Introduction

The level of photosynthetic pigments (chlorophylls and carotenoids) in leaves is tightly coordinated with chloroplast development and can change during development or in response to environmental stress. Older leaves from Arabidopsis contain enlarged chloroplasts that can sustain the steady-state turnover of pigments [7, 8, 34]. However, the level of chlorophylls and carotenoids is approximately 40% higher in younger leaves comparison to older leaves of Arabidopsis [23]. Younger Arabidopsis leaves, in comparison to older leaves, harbour actively dividing and expanding cells, leading to a net increase in total cell number and chloroplast capacity per area to store pigments for photosynthesis [21, 30]. This observation correlates with higher rates of photosynthesis in recently emerged leaves compared to mature leaves of Arabidopsis [65]. The younger leaves of Arabidopsis are more tolerant to the excessive light exposure compared to mature leaves [11, 20], and unlike older leaves they can modify their pigment levels upon exposure to elevated CO2 [23]. Develo** chloroplasts within the actively dividing cells in younger leaves can alter their biogenesis in response to environmental change, while older leaves that contain fully expanded cells with mature chloroplasts are turned over slowly [21]. This plasticity intrinsic to young Arabidopsis leaves could be utilised to develop an in planta biossay to decipher how environmental, chemical, and/or genetic perturbations regulate chloroplast biogenesis and development.

In leaves, chloroplasts develop from either the etioplast or proplastid [15, 62]. Photosynthetic complexes in the chloroplast thylakoid antennae require the assembly of chlorophylls, carotenoids (lutein, β-carotene, violaxanthin, and neoxanthin), nucleus-encoded protein subunits, and redox-active co-factors (e.g. hemes and iron–sulfur clusters) to contribute to electron transfer reactions during photosynthesis as well as facilitate light harvesting and photo-protection [6]. Blocking carotenoid biosynthesis in foliar tissues impacts plastid development by disrupting thylakoid formation, triggers changes in photosynthesis-associated nuclear gene expression [49]. Norflurazon (NFZ) is commonly used to inhibit PHYTOENE DESATURASE (PDS) activity and block downstream carotenoid accumulation (Fig. 1A). The supply of substrates from the methylerythritol 4-phosphate (MEP) pathway continue to facilitate phytoene biosynthesis in Arabidopsis leaves, etiolated seedling and/or shoot derived calli, despite the NFZ-induced impairment in plastid development [50, 58, 63, 64]. The NFZ-treated tissues accumulate chlorophyll biosynthesis intermediate metabolites (e.g. protoporphyrin IX; Mg-ProtoIX) that act as retrograde signals to downregulate photosynthesis-associated nuclear gene expression in leaves [37]. Retrograde metabolites generated by the chloroplast provide “biogenic control” during early chloroplast differentiation from proplastids or etioplasts in emerging leaves, and/or “operational control” in mature leaf chloroplasts in response to environmental stimuli [52, 71]. What remains unknown is if NFZ also perturbs the biosynthesis of a downstream carotenoid-derived signal that regulates chloroplast biogenesis in young leaves.

Fig. 1
figure 1

Optimisation of a pigment-based signalling bioassay in Arabidopsis detached rosettes. A Pathway for carotenoid biosynthesis and catabolism into an apocarotenoid signal (ACS) or phytohormone such as strigolactone (SL) and abscisic acid (ABA). Norflurazon (NFZ) inhibits PDS activity and aryl-C3N hydroxamic acid (D15) impairs CCD activity. Green arrows and red lines represent positive and negative regulation, respectively. Blue lines denote a pathway towards the generation of a carotenoid cleavage product specified in the grey box. Mutants used in this study include; ζ-carotene isomerase (z-iso) and carotenoid chloroplast regulatory 2 (ccr2), lutein deficient 2 (lut2), nonphotochemical quenching 1 (npq1), nonphotochemical quenching 2 (npq2), abscisic acid deficient 4 (aba4), more axillary branching 3 (max3). Abbreviations: GERANYLGERANYL PYROPHOSPHATE (GGPP), PHYTOENE SYNTHASE (PSY), PHYTOENE DESATURASE (PDS), ζ-CAROTENE DESATURASE (ZDS), ZETA-CAROTENE ISOMERASE (Z-ISO), CAROTENOID ISOMERASE (CRTISO), LYCOPENE EPSILON CYCLASE (LCY), and LYCOPENE BETA CYCLASE (bLCY), ZEAXANTHIN EPOXIDASE (ZE), VIOLAXANTHIN DEEPOXIDASE (VDE), NEOAXANTHIN SYNTHASE (NXS), CAROTENOID CLEAVAGE DIOXYGENASE (CCD). B Visual display of the whole rosette bioassay showing the rosettes incubating on kim wipes saturated with NFZ contained with a petri dish. The numbered rosette shows the leaf position by chronological age (1 to 15; oldest to youngest). Three week old Arabidopsis rosettes were treated with different NFZ concentrations (0–100 μM) for 24 h (D–F) and various time points over a 24 h period (G–I). Arabidopsis trays were kept in dark for 4–5 h before transferring Arabidopsis plants to NFZ under continuous light (130–150 µmol m−2 s−1, cool fluorescent lamps) at 22 °C. Mature (leaf 1–2; old) and recently emerged (leaf 9–11, young) leaves were collected after treatments and pigment levels quantified. Absolute concentrations of Phytofluene (C), Phytoene (D), Total carotenoids (E), and Total chlorophylls (F) in response to the different concentration of NFZ. Absolute concentrations of Phytoene (G), Total carotenoids (H), and Total chlorophylls (I) in response to the different NFZ incubation times over a 24 h period. Plots represent the mean values with standard error of means (n = 3–4; C) from a representative dataset of at least two independent experimental repetitions. Letter codes in the plots indicate the level of statistical variation (p < 0.05) in carotenoid content within and across the test groups determined by One-Way ANOVA adopting Holm-Sidak post-hoc multiple comparisons

Full size image

Catabolism of carotenoids is a continuous process in leaves occurring via enzymatic and non-enzymatic oxidative cleavage [7, 8, 63]. Carotenoids can be cleaved enzymatically by CAROTENOID CLEAVAGE DIOXYGENASE (CCD) and 9-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED) to generate apocarotenoids such as strigolactone (SL) and abscisic acid (ABA) phytohormones, respectively (Havaux, 2014, [56, 63]. SL and ABA control developmental and physiological processes such as shoot bud outgrowth and stomatal closure respectively, yet a function in modulating chloroplast development remains less clear [27, 48]. Exogenous application of apocarotenoids such as β-cyclocitral, anchorene, loliolide, β-cyclogeranic acid, and β-ionone have been shown to regulate nuclear gene expression, pigment accumulation in plastids, and/or stress acclimation responses in plant tissues [27, 48]. Some β-carotene-derived apocarotenoids require enzymatic cleavage by CCD1 or CCD4, mutants of which accumulate β-carotene in seeds and/or senescing leaves [4, 31, 59]. Zeaxanthin can be cleaved by a CCD subfamily member to generate zaxinone in rice that regulates growth [68], or oxidatively cleaved into apocarotenoids that exert an ABA-independent regulation upon gene expression [41]. What remains unknown is if mutants that perturb xanthophyll biosynthesis, or CCD mediated carotenoid catabolism, can alter chlorophyll levels in young and/or old leaf types.

An unidentified apocarotenoid signal (ACS) generated during acyclic cis-carotene biosynthesis has been shown to regulate nuclear gene expression and chloroplast biogenesis in Arabidopsis tissues [5, 14, 26]. The loss of ZETA-CAROTENE DESATURATE (ZDS) function causes lethality following photomorphogenesis, yet the albino seedlings accumulate cis-carotenes that were linked to the control of plastid development and formation of needle-like leaf phenotype [5, 24, 26]. The loss-of-function in ZETA-CAROTENE ISOMERASE (Z-ISO) causes cis-carotenes to accumulate in etiolated tissues and can delay chlorophyll biosynthesis during seedling photomorphogenesis [9, 18]. Also, the loss-of-function in CAROTENOID ISOMERASE (CRTISO) activity in Arabidopsis causes cis-carotene accumulation during seedling skotomorphogenesis, as well as in newly emerged leaves from plants grown under a shorter photoperiod, and triggers to accumulate an unknown cis-ACS that regulates plastid development and photosynthetic nuclear gene expression in a retrograde-like manner [14, 50]. Light-mediated photoisomerization of cis-carotenes compensates for the lack of isomerase activity in foliar tissues, presumably by reducing substrate availability required to make cis-ACS. What remains untested is if a cis-ACS can be generated under longer photoperiods to regulate chlorophyll levels accordingly in leaf-age specific manner.

In this paper, we demonstrate that extended darkness, cold exposure, and NFZ treatment reduce chlorophyll and carotenoid accumulations in young, but not old leaves of Arabidopsis. An Arabidopsis foliar pigment-based signalling bioassay was established to decipher key steps in carotenoid biosynthesis and/or catabolism that might generate an apocaroteoid signal controlling chloroplast development and chlorophyll levels in younger leaves. Genetic and chemical inhibitors of carotenogenesis were used to differentiate between the effects of carotenoid- and chlorophyll-derived signals respectively. We assume a change in chlorophyll levels in younger leaves could reflect a change in chloroplast biogenesis. We reveal new insights into how carotenoid isomerisation is the key rate-limiting step in the pathway mediating production of a signal that controls chlorophyll accumulation in the younger foliar tissues of Arabidopsis grown under a long photoperiod.

Results

Norflurazon inhibition of PDS activity reduces chlorophyll levels in young expanding leaves

An in-planta pigment-based signalling bioassay was developed using detached whole rosettes from Arabidopsis treated with different concentrations and durations of NFZ that inhibits carotenoid biosynthesis (Fig. 1A, B). Under control growth conditions, chlorophyll and carotenoid levels were significantly higher in younger (leaves 9 to 13) compared to old (leaves 1 to 4) leaf types (Fig. 1B–D). All NFZ concentrations (1 μM to 100 μM) caused phytoene to accumulate in both young and old leaf types, yet detectable levels of phytofluene were only apparent in young leaves at lower concentrations (1 and 10 μM) (Fig. 1E, F). At lower NFZ concentrations (1 µM), young leaves showed a reduction in total carotenoids, but not total chlorophylls (Fig. 1C, D). Total chlorophyll and carotenoid levels were significantly reduced in young leaves exposed to 5, 10, 50 and 100 μM of NFZ, yet their levels remained almost unchanged in older leaves. The absence of phytofluene and reduced chlorophyll levels in younger leaves exposed to 50 μM NFZ indicated this concentration was best suitable to further optimise the duration of NFZ treatement.

The impact of three durations (8, 20, 24 h) of NFZ treatment (50 μM) on pigment levels were assessed in young and old leaves. Phytoene levels were two- to threefold higher in young relative to old leaves after 8, 20 and 24 h of NFZ treatment (Fig. 1G). Detectable levels of phytoene could be observed within 4 h of NFZ treatment in younger leaves (data not shown). After 8 h of NFZ treatment, the total chlorophyll and carotenoid levels remained higher in younger leaves, whereas a significant reduction was observed in young leaves after 20–24 h (Fig. 1H, I). Therefore, 24 h of treatment with 50 uM NF shows a clear reduction in chlorophylls in young, but not old mature leaves, thereby providing a in planta pigment-based bioassay to decipher which environmental factors and what rate-limiting steps in carotenogenesis impact plastid development.

Exposure of young leaves to cold and darkness reduces pigment levels

The impact of warm (32 °C) and cold (7 °C) temperatures, and extended darkness (24 h) on plastid developed was examined using the pigment-based signalling bioassay. None of these treatments affected chlorophyll or carotenoid levels in older mature leaves (Fig. 2A–F). Similarly, these treatments did not cause phytoene to accumulate in any leaf types in the absence of NFZ (Fig. 2G–I). In contrast, young leaves exposed to the cold and darkness showed reduced chlorophyll and carotenoid levels that became equivalent to that of old leaves (Fig. 2B, C, E, F), whereas warmer temperature slightly decreased chlorophyll levels and had no significant impact on carotenoid content (Fig. 2A, D). Therefore, young leaves were highly amenable to alter their pigment levels in response to cold and darkness, while old leaves remained resilient to any environmental change.

Fig. 2
figure 2

Chlorophyll and carotenoid content in Arabidopsis rosette leaves exposed to changes in temperature and extended darkness with or without norflurazon. Total chlorophyll (A–C), Total carotenoid (D–F), and Phytoene (G–I) content in young and old leaves from the Arabidopsis rosettes exposed to warm (32 °C; A, D, G), cold (7 °C; B, E, H), and darkness (C, F, I) in the presence or absence of NFZ. Error bars display standard error of the mean (n = 4). Dataset is representative of two independent experiments. Letter codes denote statistical variation (p < 0.05) determined by Two-Way ANOVA with Holm-Sidak post-hoc multiple comparison

Full size image

The impact of environmental change on pigment levels in aging leaf types was further investigated in combination with NFZ. Both young and old leaves from the NFZ-treated plants accumulated phytoene under warmer and colder conditions, but phytoene did not accumulate in dark exposed leaves (Fig. 2G–I). The levels of phytoene were consistently higher in young compared to older leaves. Compared to the standard 22 °C growth temperature, warmer and colder temperatures significantly enhanced and reduced phytoene accumulation respectively, in both leaf types (Fig. 2G–I). Intriguingly, none of the dark-exposed leaves accumulated any phytoene during NFZ treatment (Fig. 2I). Compared to their respective control without NFZ, total chlorophylls and carotenoids were significantly reduced in young, but not old leaves from plants treated with NFZ and exposed to 32 °C, 7 °C, and darkness (Fig. 2A–F). The trends with or without NFZ were rather similar. Overall, NFZ in combination with warm, cold or dark treatments does not create an obvious additive change on total chlorophyll or carotenoid levels in either leaf-type, despite higher, lower and absent levels of phytoene in NFZ-treated leaf tissues exposed to warm, cold and dark treatments respectively.

Perturbing strigolactone, abscisic acid or xanthophyll biosynthesis does not alter chlorophyll content in young leaves

We investigated if blocking SL, ABA, and xanthophyll biosynthesis could impact chlorophyll biosynthesis in young leaves. Total chlorophyll and carotenoid levels in young leaves remained high relative to old leaves in the loss-of-function in single (ccd1, ccd4, ccd7) or double (ccd1 ccd4, ccd7 ccd 4, ccd 1 ccd 7) mutants that impaired CAROTENOID CLEAVAGE DIOXYGENASE (CCD) activity (Fig. 3A, B). Therefore, it appears unlikely that SL generated from CCD7 cleavage, or an ACS produced from cleavage by CCD1 and/or CCD4, regulates chlorophyll levels in young leaves.

Fig. 3
figure 3

Chlorophyll and carotenoid content in young and old leaves from carotenoid cleavage dioxygenase (ccd), xanthophyll, SL and ABA mutants. The average total chlorophyll (A, C), and carotenoid (B, D) content in young and old leaves from WT and mutants are displayed with error bars denoting the standard error (n = 4). Data is representative of two experimental repetitions. Letters denote statistical variation (p < 0.05) within leaf types across different germplasm determined using a Two-Way ANOVA and post-hoc Holm-Sidak multiple comparison. ccd1; carotenoid cleavage dioxygenase, ccd4; carotenoid cleavage dioxygenase 4, ccd7; carotenoid cleavage dioxygenase 7 (max3), ccd8; carotenoid cleavage dioxygenase 8 (max4); npq1; nonphotochemical quenching 1 (violaxanthin deepoxidase), npq2; nonphotochemical quenching 2 (zeaxanthin deepoxidase), aba4; abscisic acid deficient 4 (neoxanthin synthaseS), lut2; lutein defecient 2 (epsilon-lycopene cyclase)

Full size image

Mutants that block the production of lutein (lut2), violaxanthin and neoxanthin (npq2), lutein, violaxanthin and neoxanthin (npq2 lut2), lutein and neoxanthin (aba4 npq1 lut2), or hyperaccumulate zeaxanthin (npq2, npq2 lut2), did not affect the higher chlorophyll levels in younger relative to older leaves (Fig. 3C, D). There were differences in the total carotenoid content among the different mutation combinations, however, it was always higher in young relative to older leaves mirroring the same trend observed in chlorophyll levels. Therefore, perturbations in xanthophylls that are required for canonical ABA biosynthesis does not appear to affect chlorophyll accumulation in young Arabidopsis leaves.

Carotenoid isomerase activity regulates chlorophyll levels in young leaves

We investigated if the major rate-limiting step in carotenoid biosynthesis enabled by PSY could regulate pigment levels in leaves. Like WT, the content of individual, as well as total chlorophylls and carotenoids, were higher in young compared to old leaves from a transgenic line overexpressing PSY (35S::AtPSY#23) [46] (Fig. 4A–J). However, there were no major differences in pigment levels between PSY-OE and WT in the respective leaf types, with the exception for a subtle reduction in neoxanthin in older leaves from PSY-OE. Therefore, overexpression of PSY did not impact chlorophyll levels in young or old leaves.

Fig. 4
figure 4

Chlorophyll and carotenoid content in young and old leaves of Arabidopsis germplasm that alter cis-carotene biosynthesis. A Chlorophyll b, B Chlorophyll a, C Total chlorophyll, D Lutein, E β-carotene, F Violaxanthin, G Neoxanthin, H Antheraxanthin, I Zeaxanthin, and J Total carotenoids were quantified in young and old leaves of different germplasm. Mean values are displayed with standard error (n = 4) being a representative dataset from two experimental repetitions. Letters denote statistical variation (p < 0.05) within leaf types across different germplasm determined using a Two-Way ANOVA and post-hoc Holm-Sidak multiple comparison. ziso and crtiso are loss of function mutants in ζ-carotene isomerase and carotenoid isomerase, respectively. PSY-OE enables higher PSY activity (35S::AtPSY#23) [46] and 35S::AtCRTISO restored CRTISO activity to the loss-of-function ccr2.1 mutant [16]

Full size image

Next, we investigated if the loss-of-function in z-iso or crtiso mutants, that accumulate acyclic cis-carotenes under light limiting conditions, could trigger a change in chlorophyll levels in young leaves from plants grown under long photoperiod. The chlorophyll content in z-iso young leaves was higher than old leaves (Fig. 4A–C). The level of lutein, β-carotene, violaxanthin, and, hence, total carotenoids was slightly lower in young leaves of z-iso compared to the young leaves from the WT. The carotenoid content in old leaves from both z-iso and WT were identical (Fig. 4D–J). The loss-of-function of CRTISO (ccr2.1) caused a reduction in total chlorophyll content in young leaves, such that it was similar to old leaves (Fig. 4C). The young leaves from crtiso showed lower chlorophyll b content compared to WT, whereas chlorophyll a content was severely reduced and identical to WT older leaves. The level of chlorophylls in older leaves of crtiso and WT were similar (Fig. 4A, B). Total carotenoid content was significantly lower in young leaves from crtiso relative to WT, yet carotenoid levels were similar in older leaves (Fig. 4J). Transgenic overexpression of CRTISO (35S::AtCRTISO pMDC32:CRTISO: CRTISO-OE) in the crtiso mutant (ccr2.1) restored total chlorophyll and carotenoid levels in young leaves back to WT levels (Fig. 4C, J). Unlike WT, the level of lutein and β-carotene were similar in young and old leaves from crtiso (Fig. 4D, E). Whereas, vioaxanthin, antheraxanthin, zeaxanthin and neoaxanthin were all significantly higher in younger compared to older leaves from crtiso. Therefore, the reduction in chlorophyll in young but not older leaves of crtiso, reveals that photoisomerization of cis-carotenes cannot maintain the higher chlorophyll levels normally quantified in young WT leaves.

NFZ and carotenoid isomerase activity regulate chlorophyll levels differently in young leaves

We next assessed whether NFZ treatment and crtiso have synergistic effects on pigmentation in young leaves. NFZ-treatment further elevated phytoene levels in both young and old leaves of crtiso compared to WT (Fig. 5A). Curiously, phytoene content was significantly higher in young, relative to older leaves from crtiso and WT plants treated with NFZ revealing there is a continued isoprenoid supply for carotenoid biosynthesis. NFZ caused a reduction of chlorophylls in young leaves from WT, that was even more pronounced in young crtiso leaves displaying chlorophyll levels below that of older leaves (Fig. 5B–D). Similarly, total carotenoid content in young leaves from crtiso plants treated with NFZ were significantly lower than older leaves, while young leaves form NFZ treated WT plants showed carotenoid levels similar to older leaves (Fig. 5K). Hence, NFZ and crtiso might affect chlorophyll levels and perhaps chloroplast biogenesis by independent signalling pathways.

Fig. 5
figure 5

Norflurazon-induces additive changes in chlorophyll and carotenoid content in the carotenoid isomerase mutant leaves. A Phytoene, B Chlorophyll b, C Chlorophyll a, D Total chlorophyll, E Lutein, F β-carotene, G Violaxanthin, H Antheraxanthin, I Zeaxanthin, and J Neoxanthin, K Total carotenoids in young and old leaves from crtiso and WT plants exposed to norflurazon. Mean values are displayed with standard error (n = 4) being a representative dataset from two experimental repetitions. Letters denote statistical variation (p < 0.05) within leaf types across different germplasm determined using a Two-Way ANOVA and post-hoc Holm-Sidak multiple comparison. crtiso; carotenoid isomerase

Full size image

The impact of NFZ on individual carotenoid levels in young leaves from WT and crtiso treated with NFZ were assessed to determine how they impact the carotenoid biosynthetic pathway. NFZ reduced β-carotene levels in young leaves from both WT and crtiso to levels below that observed in older leaves (Fig. 5F). However, while NFZ reduced lutein levels in WT young leaves, it further reduced lutein content in the crtiso mutant to levels below that of older leaves, revealing an additive effect (Fig. 5E). Violaxanthin levels were reduced in young WT and crtiso leaves from plants treated with NFZ; even though the levels were substantially higher in older leaves from the crtiso mutant (Fig. 5G). NFZ treated younger leaves contained more antheraxanthin and zeaxanthin compared to older leaves, which was further elevated almost threefold in the crtiso mutant, evidence of continued carotenoid biosynthesis or reduced catabolism (Fig. 5H, I). The levels of neoxanthin were similar in young and older leaves from WT and crtiso plants treated with NFZ (Fig. 5J). The different impacts of NFZ on individual carotenoid levels in young leaves from WT and crtiso highlight ongoing and likely impaired chloroplast biogenesis, as well as continued isoprenoid substrate supply for carotenoid biosynthesis. The differential effects of NFZ treatment on accumulation of chlorophylls and carotenoids in crtiso relative to WT, reveal that they might signal different pathways to regulate chloroplast development.

Discussion

The higher pigment content in young relative to old Arabidopsis leaves results from a greater cell and hence chloroplast density, that undergo rapid differentiation, division and expansion in emerging leaves providing them with plasticity to change in response to environmental, chemical and/or genetic perturbations [21, 23, 30, 34]. We demonstrate that extended darkness and cold exposure for 24 h can reduce chlorophyll by 20–50% to match levels as displayed by the more resilient older leaves. The optimised pigment-based signalling bioassay allowed detached Arabidopsis rosettes to be exposed to chemicals such as NFZ, that in addition to inhibiting carotenoid biosynthesis, trigger a plastid derived signal that can impair plastid biogenesis and reduce chlorophyll levels in young, but not old leaf types. Mutations that disrupted xanthophyll biosynthesis and degradation into downstream phytohormones such as SL and ABA, as well as other apocarotenoids did not affect chlorophyll levels in young leaves. An unidentified acyclic cis-carotene derived ACS produced in tissues from Arabidopsis plants lacking function of the CAROTENOID ISOMERASE was recently shown to regulate chloroplast biogenesis in newly emerged leaves that manifested as a virescent phenotype in plants grown under a shorter photoperiod [14]. Here we demonstrate that crtiso mutant plants grown under a longer photoperiod have lower chlorophyll levels indicating that photoisomerisation can rate-limit the generation of a cis-ACS that perturbs plastid development. NFZ treatment of WT and crtiso mutant young leaves, revealed similar, opposite, as well as additive effects on individual pigment accumulations in young leaves. We propose that carotenoid isomerisation controls an unidentified cis-ACS that mediates a different signalling process to that elicited by NFZ (e.g. chlorophyllide or Mg-ProtoIX) in controlling chlorophyll biosynthesis and perhaps chloroplast development in young leaves of Arabidopsis.

Norflurazon and environmental factors impede pigmentation in young emerging leaves

Our pigment-based signalling bioassay showed that NFZ caused a two- to threefold higher accumulation of phytoene in young compared to old leaves in agreeance with previous reports [8]. Despite a presumable impairment in plastid biogenesis in young leaves containing dividing cells and develo** plastids, phytoene biosynthesis continued revealing a sufficient substrate availability from the MEP pathway. Inhibition of carotenoid biosynthesis by NFZ was previously shown to initially enhance pathway flux, presumably compensating for the short supply of β-carotene [8]. The fact that total pigment levels in old leaves following NFZ treatment were similar to the control revealed less plasticity and resilience in mature chloroplasts to maintain chlorophyll. The capacity for pigment accumulation in leaves varies by chloroplast developmental gradients along a given leaf axis (e.g. mature plastids at the tip in expanding cells, and differentiating plastids at the base of dividing cells), as well as between leaves of different ages (e.g. smaller/fewer plastids in young immature leaves undergoing cell division and expansion, and larger/numerous plastids in old mature leaves undergoing steady state turnover) [21, 30, 34]. There are proplastid to chloroplast transitions occurring within the shoot apical meristem (SAM) of the shoot apex, where flanking leaf primordia emerge as young leaves containing chloroplasts and leucoplasts with develo** grana and thylakoids [17]. While mature chloroplasts can undergo a slow steady-state carotenoid turnover [7], this was not evident within our 24 h bioassay. Therefore, we attribute the NFZ-induced concurrent decrease in chlorophyll and carotenoid content in young leaves to a perturbation in chloroplast biogenesis in rapidly dividing and expanding cell types.

Extended darkness reduced total chlorophyll and carotenoid levels in young leaves mimicking the pattern exerted by NFZ. In Arabidopsis, carbon stored in the chloroplasts during the day as starch are remobilized during the night to support sugar metabolism, and excessive accumulation of sugars in the maltose excess 1 mutant (mex1) cause chloroplast dysfunction to signal a retrograde signal and trigger chloroplast degradation [66]. Perhaps an extended period of darkness triggers the accumulation of sugars that cause a similar degradation of pigments in young leaves. The recently emerged leaves of Arabidopsis comprise smaller dividing cells containing fewer, smaller-sized chloroplasts undergoing differentiation and biogenesis that could become interrupted by a plastid-derived signal generated during extended darkness. Indeed, chloroplast division/replication can become restricted in spinach leaf discs cultured in the dark or under low intensity green light [53]. Whereas, the enlarged mature cells within older leaves that comprise numerous mature chloroplasts with well-developed thylakoid grana stacks, retain their chlorophylls embedded within the thylakoids and hence remain unaffected by darkness [30, 34, 40, 55]. The biosynthesis and degradation of carotenoids and chlorophylls continuously take place in leaves during light exposed conditions as evident from the carbon isotope labelling with 14CO2 in Arabidopsis [7]. However, dark exposure of pepper leaves downregulated the expression of PSY and PDS thereby stalling carotenoid biosynthesis [64]. In concert, there was an absence of phytoene accumulation in both young and old Arabidopsis leaves from the NFZ treated leaves subject to darkness. Whether darkness and NFZ reduce chlorophyll accumulation and impair chloroplast biogenesis in young leaves by similar signalling mechanisms remains unclear. We propose that darkness blocks the first committed step in carotenoid biosynthesis and/or stalls the supply of isoprenoid substrates from the MEP pathway.

Low temperature affects a broad spectrum of cellular components in plants, including chloroplast development and metabolism [29, 30, 34] making them amenable to metabolite signals that control chloroplast biogenesis [21, 23]. SLs were shown to positively regulate photosynthesis related genes in tomato [47]. Yet, the chlorophyll levels in younger leaves also remained consistently stable, and higher than older leaves in CCD mutant combinations. In conclusion, it appears that SL or β-apocarotenoid signals are unlikely to regulate chlorophyll accumulation, and hence chloroplast biogenesis in either young emerging or older mature Arabidopsis leaves from plants grown under a long photoperiod.

Xanthophylls such as lutein, violaxanthin, and neoxanthin are abundant carotenoids found in photosynthetic leaves, whereas antheraxanthin and zeaxanthin are crucial to maintain the functional integrity of chloroplasts during excessive light or heat stress [22, 61]. Violaxanthin and neoxanthin are precursors of abscisic acid that regulates guard cell closure in stomata, mediates stress acclimation and plant development [1B) were collected after 24 h of treatment and stored in -80 °C prior to quantifying pigments. For the dark incubation experiments, leaf tissues were harvested under a green LED light.

Pigment extraction and quantification

Pigment extraction, quantification and analysis was performed as previously described [22, 23]. In brief, frozen tissues were milled in TissueLyser® (QIAGEN; 2 min, 20 Hz) using stainless steel beads (~ 3 mm diameter) until finely powdered. Pigments were extracted in 1 ml of acetone and ethyl acetate (60:40 v/v) containing 0.1% (w/v) butylated hydroxytoluene. The mixture was vortexed, centrifuged (15,000 rpm for 5 min at 4 °C) and the upper ethyl acetate phase analysed using a HPLC (Agilent 1260 Infinity) equipped with YMC-C30 (250 × 4.6 mm, S-5 μm) column and Diode Array Detector (DAD) detector. A 35-min reverse phase method was used to separate carotenoids. This consisted of a 5 min isocratic run of 100% solvent A (methanol: triethylamine, 1000:1 v/v) followed by 20 min ramp to 100% solvent B (methyl tert-butyl ether) and 2 min isocratic run of 100% solvent B with a solvent flow rate of 1 ml/min. 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), 340 nm (phytofluene) and 286 nm (phytoene). Absolute quantification and determination of composition of pigments was performed as described [1, 2, 14]. Quantification of phytoene and phytofluene was expressed peak area per milligram (mg) fresh weight.

Data analysis

One- or Two-Way ANOVA was performed using the Holm-Sidak post-hoc multiple comparisons to determine significant interactions within, and across, the test groups in response to the various treatment conditions.