-CAROTENE DESATURASE; ZISO, ζ-CAROTENE ISOMERASE; CRTISO, CAROTENOID ISOMERASE.
Figure 1—figure supplement 2. A shorter photoperiod promotes yellow leaf virescence affecting chlorophyll levels and carotenoid composition in ccr2.

Figure 1—figure supplement 2.

(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 photoperiod and representative images taken after 14 (B) and 21 (C) days of growth. (D) Chlorophyll content in immature leaves that recently emerged from WT and ccr2 rosettes 14 DAG. Values represent the average and standard deviations of total chlorophyll content (µg/gfw) from a single leaf sector (n = 2–7 plants). Lettering denotes significance by ANOVA using a post-hoc Tukey test (p<0.05). (E) Percentage carotenoid composition (relative to total) in green (WT and ccr2) and yellow (ccr2) virescent leaves developed one week after a 16 hr to 8 hr photoperiod shift. Values represent average and standard error of means are displayed (n = 5, single leaf from five plants). Lettering denotes significance by ANOVA using a post-hoc Tukey test (p<0.05). Neoxanthin (neo), violaxanthin (viol), antheraxanthin (anth), lutein (lutein), zeaxanthin (zea), β-car (β-carotene), Green Leaf (GL), Yellow Leaf (YL).