ABSTRACT
Carotenoids are core plastid components, yet a regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a (-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the transcriptional repressor of photomorphogenesis, DEETIOLATED1 (DET1), to post-transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5) protein levels. The apocarotenoid signal and det1 complemented each other to restore POR levels and PLB formation, thereby controlling plastid development.
One-sentence summary Carotenoids are not just required as core components for plastid biogenesis, they can be cleaved into an apocarotenoid signal that regulates etioplast and chloroplast development during extended periods of darkness.
- Abbreviations
- ccr
- carotenoid and chloroplast regulation
- rccr2
- revertant of ccr2
- DAG
- days after germination
- YL
- yellow leaf
- GL
- green leaf
- NF
- norflurazon
- ACS
- apocarotenoid signal
INTRODUCTION
Carotenoids are a diverse group of hydrophobic isoprenoid pigments required for numerous biological processes in photosynthetic organisms and are essential for human health (Cazzonelli, 2011; Baranski and Cazzonelli, 2016). In addition to providing plant flowers, fruits and seeds with distinct colours, carotenoids have accessory roles in facilitating the assembly of the light harvesting complex, light capture during photosynthesis and photoprotection during high light and/or temperature stress (Nisar et al., 2015; Baranski and Cazzonelli, 2016). The current frontiers are to discover the regulators of carotenoid biosynthesis, storage, and catabolism and apocarotenoids that in turn regulate plant development and photosynthesis (Cazzonelli and Pogson, 2010; Havaux, 2014; Baranski and Cazzonelli, 2016; Hou et al., 2016).
In higher plants, cis-carotene biosynthesis is initiated by the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to form phytoene, which is catalyzed by the rate-limiting enzyme phytoene synthase (PSY) (von Lintig et al., 1997; Li et al., 2008; Rodriguez-Villalon et al., 2009; Welsch et al., 2010; Zhou et al., 2015) (Supplementary Figure 1A). Next, phytoene desaturase (PDS), ζ-carotene desaturases (ZDS), ζ-carotene isomerase (ZISO) and cis-trans-carotene isomerase (CRTISO) convert the colourless phytoene into the pinkish-red coloured all-trans-lycopene (Bartley et al., 1999; Isaacson et al., 2002; Park et al., 2002; Dong et al., 2007; Chen et al., 2010; Yu et al., 2011). In the dark, the isomerisation of tri-cis-ζ-carotene to di-cis-ζ-carotene and tetra-cis-lycopene to all-trans-lycopene has a strict requirement for ZISO and CRTISO activity respectively (Park et al., 2002; Chen et al., 2010). However, light-mediated photoisomerisation in the presence of a photosensitiser can substitute for a lack of isomerase activity (Giuliano et al., 2002; Vijayalakshmi et al., 2015; Alagoz et al., 2018).
The carotenoid biosynthetic pathway branches after lycopene to produce α/β-carotenes (Cunningham et al., 1993; Cunningham et al., 1996; Pecker et al., 1996; Ronen et al., 1999). Next, α-carotene and β-carotene are further hydroxylated to produce the oxygenated carotenoids called xanthophylls (e.g. lutein, violaxanthin and zeaxanthin), which comprise the most abundant carotenoids found in photosynthetic leaves. Carotenoids are precursors for apocarotenoids (carotenoid cleavage products) such as phytohormones abscisic acid (ABA) and strigolactone (SL) as well as other apocarotenoids that function in root-mycorrhizal interactions, leaf development, acclimation to environmental stress and retrograde signaling (Havaux, 2014; Walter et al., 2015; Chan et al., 2016; Hou et al., 2016). The carotenoid cleavage dioxygenase and nine-cis-epoxy-carotenoid dioxygenase (CCD/NCED) family cleave carotenoids to yield apocarotenoids (Hou et al., 2016). The CCDs have substrate preferences depending on the tissue and nature of the assay (Walter and Strack, 2011; Harrison and Bugg, 2014; Bruno et al., 2016). The five members of the NCED sub-group are exclusively involved in cleavage of violaxanthin and neoxanthin to form ABA (Finkelstein, 2013). The four CCDs have well defined roles in carotenoid degradation in seeds (CCD1 and CCD4) and the synthesis of strigolactones (CCD7/MAX3 and CCD8/MAX4) (Auldridge et al., 2006; Gonzalez-Jorge et al., 2013; Ilg et al., 2014; Al-Babili and Bouwmeester, 2015). Non-enzymatic oxidative cleavage of carotenoids can also generate apocarotenoids by singlet oxygen (1O2)-mediated photo-oxidation or by lipoxygenase and peroxidase-mediated co-oxidation (Leenhardt et al., 2006; Gonzalez-Perez et al., 2011). Non-enzymatic carotenoid degradation acts preferentially on selective molecules such as β-carotene and its apocarotenoid derivatives.
cis-carotenes such as phytoene, phytofluene and tetra-cis-lycopene are reported to be resistant to non-enzymatic degradation (Schaub et al., 2018), although there are some reports that CCDs cleave specific cis-carotenes in vitro (Bruno et al., 2016). Whether there is a physiological relevance for a cis-carotene derived cleavage product or apocarotenoid signal (ACS) in vivo, remains unclear. A hunt is on to identify a cis-carotene cleavage product that functions as a retrograde signal to regulate nuclear gene expression (Kachanovsky et al., 2012; Fantini et al., 2013; Avendano-Vazquez et al., 2014; Alvarez et al., 2016). CCD4 is implicated in the generation of a cis-carotene-derived apocarotenoid signal that regulates leaf shape, chloroplast and nuclear gene expression in the Arabidopsis clb5/zds (chloroplast biogenesis-5 / ζ-carotene desaturase) mutant (Avendano-Vazquez et al., 2014). A metabolon regulatory loop around all-trans-ζ-carotene was proposed in tomato fruit that can sense cis-carotene accumulation, their derivatives or the enzymes themselves (Fantini et al., 2013). The accumulation of cis-carotenes in tomato fruit have also been linked to the metabolic feedback-regulation of PSY transcription and translation (Kachanovsky et al., 2012; Alvarez et al., 2016). Therefore, cis-carotenes themselves or their cleavage products appear to have some functional roles, of which the targets and regulatory mechanism(s) remains unknown.
Determining a mechanistic function for cis-carotenes in planta has been challenged by low levels of cis-carotene accumulation in wild type tissues. Although, when the upper carotenoid pathway is perturbed (Alagoz et al., 2018), seedling lethality (psy, pds and zds), impaired chlorophyll and cis-carotene accumulation (ziso and crtiso) as well as a reduction in lutein (crtiso) become apparent (Isaacson et al., 2002; Park et al., 2002). ziso mutants in maize (y9) and Arabidopsis (zic) display pale-green zebra-striping patterns and a delay in cotyledon greening respectively, that resemble a leaf variegation phenotype (Janick-Buckner et al., 2001; Li et al., 2007; Chen et al., 2010). Similarly, crtiso loss-of-function in tomato (tangerine), melon (yofi) and rice (zebra) mutants show varying degrees of unexplained yellow leaf variegation (Isaacson et al., 2002; Park et al., 2002; Chai et al., 2010; Galpaz et al., 2013), the causes of which were assumed to relate to perturbed photosystem biogenesis and operation.
During skotomorphogenesis prolamellar bodies (PLB) develop in etioplasts of seedling tissues. The PLB is a crystalline agglomeration of protochlorophyllide (PChlide), POR enzyme and fragments of pro-thylakoid membranes that provide a structural framework for the light-catalysed conversion of PChlide into chlorophylls by POR within picoseconds in conjunction with the assembly of the photosynthetic apparatus (Sundqvist and Dahlin, 1997; Sytina et al., 2008). The de-etiolation of seedlings upon exposure to light activates a sophisticated network consisting of receptors, genetic and biochemical signals that trigger photomorphognesis. Changes in light-induced morphogenesis include: short hypocotyls; expanded and photosynthetically-active cotyledons with developing chloroplasts; and self-regulated stem cell populations at root and shoot apices (Arsovski et al., 2012; Lau and Deng, 2012). Mutants that block skotomorphogenesis and instead promote photomorphogenesis in the dark, such as DETIOLATED1 (DET1) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) lack POR and thus fail to assemble PLBs (Chory et al., 1989; Sperling et al., 1998; Datta et al., 2006)(Supplementary Figure 1B). This is a consequence of DET1 modulating the levels of PHYTOCHROME INTERACTING FACTOR 3 (PIF3; constitutive transcriptional repressor of photomorphogenesis) and ELONGATED HYPOCOTYL 5 (HY5; positive transcriptional regulator of photomorphogenesis) to control PORA and PhANG expression (Stephenson et al., 2009; Lau and Deng, 2012; Xu et al., 2016; Llorente et al., 2017). Thus, in the dark wild-type plants accumulate PIF3, but lack HY5, conversely det1 lacks PIF3 and accumulates HY5 protein (Supplementary Figure 1B).
PLB formation occurs in carotenoid deficient mutants. Norflurazon treated wheat seedlings grown in darkness can still form a PLB, however it is somewhat aberrant having a looser attachment of POR to the lipid phase and there is an early dissociation from the membranes during photomorphogenesis (Denev et al., 2005). In contrast, ccr2 is similar to cop1/det1 mutants in that it lacks a PLB in etioplasts, yet it is unique in having normal PChlide and POR protein levels (Park et al., 2002). The associated hyper accumulation of cis-carotenes led to the untested hypothesis that cis-carotenes structurally prevent PLB formation in etioplasts of dark germinated ccr2 during skotomorphogenesis and this in turn delayed cotyledon greening following illumination (Park et al., 2002; Datta et al., 2006; Cuttriss et al., 2007). However, it was never apparent why other linear carotenes, such as 15-cis-phytoene and all-trans-lycopene, permitted PLB formation, raising the question as to whether there were regulatory functions for the cis-carotenes that accumulate in ccr2.
In this paper, we describe how changes in photoperiod are sufficient to perturb or permit plastid development in ccr2, the former leading to leaf variegation. A revertant screen of ccr2 revealed new connections between a cis-carotene-derived signaling metabolite, PLB formation, skotomorphogenesis and chloroplast development. We demonstrate how an unidentified apocarotenoid signal acts in parallel to DET1 to regulate PLB formation and post-transcriptionally control POR, PIF3 and HY5 protein levels in order to fine-tune plastid development in tissues exposed to extended periods of darkness.
RESULTS
A shorter photoperiod perturbs chloroplast biogenesis and promotes leaf variegation
The crtiso mutants have been reported to display different leaf pigmentation phenotypes (resembling variegations of yellow and green sectors) in a species-dependant manner, with rice and tomato showing changes in pigmentation, but not Arabidopsis. To address if this is species-dependent we investigated if light regimes affected leaf pigment levels and hence plastid development in Arabidopsis crtiso mutants. Growing ccr2 plants at a lower light intensity of 50 E during a long 16 h photoperiod did not cause any obvious changes in morphology or leaf variegation (Supplemental Figure 2A). Whereas, reducing the photoperiod to 8 h resulted in the newly emerged ccr2 leaves to appear yellow in variegation (Supplemental Figure 2B) due to a substantial reduction in total chlorophyll (Supplemental Figure 2D). As development progressed the yellow leaf (YL) phenotype became less obvious and greener leaves (GL) developed (Supplemental Figure 2C). Therefore, by reducing the photoperiod we were able to replicate in Arabidopsis previous reports in tomato and rice of leaf variegation (Isaacson et al., 2002; Chai et al., 2010).
Next, we demonstrated that day length affects plastid development in newly emerged leaf tissues undergoing cellular differentiation. We replicated the YL phenotype by shifting three weeks old ccr2 plants from a long 16-h to shorter 8-h photoperiod (Figure 1A-B). The newly emerged leaves of ccr2 appeared yellow and virescent, while leaves that developed under a 16-h photoperiod remained green similar to wild type (Figure 1B). Consistent with the phenotype, the yellow sectors of ccr2 displayed a 2.4-fold reduction in total chlorophyll levels, while mature green leaf sectors formed prior to the photoperiod shift had chlorophyll levels similar to that of WT (Figure 1C). The chlorophyll a/b as well as carotenoid/chlorophyll ratios were not significantly different (Figure 1C). Consistent with the reduction in chlorophyll, total carotenoid content in yellow sectors of ccr2 was reduced due to lower levels of lutein, β-carotene and neoxanthin (Figure 1D). The percentage composition of zeaxanthin and antheraxanthin was significantly enhanced in yellow sectors, perhaps reflecting a greater demand for xanthophyll cycle pigments that reduce photooxidative damage (Supplemental Figure 2E). Transmission electron microscopy (TEM) revealed that yellow ccr2 leaf sectors contained poorly differentiated chloroplasts lacking membrane structures consisting of thylakoid and grana stacks, as well as appearing spherical in shape, rather than oval when compared to green leaf tissues from WT or ccr2 (Figure 1E).
(A) Three-week-old wild type (WT) and ccr2 plants growing under a 16-h light photoperiod.
(B) Three-week-old plants were shifted from a 16-h to 8-h 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 h to 8 h. Standard error is shown for TChl (n=5, single leaf from 5 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 h to 8 h. Values represent average and standard error bars are displayed (n=5, single leaf from 5 plants). Lettering denotes significance (ANOVA; p < 0.05). Neoxanthin (neo), violaxanthin (viol), antheraxanthin (anth), lutein (lutein), zeaxanthin (zea), β-car (β-carotene), 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.
(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-h photoperiod and then shifted to a shorter 8-h photoperiod for one week. Representative images showing newly emerged and expanding leaves from multiple experimental and biological repetitions (n > 20 plants per line) are displayed. Genetic alleles tested include Col-0 (WT), ccr2.1 (carotenoid isomerase), lut2.1 (epsilon lycopene cyclase), aba1-3 (Ler background) (zeaxanthin epoxidase), max4/ccd8 (carotenoid cleavage dioxygenase 8), ccr1.1/sdg8 (set domain group 8) and ziso1-3 (ζ-carotene isomerase).
(B) Carotenoid profiles in rosette leaves from three-week-old plants grown under a 16-h photoperiod and subjected to 6-d of extended darkness.
(C) Carotenoid profiles in three-week-old rosette leaves from plants grown under a constant 8-h light photoperiod. Pigments were profiled in a yellow leaf (YL) and green leaf (GL) from WT and ccr2.
(D) Carotenoid profiles in newly emerged floral bud and rosette leaf tissues harvested from four-week-old plants growing under a 16-h photoperiod. Carotenoid profile traces of various tissue extracts from wild type (WT) and ccr2 show pigments at wavelengths close to the absorption maxima of A440nm (Neoxanthin; N, violaxanthin; V, antheraxanthin; A, lutein; L, zeaxanthin; Z, β-carotene isomers; β-C, chlorophyll a; Chl a, chlorophyll b; chl b, tetra-cis-lycopene; plyc, neurosporene isomers; neuro, and ζ-carotene; ζ-C), A348nm (phytofluene; pflu) and A286nm (phytoene; phyt). HPLC profile y-axis units are in milli-absorbance units (mAU). HPLC traces are representative of multiple leaves from multiple experimental repetitions and retention times vary due to using different columns.
The leaf variegation phenotype correlated with cis-carotene accumulation
We next investigated the relationship between photoperiod, perturbations in carotenogenesis and plastid development. Green leaf tissues from ccr2 have an altered proportion of β-xanthophylls at the expense of less lutein, yet plants grown under a longer photoperiod show normal plastid development (Park et al., 2002). This raised a question: does reducing the photoperiod limit the photoisomerisation of tetra-cis-lycopene to all-trans-lycopene thereby altering lutein, ABA and/or strigolactone biosynthesis? To address this, ccr2, lycopene epsilon cyclase (lut2; lutein deficient 2), zeaxanthin epoxidase (aba1-3; aba deficient 1) and carotenoid cleavage dioxygenase 8 (max 4; more axillary branching 4) mutants were shifted from a 16-h to 8-h photoperiod (Figure 2A). ccr2 showed a clear yellow variegation phenotype, while the other mutants produced green leaves similar to that of WT. Therefore, we could not attribute the yellow leaf colour variegation to a reduction in lutein or a perturbation of SL or ABA biosynthesis. Next, we tested if the ccr2 yellow leaf phenotype was linked to the accumulation of cis-carotenes in the pathway upstream of all-trans-lycopene. Mutations in PSY, PDS and ZDS cause leaf bleaching and are not viable in soil. Alternatively, carotenoid chloroplast regulator 1 (ccr1 or otherwise known as sdg8; set domain group 8) and (ζ-carotene isomerase (ziso) mutants are viable and accumulate cis-carotenes in etiolated tissues (Cazzonelli et al., 2009b; Chen et al., 2010). Indeed, both ccr1 and ziso displayed a partial yellow leaf phenotype near the zone of cellular differentiation (e.g. petiole-leaf margin), however unlike ccr2 the maturing leaf tissues restore greening rapidly such that ziso was more similar to WT than ccr2 (Figure 2A).
This raised a question: does a shorter photoperiod lead to the accumulation of cis-carotenes in newly emerged leaf tissues of ccr2 displaying altered plastid development? First, we tested if an extended dark period (6 days) would result in the accumulation of cis-carotenoids in mature (3 weeks) rosette leaf tissues. Compared to adult WT prolonged darkness resulted in notable yellowing of ccr2 leaves and clearly discernible accumulation of tetra-cis-lycopene, neurosporene isomers, ζ-carotene, phytofluene and phytoene (Figure 2B). We next shifted three-week-old plants from a 16-h to 8-h photoperiod and the yellow sectors from newly emerged ccr2 leaves accumulated detectable levels of cis-lycopene, neurosporene isomers, ζ-carotene, phytofluene and phytoene (Figure 2C). Interestingly, even when plants were grown under a 16-h photoperiod, we could detect phytofluene and phytoene in floral buds as well as newly emerged rosette leaves from ccr2, and at trace levels in WT (Figure 2D). In addition, a higher ratio of phytofluene and phytoene relative to β-carotene was observed in newly emerged ccr2 tissues, which coincided with a lower percentage of lutein when compared to older tissues (Supplemental Table 1).
A cis-carotene derived ACS acts in parallel to DET1 to control PLB formation
Second site genetic reversion restored plastid development in ccr2
We undertook a revertant screen to identify genes and proteins that could complement the plastid development in ccr2, while still maintaining a perturbed carotenoid profile. Seeds were mutagenized using ethyl-methane sulfonate (EMS), grown and collected from pools of 5-10 M1 plants. Approximately 40,000 M2 seedlings from 30 stocks of pooled seeds were screened for the emergence of immature green rosette leaves when grown under a 10-h photoperiod. Twenty-five revertant lines reproducibly displayed green immature leaves in response to a photoperiod shift, as exemplified by rccr2154 and rccr2155 (Figure 3A). Leaf tissues of all rccr2 lines contained reduced lutein and xanthophyll composition similar to ccr2 (Figure 3B). When grown under a shorter photoperiod, rccr2 lines produced greener rosettes with less yellow colour variegation compared to ccr2 and chlorophyll levels were similar to WT (Figure 3C-D).
(A) Representative images of rccr2155 and rccr2154 rosettes one week after shifting two-week old plants from a 16-h to 8-h photoperiod.
(B) Percentage lutein relative to total carotenoids in immature leaves from WT, ccr2 and rccr2 lines.
(C) The degree of leaf variegation detected in rosettes following a reduction in photoperiod. Leaf variegation (% of yellow relative to RGB) in WT, ccr2, rccr2154 and rccr2155 rosettes was quantified using the Lemnatec Scanalyser system and software.
(D) Total chlorophyll content in rosette leaves from WT, ccr2, rccr2154 and rccr2155 plants exposed to a shorter photoperiod.
(E) Segregation ratios of rccr2154 and rccr2155 after backcrossing to the ccr2 parent in both Columbia (Col-0) and Landsberg erecta (Ler) ecotypes. (NGS; next generation sequencing)
(F) and (G) NGS of pooled leaf gDNA from a segregating population of rccr2155 (F) and rccr2154 (G) plants revealed less recombination surrounding SNPs at 3606630 bp and 6347991 bp, respectively. Error bars denote standard error of means (SEM) and stars represent statistical significance (ANOVA; p < 0.05).
In order to establish a segregating population for next generation mapping (NGM) rccr2 lines were backcrossed to the original ccr2 parent (Col-0) and/or a ccr2 line established in the Landsberg erecta background (Lccr2). All rccr2 lines were recessive for the reversion of shorter photoperiod dependent yellow leaves (e.g. rccr2154 and rccr2155; Figure 3E). Next generation sequencing (NGS)technologies were used to deep sequence the genomic DNA (gDNA) from leaves of homozygous (M2) plants to identify non-recombinant deserts in chromosome 1 (3605576 bp) and chromosome 4 (6346463 bp) for both rccr2155 and rccr2154, respectively (Figure 3F-G). Both non-recombinant deserts contained SNPs displaying a discordant chastity value of approximately 1.0 representing the causal mutation of interest (Austin et al., 2011).
An epistatic interaction between ziso and ccr2 revealed specific cis-carotenes perturb PLB formation
rccr2155 lacked recombination at the bottom arm of chromosome 1 surrounding a single nucleotide polymorphism (G-A mutation at 3606630 bp) within exon 3 of the ZISO gene (639 bp of mRNA), hereafter referred as ccr2 ziso-155 (Figure 4A). This polymorphism caused a premature stop codon leading to a truncated ZISO protein (212 instead of 367 amino acids). The overexpression of the functional ZISO cDNA fragment in ccr2 ziso-155 restored the leaf variegation phenotype displayed by ccr2 plants grown under an 8-h photoperiod (Figure 4B). A double mutant generated by crossing ccr2 with ziso1-4 further confirmed the loss-of-function in ziso can restore plastid development in newly emerged immature leaves of ccr2. Carotenoid analysis of immature leaf tissues of ccr2 ziso-155 revealed reduced lutein and xanthophyll composition similar to ccr2, indicating that the complementation of the YL was not due to a change in xanthophyll levels (Figure 3B). The epistatic nature between ziso and crtiso revealed that a specific cis-carotene downstream of ZISO activity perturbed plastid development.
Analysis of the cis-carotene profile in etiolated cotyledons showed that ccr2 ziso1-4 had an identical carotenoid profile to that of ziso in that it could only accumulate 9,15,9’-tri-cis-ζ-carotene, phytofluene and phytoene (Figure 4C). In contrast, ccr2 accumulated lower levels of these three compounds, yet higher quantities of 9, 9’-di-cis ζ-carotene, 7,9,9’-tri-cis-neurosporene and 7,9,9’,7’-tetra-cis-lycopene, all of which were undetectable in a ziso background (Figure 4C). Therefore, ziso blocks the biosynthesis of neurosporene isomers, tetra-cis-lycopene and 9, 9’-di-cis ζ-carotene under shorter photoperiods, and they themselves or their cleavage products appear to disrupt plastid development 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 confirmed by Sanger sequencing that results in a premature stop codon (TGA) in exon 3.
(B) Rosette images of WT, ccr2, ccr2 ziso-155, and ccr2 ziso-155::ZISO-OE#5 showing leaf pigmentations in newly emerged leaves following a reduction in photoperiod. Images are representative of 84/89 T4 generation ccr2 ziso-155 plants and six independent lines of ccr2 ziso-155::ZISO-OE.
(C) Carotenoid profiles of dark grown cotyledons from WT, ccr2, ziso1-4, and ccr2 ziso1-4. Wavelengths close to the absorption maxima of A440nm (major carotenoids and ζ-carotene isomers), A348nm (phytofluene) and A286nm (phytoene) are shown. Neoxanthin (N); violaxanthin (V); lutein (L); β-carotene (β-C); neurosporene (1 and 2); tetra-cis-lycopene (3); pro-neurosporene (4); ζ-carotene (5); phytofluene (6); phytoene (7).
(D) Transmission electron micrographs of a representative etioplast from 5-d-old dark grown cotyledons. The etioplasts of WT, ziso and ccr2 ziso-155 show well-developed PLBs, while ccr2 does not have any. Images are representative of 15 plastids from at least 5 TEM sections.
(E) Total chlorophyll levels in cotyledons following de-etiolation. WT, ccr2, ziso1-4, ccr2 ziso-155, and ccr2 ziso1-4 were grown in darkness for 4-d, exposed to continuous white light and chlorophyll measured at 0, 24, 48 and 72-h. Letters within a time point denote statistical analysis by ANOVA with a post-hoc Tukey test (n > 20 seedlings). Error bars denote standard error of means (SEM).
How are the specific cis-carotenes disrupting plastid development? To answer this question, we first examined etiolated cotyledons of WT, ccr2, ziso and ccr2 ziso-155. We confirmed ccr2 lacked a PLB in all sections examined (Figure 4D, Supplemental Table 2). We observed 65% of ziso etioplasts contained PLBs (Figure 4D, Supplemental Table 2). Intriguingly, the vast majority (>94%) of etioplasts examined from ccr2 ziso-155 and ccr2 ziso1-4 contained a PLB (Figure 4D, Supplemental Table 2). Cotyledon greening of de-etiolated seedlings revealed a significant delay in chlorophyll accumulation for both ccr2 and ziso1-4 when compared to WT after 24, 48 and 72 h of continuous white light (Figure 4E). The reduced levels of chlorophyll in ziso were not as severe as ccr2, consistent with ziso showing a slight virescent phenotype in comparison to ccr2 (Figure 2A). Cotyledons of the ccr2 ziso-155 and ccr2 ziso1-4 double mutants accumulated levels of chlorophyll similar to that of WT, 48 and 72 h following de-etiolation (Figure 4E). We conclude that a specific cis-carotene produced in ccr2 prevents PLB formation during skotomorphogenesis and perturbs chloroplast development.
The activation of photosynthesis associated nuclear gene expression restores PLB formation in ccr2
The transcriptomes of WT, ccr2 and ccr2 ziso-155 etiolated seedlings (ES), yellow emerging juvenile leaves (JL) from ccr2, and green JL leaves from WT and ccr2 ziso-155 were assessed using RNA sequencing analysis. Compared to WT there were 2-to 4-fold less differentially expressed (DE) genes in ccr2 (ES;191 and JL;1217) than for ccr2 ziso-155 (ES;385 and JL;5550). Gene ontology (GO) analysis revealed a DE gene list significantly enriched in metabolic processes and stress responses in both tissue types of ccr2. Etiolated tissues of ccr2 showed DE genes enriched in photosynthetic processes (17/191; FDR < 3.8xE−06) that were not apparent in ccr2 ziso-155, which had DE genes more responsive to a stimulus (134/382; FDR < 3.7xE−7) involving hormones and abiotic stress (Supplemental Table 3). Juvenile leaves of both ccr2 and ccr2 ziso-155 showed a significant enrichment in DE genes also responsive to a stimulus (470/1212; FDR < 2.4xE−34 and 1724/5510; FDR < 5.4xE−43, respectively) involving several hormones and stress. Even more intriguing was the enhanced enrichment of DE genes specific to ccr2 ziso-155 juvenile leaves that were involved in biological regulation (1623/5510; FDR < 4.2xE−30) and epigenetic processes (184/5510; FDR < 3.1xE−11) such as DNA methylation, histone modification and gene silencing (Supplemental Table 4).
We utilised Genevestigator to compare DE genes in etiolated seedlings of ccr2 and ccr2 ziso-155 with that of mutant germplasm growing on MS media +/-chemical treatments in an attempt to identify co-or contra-changes of gene expression (>20% overlap) (Supplemental Table 3). Norflurazon, a carotenoid inhibitor of PDS activity and inducer of a retrograde signal(s) was able to induce 30-35% of DE genes in ccr2, which was not apparent in ccr2 ziso-155 (12-14%). An unexpected finding was the DE genes in ccr2 shared 31-42% in common with the cop9 and cop1 mutants, which ccr2 ziso-155 contra-regulated in cop9, but not cop1. Genes regulated during light-mediated germination were contra-expressed in ccr2 (28-48%), yet co-expressed in ccr2 ziso-155 (44-48%).
We next searched for differentially expressed genes in ccr2 that were attenuated or contra-expressed in the ccr2 ziso-155. Twenty contra-expressed genes were identified to be enriched in process related to photosynthesis, pigment biosynthesis and light regulation (5/20; FDR < 1.2xE−4) (Supplemental Table 5). Photomorphogenesis associated nuclear gene (PhMoANG) expression (e.g. DET1, COP1) was up-regulated in ccr2, yet down-regulated in ccr2 ziso-155. This finding is consistent with the fact that DE genes miss-expressed in ccr2 ziso-155 leaf tissues were enriched in chromatin modifying processes. det1.1 mutants were shown to have reduced PIF3 transcripts, and higher HY5 protein levels that activate downstream PhANG expression (Supplemental Table 6)(Lau and Deng, 2012). Indeed, our comparative analysis of contra-expressed genes in ccr2 ziso-155 revealed the down-regulation of PIF3, up-regulation of HY5 and PHANG expression (e.g. DXS, CLB6, LHCB1, LHCB2, RBCS, GUN5) (Supplemental Table 6). It is not unusual to observe miss-regulation of PhANG expression in mutants having impaired plastid development (Ruckle et al., 2007; Woodson et al., 2011). In summary, the repression of negative regulators of photomorphogenesis, correlates well with the up-regulation of PhANG expression in ccr2 ziso-155 and links cis-carotene accumulation to gene targets plastid development.
Activation of photomorphogenesis by det1-154 restores plastid development in ccr2
We searched the SNP deserts of the remaining 24 rccr2 lines for genes that could link cis-carotene signalling to regulators of photomorphogenesis. rccr2154 was mapped to a mutation in de-etiolated 1 (det1), hereafter referred as ccr2 det1-154, which restored plastid development in immature ccr2 leaves (Figure 3). Sequencing of the det1-154 gDNA identified a G to A point mutation at the end of exon 4. Sequencing of the det1-154 cDNA revealed the removal a 23 amino acid open reading frame due to alternate splicing (Figure 5A). Quantitative PCR analysis confirmed that the shorter DET1-154 transcript (spliced and missing exon 4) was highly enriched (approx. 200 fold) in ccr2 det1-154, while the normal DET1-154 transcript (contains exon 4) was repressed in ccr2 det1-154 (Supplemental Figure 3A). The phenotypes of ccr2 det1-154 and det1-154 were intermediate to that of det1-1 (Chory et al., 1989) showing a smaller rosette with a shorter floral stem height and reduced fertility relative to the WT (Supplemental Figure 3B). The overexpression of the full length DET1 transcript (CaMV35s::DET1-OE) in ccr2 det1-154 restored the virescent phenotype in ccr2 leaves from plants grown under an 8-h photoperiod (Figure 5B). Therefore, alternative splicing of det1 and removal of exon 4 appeared sufficient to restore plastid development in ccr2 leaves grown under a shorter photoperiod.
(A) Schematic structure of the wild type DET1 gDNA, DET1 protein, SNP confirmation 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 following a 16 h to 8 h photoperiod shift assay. 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 h 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.
We investigated how det1-154 can restore plastid development in ccr2. ccr2 det1-154 mature leaves contained less carotenoids and chlorophylls compared to ccr2 (Supplemental Figure 3C). That is, the xanthophylls and β-carotene were all significantly reduced by det1-154. det1-154 also reduced total cis-carotene content in ccr2 etiolated cotyledons (Figure 5C; Supplemental Figure 3D). However, only tri-cis-ζ-carotene, pro-neurosporene and tetra-cis-lycopene were significantly reduced in ccr2 det1-154, phytoene and phytofluene levels were not significantly different to ccr2 (; Supplemental Figure 3D). ccr2 prevented PLB formation during skotomorphogenesis, yet displayed no obvious phenotype that resembled photomorphogenic mutants (Table 1). TEM confirmed that the dark-grown cotyledons from etiolated ccr2 det1-154 seedlings showed PLBs in 69% of etioplasts examined during skotomorphogenesis (Table 1; Supplemental Figure 3E). The restoration of a PLB in ccr2 det1-154 dark grown seedlings coincided with a restoration of cotyledon greening following de-etiolation (Figure 5E). In leaves and etiolated cotyledons, det1 mutants reduced total carotenoid and/or chlorophyll content when compared to WT (Supplemental Table 7). That is, the xanthophylls and β-carotene were all significantly reduced in det1 mutants. We detected traces of phytoene and phytofluene in emerging leaves and in addition tri-cis-ζ-carotene at higher levels in etiolated cotyledons of det1 mutants (Supplemental Table 7). det1-154 activated photomorphogenesis in ccr2 as evident by etiolated seedlings having characteristic shorter hypocotyl, no apical hook and opened large cotyledons similar to det1-1 (Figure 5D), which agrees with our transcriptomic data whereby ccr2 det1-154 lead to a repression of det1 and activation of PhANGs (Supplemental Table 6). Therefore, the reduction of the full length DET1 mRNA in ccr2 caused a reduction in specific cis-carotenes and restored PLB formation (Table 1).
D15 inhibition of carotenoid cleavage activity reveals a cis-carotene cleavage product controls PLB formation
A question remained as to whether the accumulation of specific cis-carotenes lead directly to PLB perturbation as hypothesised (Park et al., 2002), or production of an apocarotenoid signal that could regulate PLB formation. We crossed ccr2 to carotenoid cleavage dioxygenase loss-of-function mutants; ccd1, ccd4, ccd7 (max3) and ccd8 (max4) and tested if plants exposed to a shorter photoperiod would revert the virescent leaf phenotype of ccr2. We analysed more than 10 plants for each of the ccr2 ccd double mutant lines and observed a perturbation in plastid development in >93% of plants, each displaying clearly visible yellow virescent leaves similar to ccr2 (Supplemental Figure 4A-B). We concluded that no single ccd mutant was sufficient to block the production of any cis-carotene derived cleavage product. However, there is a degree of functional redundancy among family members, as well as multiple cleavage activities and substrate promiscuity (Hou et al., 2016).
To address this challenge we decided to utilise the aryl-C3N hydroxamic acid compound (D15), which is a specific inhibitor (>70% inhibition) of 9,10 cleavage enzymes (CCD) rather than 11,12 cleavage enzymes (NCED) (Sergeant et al., 2009; Van Norman et al., 2014). We imaged etioplasts from WT and ccr2 etiolated seedlings treated with an optimal concentration of D15 (Van Norman et al., 2014). The majority (86%) of D15-treated ccr2 etioplasts displayed a PLB, whilst in control treatments ccr2 etioplasts showed no discernible PLB (Figure 6A; Supplemental Table 2). Total PChlide levels in WT and ccr2 after D15 treatment were similar (Figure 6B). As expected, etiolated ccr2 seedlings grown on D15-treated MS media accumulated chlorophyll in cotyledons within 24-h of continuous light treatment following de-etiolation in a manner similar to WT (Figure 6C). D15 significantly enhanced di-cis-ζ-carotene and pro-neurosporene, yet reduced tetra-cis-lycopene in etiolated cotyledons of ccr2 (Figure 6D). In WT etiolated cotyledons, D15 significantly enhanced violaxanthin, neoxanthin and antheraxanthin content, which has previously been shown to also occur in Arabidopsis roots (Van Norman et al., 2014)(Figure 6E). Treatment of dark and light grown wild type seedlings with D15 did not cause adverse pleiotropic effects on germination, hypocotyl elongation and/or plastid development in cotyledons (Figure 6, Table 1, Supplemental Table 2). Therefore, apocarotenoid formation from either cleavage of di-cis-ζ-carotene and/or pro-neurosporene in ccr2 can perturb PLB formation independent of PChlide biosynthesis.
(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 only (control; ctrl).
(B) Pchlide levels in Wild Type (WT) and ccr2 treated +/-D15. Fluorescence was measured at 638 nm and 675 nm with an excitation at 440 nm. Net fluorescence of Pchlide was calculated and normalised to protein content.
(C) D15 restores chlorophyll accumulation in ccr2 de-etiolated seedlings exposed to continuous light. Twenty seedlings from each of three biological replicates were harvested for chlorophyll determination in every 24 h under continuous light. Statistical analysis was by ANOVA with a post-hoc Tukey test (n= 20 seedlings).
(D) cis-carotene quantification in etiolated cotyledons of ccr2 treated with D15. 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). Error bars show standard error (n =4).
(E) Quantification of carotenoid levels in etiolated tissues of WT treated with D15. Neoxanthin (N); violaxanthin (V); antheraxanthin (A), lutein (L), β-carotene (β-C) and total carotenoids (T) were quantified at a 440nm absorption wavelength providing maximum detection. Star denotes significance (ANOVA, p < 0.05). Data is representative of two independent experiments.
A cis-carotene cleavage product acts downstream of DET1 to post-transcriptionally regulate protein levels
We searched for a transcriptional regulatory mechanism by which a cis-carotene cleavage product could control PLB formation during skotomorphogenesis. PORA transcript levels are relatively high in etiolated seedlings, becoming down-regulated upon exposure to white light or when photomorphogenesis is activated by det1-1 (Armstrong et al., 1995; Sperling et al., 1998). PIF3 and HY5 are key regulatory transcription factors involved in controlling downstream PhANG expression during the dark to light transition (Osterlund et al., 2000; Dong et al., 2014). A reduction in PORA and PIF3 has been shown to perturb PLB formation. In etiolated tissues of ccr2 det1-154, which harbour etioplasts containing a PLB, the transcript levels of PORA, PORB, PIF3 and HY5 were substantially reduced. D15 treatment did not affect the expression levels of these genes in either WT or ccr2 det1-154 (Figure 7A), therefore, cis-carotene cleavage does not appear to directly affect the transcriptional regulation of these genes.
(A) Transcript levels of PORA, PORB, PIF3 and HY5 in WT and ccr2 det1-154 etiolated seedlings growing on MS media +/-D15. Statistical analysis was performed using two-way ANOVA followed by a post-hoc paired t-test (p < 0.05). Error bars represent standard error of means.
(B) Representative image of a western blot showing POR protein levels. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media +/-D15 (control; Ctrl). The membrane was re-probed using anti-Actin antibody as an internal control to normalise POR protein levels among different samples.
(C) Representative image of a western blot showing DET1 protein levels. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media +/-D15 (control; Ctrl). The membrane was re-probed using anti-Actin antibody as an internal control to normalise POR protein levels among different samples.
(D) Western blot image of HY5 and PIF3 protein levels. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media (Ctrl) or media containing D15. The membrane was re-probed using anti-Actin antibody as an internal control to normalise HY5/PIF3 protein levels among different samples.
We then searched for a change in POR protein in dark grown seedlings, with or without D15, noting that wild-type and ccr2 accumulated POR (Park et al., 2002) and det1 lacked POR (Sperling et al., 1998)(Supplementary Figure 1B). Under the electrophoresis conditions used herein, the Arabidopsis PORA/B proteins were detected as a single immunoreactive signal (PORA; 37 kDa, and PORB; 36 kD) (Sperling et al., 1998; Park et al., 2002; Paddock et al., 2012) (Figure 7B). A substantial increase in POR was observed in ccr2, which was reduced back to WT levels by D15 (Figure 7B). ccr2 det1-154 accumulated wild-type levels of POR (Figure 7B), complementing the reported lack of POR in etiolated det1 tissues (Sperling et al., 1998)(Supplementary Figure 1B). Intriguingly, treatment of ccr2 det1-154 with D15 reverted POR levels back to those expected for det1. This was not due to ccr2 or D15 changing DET1 protein levels (Figure 7C). Therefore, cis-carotene cleavage mediates a signal that elevates POR accumulation in det1.
DET1 is a negative regulator of photomorphogenesis, such that det1 mutants lack PIF3 and accumulate HY5 protein levels during skotomorphogenesis (Osterlund et al., 2000; Dong et al., 2014)(Supplementary Figure 1B). We questioned if the apocarotenoid signal acted upstream of the PIF3-HY5 regulatory hub that controls PhANG expression, noting that wild-type has high levels of PIF3 and low or trace levels of HY5, with the converse in det1 (Dong et al., 2014) (Supplementary Figure 1B). PIF3 levels increased and HY5 decreased in both ccr2 and ccr2 det1-154 etiolated cotyledons and this was reverted by D15 treatment (Figure 7D). This indicates that an apocarotenoid signal can post-transcriptionally change the PIF3 / HY5 ratio in the presence or absence of DET1, indicating it is acting either in parallel with, or downstream of, DET1. The relative difference in PIF3 levels in ccr2 compared to ccr2 det1-154 in the presence of D15 would suggest the two pathways operate in parallel.
DISCUSSION
Plastid and light signalling coordinate leaf development under various photoperiods, and younger leaves display a greater plasticity to modulate their pigment levels in response to environmental change (Lepisto and Rintamaki, 2012; Dhami et al., 2018). We attribute ccr2 leaf variegation to the fine-tuning of plastid development in meristematic cells as a consequence of cis-carotene accumulation and not the generation of singlet oxygen (Kato et al., 2009; Chai et al., 2010; Han et al., 2012). Our evidence revealed that leaf variegation is linked to the hyper-accumulation of specific cis-carotenes since, ziso-155 and det1-154 as well as D15 were able to reduce cis-carotene biosynthesis in ccr2 tissues, as well as restore leaf greening in plants grown under a shorter photoperiod (Figures 4,5). A shorter photoperiod maybe a seasonal factor capable of triggering cis-carotene hyper-accumulation in newly emerged photosynthetic tissues when CRTISO activity is perturbed, and cause leaf variegation. The altered plastid development in etiolated cotyledons and younger virescent leaves from ccr2 cannot be attributed to a block in lutein, strigolactone, ABA or alteration in xanthophyll composition (Figure 2). Phytoene, phytofluene and to a lesser extent ζ-carotene were noted to accumulate in wild type tissues from different plant species (Alagoz et al., 2018). We also detected traces of these cis-carotenes in newly emerged leaves from wild type, and even more so in det1 mutants. Without the signal itself to assess the physiological function in wild-type plant tissues, we provided evidence for the existence of a cis-carotene cleavage product in ccr2 that can regulate PLB formation during skotomorphogenesis and plastid development during leaf greening independent of, and capable of compensating for mutations in DET1 (Figure 8A). We contrast how the cis-carotene derived novel apocarotenoid signal, in parallel with DET1 can post-transcriptionally control repressor and activator proteins that mediate the expression of a similar set of PhANGs in both the etioplast and chloroplast.
(A) ccr2 can accumulate poly-cis-carotenes that undergo enzymatic cleavage via CCDs to generate an apocarotenoid signal (ACS). Norflurazon (NF) treatment of ccr2 etiolated seedlings or the loss-of-function in ziso-155 block the accumulation of downstream cis-carotenes required for the biosynthesis of ACS. Chemical treatment of etiolated seedlings with D15 inhibits CCD cleavage of pro-neurosporene and/or tetra-cis-lycopene isomers into ACS.
(B) During skotomorphogenesis, ACS promotes “Factor X”. Factor X negatively affects PLB formation. Factor X could act to stabilise proteins by disrupting ubiquitination, de-ubiquitination, protease mediated protein degradation, heterodimerization of transcription factors, coactivator concentrations, and/or interact with ligand binding sites of receptors. DET1 is a repressor of photomorphogenesis that post-transcriptionally regulate PIF3 and HY5 protein levels, which control PhANG expression. det1 mutants lack POR and cannot make a PLB. ACS post-transcriptionally enhances POR protein levels, while det1 blocks Factor X, thereby allowing PLB formation in ccr2 det1-154. det1 reduces cis-carotene accumulation, and downregulates pro-neurosporene and tetra-cis-lycopene to maintain a threshold level of ACS.
(C) During photomorphogenesis, extended dark and/or shorter photoperiods, ACS manifests in newly emerged leaves from the ccr2 shoot meristem and perturbed chloroplast development and chlorophyll accumulation causing a leaf variegation phenotype. Green arrows and red lines represent positive and negative regulation, respectively. Abbreviations: PSY, phytoene synthase; PDS, phytoene desaturase, ZDS, ζ-carotene desaturase; Z1SO, ζ-carotene isomerase; CRTISO, carotenoid isomerase; det1-154, DEETIOLATED1-154; D15, inhibitor of CCD activity; CCD, carotenoid cleavage dioxygenase; ccr2, CRTISO mutant.
A cis-carotene derived cleavage product regulates plastid development
Due to their hydrophobicity and cis-configuration, cis-carotenes were proposed to function as a membrane-bound structural inhibitor of PLB formation during skotomorphogenesis (Park et al., 2002; Cuttriss et al., 2007). Instead, we demonstrate here that ccr2 generated a cis-carotene-derived cleavage product, as D15 chemical inhibition of CCD activity (Figure 8A) restored PLB formation (85%) in ccr2 etioplasts (Figure 6). This is in agreeance with evidence showing cis-carotenes are cleavable in vitro by CCD7 enzymatic activity (Bruno et al., 2016) and that CCD4 activity is necessary for generation of a cis-carotene derived apocarotenoid signal in zds/clb5, that affected leaf development (Avendano-Vazquez et al., 2014). However, loss-in-function of ccd1, ccd4, ccd7 and ccd8 were not sufficient to restore plastid development and prevent leaf variegation in ccr2 (Supplemental Figure 4). So, we conclude that there must be some redundancy among two or more CCDs in generating a ccr2 derived apocarotenoid signalling metabolite that controls plastid development.
Which cis-carotene is the precursor for the apocarotenoid signal? Treatment with NF (Figure 8A) restored PLB formation in ccr2 etioplasts (Cuttriss et al., 2007), which ruled out both phytoene and phytofluene as substrates for the generation of a cleavage product. Here we show ZISO restored PLB formation and cotyledon greening in ccr2 ruling out tri-cis-ζ-carotene and revealing that di-cis-ζ-carotene, pro-neurosporene isomers and/or tetra-cis-lycopene are likely candidates (Figure 4). ccr2 det1-154 displayed a substantial reduction in pro-neurosporene and tetra-cis-lycopene, and to a lesser extent di-cis ζ-carotene (Supplemental Figure 3). Tetra-cis-lycopene accumulated in variegated leaves from the rice zebra mutant (Han et al., 2012). However, in the presence of D15 and hence absence of any enzymatic cleavage, only di-cis-ζ-carotene and pro-neurosporene accumulated, not tetra-cis-lycopene (Figure 6). Based on the evidence to date, we consider pro-neurosporene and perhaps di-cis-ζ-carotene are preferred substrate(s) for in vivo cleavage into a signaling metabolite.
A cis-carotene cleavage product controls PLB formation independent of GUN activity
One question is whether the proposed apocarotenoid requires GUN activity to regulate PLB formation and/or PhANG expression? Given that gun1 etioplasts contain PLBs, then that aspect of the ccr2 phenotype is not GUN-related (Susek et al., 1993; Xu et al., 2016). Additionally, there were relatively few differentially expressed genes in common between ccr2 etiolated seedlings and gun1/gun5 seedlings treated with norflurazon (Supplemental Table 6) and none of the 25 revertants were in genic regions to which GUN genes are located. Norflurazon treatment of etiolated tissues does not affect PLB formation in wild type, but can restore PLB formation in ccr2 (Cuttriss et al., 2007; Xu et al., 2016). Lincomycin treatment, on the other hand can suppress PLB formation in etiolated seedlings and unlike norflurazon, affects the phenotype of pifq mutant seedlings grown in the dark. GUN1-facilitated retrograde-signaling antagonized PIF-regulated gene expression and attenuated de-etiolation phenotypes triggered by lincomycin (Martin et al., 2016). Furthermore, lincomycin also inhibited PLB formation in the pifq mutant, revealing that PIFs are not necessary for PLB formation (Martin et al., 2016). GUN1-dependent and independent signaling pathways were proposed to act upstream of HY5 to repress photomorphogenesis of cotyledons (Ruckle et al., 2007). Intriguingly, the ccr2 generated cis-carotene derived cleavage product also regulated a distinct set of genes involved in a photomorphogenic-dependent pathway. The nature by which a cis-carotene derived cleavage product regulates PLB formation by post-transcriptionally enhancing POR is quite distinct to that of GUN regulation of PhANGs.
An apocarotenoid post-transcriptionally regulates PIF3 and HY5 protein levels
Here we demonstrate that the ccr2-generated apocarotenoid acted in a retrograde manner to post-transcriptionally regulate POR protein levels of two key transcription factors, PIF3 and HY5, in ccr2 and ccr2 det1-154 backgrounds (Figure 7). Of particular interest is how the abundance all three proteins was reverted in ccr2 det1-154 to expected levels for det1 mutants by treatment with D15. (Figure 7). Previous research has shown that hy5, pif3 and pifq dark grown seedlings all contain etioplasts with PLBs (Chang et al., 2008; Stephenson et al., 2009; Martin et al., 2016). Consequently, we deduce that the lack of a PLB in ccr2 is neither a consequence of apocarotenoid regulation of PIF3 or HY5, nor a lack of POR. An alternative hypothesis proposed in our model (Figure 8B) depicts how the apocarotenoid signal and DET1 may regulate an unknown factor required for PLB formation that is independent of POR abundance.
An apocarotenoid signal regulates skotomorphogenesis and plastid biogenesis in parallel to DET1
DET1 is required for cis-carotene biosynthesis in wild type tissues, as det1 mutants accumulate phytoene, phytofluene and tri-cis-ζ-carotene (Supplemental Table 7). cis-carotenes will hyper-accumulate in etiolated cotyledons and younger leaf tissues exposed to an extended dark period when CRTISO activity becomes rate-limited, such as in the absence of SDG8, which is required for permissive expression of CRTISO in the shoot meristem (Cazzonelli et al., 2009b; Cazzonelli et al., 2009a; Cazzonelli et al., 2010)(Figure 2). SDG8 transcript levels are developmentally regulated, increasing from low basal levels after germination and declining during the dark phase of the diurnal cycle (Kim et al., 2005). Therefore, the accumulation of cis-carotenes and the apocarotenoid signal that regulates plastid biogenesis can be finely tuned with epigenetic and chromatin modifying processes that control development.
Herein we revealed how ccr2 and det1 oppositely regulate the chlorophyll biosynthetic enzyme, POR, at post-transcriptional and transcriptional levels, respectively, to control PLB formation (Figure 8). There are relatively few mutants published to date that do not produce a PLB in dark grown tissues and all, except ccr2, are due to reduced levels of PORA and/or PChlide. Arabidopsis mutants like det1-1 and cop1 mutants have less photoactive PChlide-F655 and higher total PChlide levels due to a reduction in POR that block PLB formation. Like det1-1, det1-154 exhibits all the same phenotypes and indeed D15 treatment of ccr2 det1-154 blocked PLB formation (Chory et al., 1989)(Supplementary Figure 3)(Table 1). Although, etioplasts in ccr2 dark grown cotyledons do not make a PLB, they have an abundance of POR protein and total PChlide levels are similar to wild type (Figure 6,7). Therefore, ccr2 and det1 control PLB formation via independent signalling pathways. The cis-carotene derived cleavage product acts independent of det1 to post-transcriptionally up-regulate POR protein levels and enhance PChlide thereby enabling PLB formation and etioplasts to chloroplast differentiation following de-etiolation and the normal greening of cotyledons exposed to continuous light.
DET1 encodes a nuclear protein acting downstream from the phytochrome photoreceptors to regulate light-driven seedling development and PhANG expression (Schroeder et al., 2002). DET1 interacts with COP1 and the chromatin regulator DDB1, to limit the access of transcription factors to promoters and negatively regulate the expression of hundreds of genes via chromatin interactions (Schroeder et al., 2002; Lau and Deng, 2012). Light stimulates photomorphogenesis and the rapid down-regulation of DET1 leading to a lower PIF3:HY5 protein ratio and the up-regulation of PhANG expression. Genetic mutations in cop1 and det1 also lower the PIF3:HY5 ratio thereby activating PhANG expression (Benvenuto et al., 2002) (Osterlund et al., 2000). Consistent with these findings, ccr2 det1-154 etiolated seedlings treated with D15 displayed elevated HY5 and almost negligible PIF3 protein levels, contrasting opposite to that of ccr2, revealing that the cis-carotene derived cleavage metabolite post-transcriptionally antagonises DET1 regulation of HY5 and PIF3 (Figure 8). This raised a question as to whether the ccr2-derived cleavage product could directly regulate DET1? This is unlikely for several reasons. First, ccr2 and ccr2 ziso-155 displayed closed cotyledons, an apical hook and normal hypocotyl length revealing that the cis-carotene derived cleavage metabolite does not activate photomorphogenesis (Table 1). Second, DET1 protein levels were relatively unchanged in WT, ccr2 and ccr2 det1-154, regardless of D15 chemical inhibition. In conclusion, we deduce that the apocarotenoid signal acts in parallel with DET1 to regulate POR, PIF3 and HY5 protein accumulation and thus regulate etioplast development during skotomorphogenesis and chloroplast development under extended periods of darkness.
METHODS
Mutants used in this study
All germplasms are in the Arabidopsis thaliana ecotype Columbia (Col-0) background except where otherwise indicated. Germplasm used in this study include; ziso#11C (zic1-3: Salk_136385), ziso#12D (zic1-6; Salk_057915C), ziso#13A (zic1-4; CS859876), ccr2.1/crtiso (Park et al., 2002), ccr1.1/sdg8 (Cazzonelli et al., 2009b), lut2-1 (Pogson et al., 1996), ccd1-1 (SAIL_390_C01), ccd4 (Salk_097984c), max3-9/ccd7 (Stirnberg et al., 2002), max4-1/ccd8 (Sorefan et al., 2003), aba1-3 (Koornneef et al., 1982), det1-1 (CS6158), ccr2 det1-154, and det1-154.
Plant growth conditions and treatments
For soil grown plants, seeds were sown on DEBCO seed raising mixture and stratified for 3 d at 4 °C in the dark, prior to transferring to an environmentally controlled growth chamber set to 21 °C and illuminated by approximately 120 µmol.m−2.sec−1 of fluorescent lighting. Unless otherwise stated, plants were grown in a 16-h photoperiod. Photoperiod shift assays were performed by shifting 2-3 week old plants grown under a 16-h photoperiod to an 8-h photoperiod for one week and newly emerged immature leaves were scored as displaying either a yellow leaf (YL) or green leaf (GL) phenotype, reflecting either impaired or normal plastid development respectively.
For media grown seedlings, Arabidopsis seeds were sterilized for 3 h under chlorine gas in a sealed container, followed by washing seeds once with 70% ethanol and three times with sterilized water. Seeds were sown onto Murashige and Skoog (MS) media (Caisson Labs; MSP01) containing 0.5% phytagel (Sigma) and half-strength of Gamborg’s vitamin solution 1000X (Sigma Aldrich) followed by stratification for 2 d (4 °C in dark) to synchronise germination. Inhibition of carotenoid cleavage dioxygenase (CCD) enzyme activity was achieved by adding D15 (aryl-C3N hydroxamic acid) dissolved in ethanol to a final optimal concentration of 100 μM as previously described (Van Norman et al., 2014). Etiolation experiments involved growing seedlings in the dark at 21°C for 7 d and harvesting tissue under a dim green LED light. For de-etiolation and greening experiments, Arabidopsis seeds were stratified for 2 d and germinated in the dark at 21°C for 4 d. Seedlings were then exposed to constant light (~80 µmol.m−2.sec−1, metal-halide lamp) for 72 h at 21 °C. Cotyledon tissues were harvested at 24-h intervals for chlorophyll quantification.
Plasmid construction
pEARLEY::ZISO-OE and pEARLEY::DET1-OE binary vectors were designed to overexpress ZISO and DET1 cDNA fragments, respectively. Both genes were regulated by the constitutive CaMV35S promoter. Full length cDNA coding regions were chemically synthesised (Thermo Fisher Scientific) and cloned into the intermediate vector pDONR221. Next, using gateway homologous recombination, the cDNA fragments were cloned into pEarleyGate100 vector as per Gateway® Technology manufacturer’s instructions (Thermo Fisher Scientific). Vector construction was confirmed by restriction digestion and sanger sequencing.
Generation of transgenic plants
The ccr2 ziso-155 and ccr2 det1-154 EMS generated mutant lines were transformed by dipping Arabidopsis flowers with Agrobacteria harbouring pEARLEY::ZISO-OE or pEARLEY::DET1-OE binary vectors to generate ccr2 ziso-155::ZISO-OE and ccr2 det1154::DET1-OE transgenic lines, respectively. At least 10 independent transgenic lines were generated by spraying seedlings grown on soil with 50 mg/L of glufosinate-ammonium salt (Basta herbicide).
Chlorophyll pigment quantification
Total chlorophyll was measured as described previously (Porra et al., 1989) with minor modifications. Briefly, 20 seedlings from each sample were frozen and ground to fine powder using a TissueLyser (Qiagen). Homogenised tissue was rigorously suspended in 300 µL of extraction buffer (80% acetone and 2.5mM NaH2PO4, pH 7.4), incubated at 4°C in dark for 15 min and centrifuged at 20,000 g for 10 min. Two hundred and fifty microliters of supernatant was transferred to a NUNC 96-well plate (Thermo Fisher Scientific) and measurements of A647, A664 and A750 were obtained using an iMark Microplate Absorbance Reader (Thermo Fisher Scientific). Total chlorophyll in each extract was determined using the following equation modified from (Porra, 2002): (Chl a + Chl b) (µg) = (17.76 × (A647-A750) + 7.34 × (A664-A750)) × 0.895 × 0.25.
Carotenoid pigment analysis
Pigment extraction and HPLC-based separation was performed as previously described (Cuttriss et al., 2007; Dhami et al., 2018). Reverse phase HPLC (Agilent 1200 Series) was performed using either the GraceSmart-C18 (4-µm, 4.6 × 250-mm column; Alltech) or Allsphere-C18 (OD2 Column 5-µm, 4.6 × 250; Grace Davison) and/or YMC-C30 (250 × 4.6mm, S-5µm) columns. The C18 columns were used to quantify β-carotene, xanthophylls and generate cis-carotene chromatograms, while the C30 column improved cis-carotene separation and absolute quantification. Carotenoids and chlorophylls were identified based upon retention time relative to known standards and their light emission absorbance spectra at 440 nm (chlorophyll, β-carotene, xanthophylls, pro-neurosporene, tetra-cis-lycopene), 400 nm (-carotenes), 340 nm (phytofluene) and 286 nm (phytoene). Quantification of xanthophyll pigments was performed as previously described (Pogson et al., 1996). Quantification of cis-carotenes was performed by using their molar extinction coefficient and molecular weight to derive peak area in terms of micrograms (μg) per gram fresh weight (gfw) as previously described (Britton, 1995).
Transmission Electron Microscopy (TEM)
Cotyledons from 5-d-old etiolated seedlings were harvested in dim-green safe light and fixed overnight in primary fixation buffer (2.5% Glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.2) under vacuum, post-fixed in 1% osmium tetroxide for 1 h, followed by an ethanol series: 50%, 70%, 80%, 90%, 95% and 3 × 100% for 10 min each. After dehydration, samples were incubated in epon araldite (resin): ethanol at 1 : 2, 1 : 1 and 2:1 for 30 min each, then 3 times in 100% resin for 2 h. Samples were then transferred to fresh resin and hardened under nitrogen air at 60 °C for 2 d, followed by sectioning of samples using Leica EM UC7 ultramicrotome (Wetzlar). Sections were placed on copper grids, stained with 5% uranyl acetate, washed thoroughly with distilled water, dried, and imaged with H7100FA transmission electron microscope (Hitachi) at 100 kV. For each of the dark-grown seedling samples, prolamellar bodies were counted from 12 fields on 3 grids, and data analysed using two-way ANOVA with post-hoc Tukey HSD.
DNA-seq Library Construction, Sequencing and Bioinformatics Identification of SNPs
Genomic DNA (gDNA) was extracted using the DNeasy Plant Mini Kit (Qiagen). One microgram of gDNA was sheared using the M220 Focused-Ultrasonicator (Covaris) and libraries were prepared using NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs) followed by size selection (~320 bp) using AMPure XP Beads (Beckman Coulter). Paired-end sequencing was performed using the Illumina HiSEQ1500. After sequencing, the raw reads were assessed for quality using the FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and subjected to trimming of illumina adapters and filtering of low quality reads with AdapterRemoval programme (Lindgreen, 2012). The reads were mapped to the Arabidopsis thaliana (TAIR9) genome with BWA mapper (Li and Durbin, 2009). The resultant BWA alignment files were converted to sorted bam files using the samtools v0.1.18 package (Li et al., 2009) and were used as input for the subsequent SNP calling analyses. The SNPs were called and analysed further on both the parent and mutant lines using NGM pipeline (Austin et al., 2011) and SHOREmap (Schneeberger et al., 2009). For the NGM pipeline, SNPs were called using samtools (v0.1.16) as instructed and processed into ‘.emap’ files using a script provided on the NGM website. The .emap files were uploaded to the NGM web-portal to assess SNPs with associated discordant chastity values. To identify mutant specific SNPs, SNPs from parental lines were filtered out and EMS-induced homozygous SNPs were defined based on the discordant chastity metric. For SHOREmap, the SHORE software (Ossowski et al., 2008) was used to align the reads (implementing BWA) and call the SNPs (Hartwig et al., 2012). SHOREmap backcross was then implemented to calculate mutant allele frequencies, filter out parent SNPs and define the EMS mutational changes. Where appropriate, custom scripts were used to identify mutant specific EMS SNPs, filter out parent SNPs and annotate the region of interest. The SNPs and InDels were localized based on the annotation of gene models provided by TAIR database (http://www.arabidopsis.org/). The polymorphisms in the gene region and other genome regions were annotated as genic and intergenic, respectively. The genic polymorphisms were classified as CDS (coding sequences), UTR (untranslated regions), introns and splice site junctions according to their localization. SNPs in the CDS were further separated into synonymous and non-synonymous amino substitution. The GO/PFAM annotation data were further used to functionally annotate each gene.
RNA-seq Library Construction, Sequencing and Differential Gene Expression Analysis
Total RNA was extracted from Arabidopsis leaf tissues grown under an 8-h photoperiod or cotyledons from etiolated seedlings grown in dark for 7 d by TRIzol (Thermo Fisher Scientific) followed by DNase treatment at 37 °C for 30 min. RNA was recovered using x1.8 Agencourt RNAClean XP magnetic beads (Beckman Coulter). RNA (1 µg) libraries were constructed using 1llumina TruSeq Stranded mRNA Library Prep Kit (ROCHE) followed by bead size selection (~280 bp) using AMPure XP Beads and libraries sequenced using the Illumina HiSEQ2000. Fifteen million reads were obtained from sequencing each library and 21365 to 23840 mRNA transcripts were identified. Quality control was performed with FASTQC v.0.11.2. Adapters were removed using scythe v.0.991 (flags -p 0.01 for the prior), reads trimmed with sickle v.1.33 (flags q 20; quality threshold and −l 20 for minimum read length after trimming) and aligned to the Arabidopsis genome (TAIR10) using the subjunc v.1.4.6 aligner (-u and -H flags to report reads with a single, unambiguous mapping location) (Liao et al., 2014). The number of reads mapping per gene were summarised using feature Counts (v.1.4.6 with flags -s 2, -P and -c) to map reverse stranded and discard read pairs mapping to different chromosomes (Liao et al., 2014). Statistical testing for relative gene expression was performed in R using edgeR v.3.4.2 (Robinson and Smyth, 2007, 2008; Robinson et al., 2010; Robinson and Oshlack, 2010; McCarthy et al., 2012), Voom (Law et al., 2014) in the limma package 3.20.1 (Smyth, 2004, 2005). Transcripts were considered differentially expressed when a fold change > 2 and FDR adjusted p < 0.05. The bioinformatics analysis pipeline from fastq to summarised counts per gene is available at https://github.com/pedrocrisp/NGS-pipelines. RNAseq data sets was deposited into a permanent public repository with open access (https://www.ncbi.nlm.nih.gov/sra/PRJNA498324).
Protein extraction and western blot analysis
For protein extraction, fifty to one hundred milligrams of etiolated Arabidopsis cotyledons (7-d-old) were harvested under dim-green safe light and ground to fine powder. Total protein was extracted using a TCA-acetone protocol (Mechin et al., 2007) with minor modification and pellets were resuspended in 100 μL – 200 μL solubilization buffer. The concentration of total protein was measured using Bradford reagent (Bio-Rad) and adjusted to 2 μg/μL. A serial dilution was used to determine western blot sensitivity for each antibody and determine the optimal concentration for quantification. Five micrograms of total protein run on a gel was transferred to a PVDF membrane (Bio-Rad) and incubated with anti-POR polyclonal antibody (Agrisera Antibodies AS05067, 1:2000), anti-PIF3 polyclonal antibody (Agrisera Antibodies, 1:2000) or anti-HY5 antibody (Agrisera Antibodies AS05067, 1:1000) for 2 h. To examine DET1 protein levels, 10 μg of total protein was loaded to the gel and anti-DET1 polyclonal antibody (Agrisera Antibodies AS153082) was used at a 1:1000 dilution. Membranes were washed and incubated with HRP-conjugated Goat anti-Rabbit IgG (Agrisera Antibodies, 1:2500) for 90 min. Membranes were re-probed using anti-Actin polyclonal antibody (Agrisera Antibodies AS132640, 1:3000) and HRP-conjugated Goat anti-Rabbit IgG (Agrisera Antibodies, 1:2500) for internal protein normalisation.
Protochlorophyllide Quantification
Protochlorophyllides (Pchlides) were extracted and measured using published methods (Kolossov and Rebeiz, 2003) with modifications. Around 100 mg of etiolated Arabidopsis seedlings (7-d-old) were harvested under dim-green safe light, frozen and ground to fine powder. Two milliliters of 80% ice-cold acetone was added to each sample and the mixture was briefly homogenized. After centrifugation at 18,000 g for 10 min at 1 °C, supernatant was split to 2 × 1 mL for Pchlides and protein extraction. Fully esterified tetrapyrroles were extracted from the acetone extracts with equal volume followed by 1/3 volume of hexane. Pchlides remained in the hexane-extracted acetone residue were used for fluorescence measurement with a TECAN M1000PRO plate reader (Tecan Group) and net fluorescence were determined as previously described (Rebeiz et al., 1975). Protein extraction was performed using 80% acetone and 10% TCA; protein concentration was used to normalize the net fluorescence of Pchlides.
Real-Time PCR Analysis
The total RNA was extracted using Spectrum™ Plant Total RNA kit as per manufacturer’s protocol (Sigma-Aldrich). The qRT-PCR was performed with mixture of 2 μL of primer mix (2 μM from each F & R primer), 1 μL 1/10 diluted cDNA template, 5 μL LightCycler 480 SYBR Green I Master mix and distilled water up to a total volume of 10 μL. Relative transcript abundance was quantified using LightCycler 480 as per instructions (Roche). For each sample, three technical replicates for each of three biological replicates were tested. The relative gene expression levels were calculated by using relative quantification (Target Eff Ct(Wt-target) / Reference Eff Ct(Wt-target)) and fit point analysis (Pfaffl, 2001). Protein Phosphatase 2A (At1g13320) was used as housekeeper reference control for all experiments (Czechowski et al., 2005). All primer sequences are listed in Supplemental Table 8. Statistical analysis was performed using Two-Way ANOVA.
SUPPORTING INFORMATION
A) A pathway for cis-carotene biosynthesis. The carotenoid isomerase mutant (ccr2) accumulates cis-carotenes in etiolated seedlings. Norflurazon (NF) inhibits cis-carotene accumulation.
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 protein levels of PIF3, which reduce PhANG expression. Upon de-etiolation, DET1 and PIF3 protein levels decline and det1 mutants accumulate HY5 protein, which promotes the expression of PhANGs. det1 mutants do not accumulate PORA proteins and do not form a PLB in etioplasts. 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: PSY, phytoene synthase; PDS, phytoene desaturase, ZDS, ζ-carotene desaturase; Z1SO, ζ-carotene isomerase; CRTISO, carotenoid isomerase
(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 h 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 (ANOVA, 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 h to 8 h photoperiod shift. Values represent average and standard error of means are displayed (n=5, single leaf from 5 plants). Lettering denotes significance (paired t-test; p < 0.05). Neoxanthin (neo), violaxanthin (viol), antheraxanthin (anth), lutein (lutein), zeaxanthin (zea), β-car (β-carotene), Green Leaf (GL), Yellow Leaf (YL).
(A) qRT-PCR confirms alternative splicing of exon 4 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 partially 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 440nm. 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).
(D) Transmission electron micrographs of a representative etioplast from 5-d-old dark grown cotyledons showing a well-developed PLB in ccr2 det1-154.
Three-week-old WT, ccr2, ccr2 ccd1, ccr2 ccd4, ccr2 ccd7, and ccr2 ccd8 (F3 homozygous double mutant lines) plants were shifted from a 16-h to 8-h photoperiod until newly formed leaves in the ccr2 rosette displayed a virescent leaf phenotype.
(A) Representative images of plants showing newly developed leaves in the rosette.
(B) Quantification of leaf variegation in individual rosettes from ccr2 ccd double mutants. Data is representative of multiple independent experiments. Statistical analysis by ANOVA with post-hoc Tukey test showed no significant difference in the number of ccr2 and ccr2 ccd plants displaying a virescent phenotype.
Immature ccr2 tissues have an altered cis-carotene and xanthophyll composition.
D15 and ziso restore PLB formation in ccr2 etiolated cotyledons.
Supplemental Table 3. Transcriptomic analysis of WT, ccr2 and ccr2 ziso-155 etiolated tissues.
Supplemental Table 4. Transcriptome analysis of WT, ccr2 and ccr2 ziso-155 immature leaf tissues
Supplemental Table 5. Significantly expressed genes regulated in ccr2 and contra-regulated ccr2 ziso-155 that are common to both etiolated and immature leaf tissues.
Contra-regulated differential gene expression in etiolated seedlings and young leaves of ccr2 ziso-155.
det1 reduced carotenoids and caused cis-carotenes to accumulate in leaves and etiolated tissues.
Primer sequences used for qRT-PCR and ccr2 det154 characterisation
AUTHOR CONTRIBUTIONS
CIC and BJP conceived ideas and designed research. CIC and XH wrote the manuscript. CIC and XH prepared figures and tables and performed the majority of experiments. YA contributed to Figures 5, 6, 7, 8. YA and ND contributed to Tables 1, S7 and Figure S3. JR produced Figure S3. MS contributed expertise in DNA bioinformatics analysis. JL contributed expertise in TEM. CIC and BJP supervised XH, JR and ND. CIC supervised YA. All authors contributed to editing the manuscript.
ACKNOWLEDGEMENTS
We would like to especially thank Rishi Aryal for his technical assistance in confirming the splicing of det1-154 and Peter Crisp for assisting XH with the bioinformatics analysis. Many thanks to Arun Yadav, Shelly Verma, William Walker, Michelle Nairn, Sam Perotti, Jacinta Watkins and Kai Chan for their assistance in maintaining plants, crossing mutant germplasm and performing HPLC. We thank Chris Mitchell for developing cis-carotene standards. We thank Philip Benfey for providing the D15 chemical inhibitor of CCD activity. Next generation sequencing was performed at the Biomolecular Resource Facility (ANU). This work was supported by Grant CE140100008 (BJP) and DP130102593 (CIC).
Footnotes
The authors declare no conflict of interest
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