A Foliar Pigment-Based Bioassay for Interrogating Chloroplast Signalling Reveals that Chlorophyll Biosynthesis Requires Carotenoid Isomerisation


 Background: Plastid-derived metabolites can signal control over nuclear gene expression, chloroplast biogenesis, and chlorophyll biosynthesis. Norflurazon (NFZ) inhibition of carotenoid biosynthesis in seedlings can elicit a protoporphyrin retrograde signal that controls chlorophyll and chloroplast biogenesis. Recent evidence reveals that plastid development can be regulated by carotenoid cleavage products called apocarotenoids. The key steps in carotenoid biosynthesis and catabolism that generate apocarotenoid signalling metabolites in foliar tissues remains to be elucidated. Here, we established an Arabidopsis foliar pigment-based bioassay using detached rosettes to differentiate plastid signalling processes in young expanding leaves containing dividing cells with active chloroplast biogenesis, from fully expanded leaves containing mature chloroplasts. Results: We demonstrate that environmental (extended darkness and cold exposure) as well as chemical (norflurazon; NFZ) inhibition of carotenoid biosynthesis can reduce chlorophyll levels in young, but not older leaves following a 24 h of rosette treatment. Mutants that disrupted xanthophyll accumulation, phytohormone biosynthesis (abscisic acid and strigolactone), or enzymatic carotenoid cleavage, did not alter chlorophyll levels in young or old leaves. Perturbations in acyclic cis-carotene biosynthesis revealed that disruption of CAROTENOID ISOMERASE (CRTISO), but not ZETA-CAROTENE ISOMERASE (Z-ISO) activity, reduced chlorophyll levels in young but not older leaves of plants growing under a long photoperiod. NFZ-induced inhibition of PHYTOENE DESATURASE (PDS) activity triggered phytoene accumulation more so in younger relative to older leaves from both WT and the crtiso mutant, indicating a continued substrate supply from the methylerythritol 4-phosphate (MEP) pathway for carotenogenesis. NFZ treatment of WT and crtiso mutant rosettes reveal similar, additive, and opposite effects on individual pigment accumulation.Conclusion: The Arabidopsis foliar pigment-based bioassay was used to differentiate signalling events elicited by environmental, chemical, genetic, and combinations thereof, that control chlorophyll biosynthesis. Genetic perturbations that impaired xanthophyll biosynthesis and/or carotenoid catabolism did not affect chlorophyll biosynthesis. The lack of CAROTENOID ISOMERISATION generated a signal that rate-limited chlorophyll accumulation, but not phytoene biosynthesis in young Arabidopsis leaves exposed to a long photoperiod. Findings generated using this new foliar pigment bioassay implicate that carotenoid isomerisation and NFZ elicit different signalling pathways to control chlorophyll homeostasis in young emerging leaves.


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 Arabidopsis leaves contain enlarged chloroplasts with more pigments that can sustain steady state turnover (Beisel et al., 2010, Beisel et al., 2011, Gugel and Soll, 2017). Yet Arabidopsis younger leaves contain approximately 40% more chlorophylls and carotenoids in comparison to older leaves (Dhami et respectively, yet a function in modulating chloroplast development remains less clear , Felemban et al., 2019. Exogenous application of apocarotenoids such as β-cyclocitral, anchorene, lollilode, 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 , Felemban et al., 2019. Some β-carotene-derived apocarotenoids require enzymatic cleavage by CCD1 or CCD4, mutants of which accumulate β-carotene in seeds and/or senescing leaves (Auldridge et al., 2006, Gonzalez-Jorge et al., 2013, Rottet et al., 2016. Zeaxanthin can be cleaved by a CCD subfamily member to generate zaxinone in rice that regulates growth , or oxidatively cleaved into apocarotenoids that exert an ABA-independent regulation upon gene expression (Jia et al., 2021). 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 unidenti ed apocarotenoid signal (ACS) generated during acyclic cis-carotene biosynthesis has been shown to regulate nuclear gene expression and chloroplast biogenesis in Arabidopsis tissues (Cazzonelli et Beltran et al., 2015). The loss-of-function in CAROTENOID ISOMERASE (CRTISO) activity in Arabidopsis causes cis-carotenes to accumulate during seedling skotomorphogenesis, as well as in newly emerged leaves from plants grown under a shorter photoperiod, and triggers accumulation of an unknown cis-ACS that regulates plastid development and photosynthetic nuclear gene expression in a retrograde-like manner (Park et al., 2002. 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 speci c 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 degradation that generate a carotenoid-derived signal that can feedback to control 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 can re ect 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 Arabidopsis foliar tissues grown under a long photoperiod.
Arabidopsis plants were grown in growth cabinets (Climatron Star700, Thermoline Scienti c, Australia) or walk-in room equipped with the controlled plant growth conditions. Debco Seed Raising and Superior germinating mix (Scotts Australia) was supplemented with 3% Osmocote slow-releasing fertiliser (Garden City Plastics Australia) and used to grow plants in 30 Arabidopsis rosette leaves at different developmental stages were collected (20-50 mg/sample) 7 to 9 hours after illumination, snap-frozen in liquid nitrogen, and stored in -80 o C prior to quantify pigment levels. The ontogeny-based numbering of rosette leaves was numbered serially as described (Boyes et al., 2001, Granier et al., 2002.

Pigment-based signalling bioassay for Arabidopsis leaves
The three-week-old soil-grown Arabidopsis plants were used in a pigment-based retrograde signal bioassay developed in-planta. Plants were kept in the dark for four hours prior to experimentation to establish a metabolic equilibrium. Dark-adapted rosettes were detached from the rootstock keeping a 5 mm portion of the hypocotyl intact and transferred onto a Kimwipe paper towel within a plastic Petri dish saturated with 10 ml of NF (50 µM; or as indicated) solution or MilliQ. Three to four plants (10 to 15 leaves) were incubated per petri dish, covered with a clear plastic lid, and incubated (24 hours: or as indicated) under the continuous light or darkness at 22 o C. The mature fully expanded (leaves 1 to 4; old) and recently emerged (leaves 9 to 13, young) leaves ( Figure 1B) were collected after 24 h of treatment and stored in -80 o C prior to quantifying pigments. For the dark incubation experiments, leaf tissues were harvested under a green LED light.

Pigment extraction and quanti cation
Pigment extraction, quanti cation and analysis was performed as previously described (Dhami et al., 2018. In brief, frozen tissues were milled in TissueLyser® (QIAGEN; 2 min, 20 Hz) using stainless steel beads (~3 mm diameter) until nely 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 minutes at 4 o C) and the upper ethyl acetate phase analysed using a HPLC (Agilent 1260 In nity) equipped with YMC-C30 (250 x 4.6mm, S-5 µm) column and Diode Array Detector (DAD) detector. A 35-minute 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 ow rate of 1 ml/min. Carotenoids and chlorophylls were identi ed based upon retention time relative to known standards and their light emission absorbance spectra at 440 nm (chlorophyll, βcarotene, xanthophylls), 340 nm (phyto uene) and 286 nm (phytoene). Absolute quanti cation and determination of composition of pigments was performed as described , Anwar et al., 2022. Quanti cation of phytoene and phyto uene was expressed as peak area per fresh weight.

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

Results
Nor urazon 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 ( Figure 1A, B). Under control growth conditions, chlorophyll and carotenoid levels were signi cantly higher in younger (leaves 9 to 13) compared to old (leaves 1 to 4) leaf types ( Figure 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 phyto uene were only apparent in young leaves at lower concentrations (1 and 10 µM) ( Figure 1E, F). At lower NFZ concentrations (1 µM), young leaves showed a reduction in total carotenoids, but not total chlorophylls ( Figure 1C, D). Total chlorophyll and carotenoid levels were signi cantly 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 phyto uene 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 2-to 3-fold higher in young relative to old leaves after 8, 20 and 24 h of NFZ treatment ( Figure 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 signi cant reduction was observed in young leaves after 20-24 hrs ( Figure 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 o C) and cold (7 o C) temperatures, and extended darkness (24 h) on plastid developed was examined using the pigment-based signalling bioassay. These treatments did not impact chlorophyll or carotenoid levels in older mature leaves (Figure 2A-F). Similarly, these treatments did not cause phytoene to accumulate ( Figure 2G-I). Young leaves exposed to the cold and darkness showed reduced chlorophyll and carotenoid levels like that of old leaves ( Figure 2B, C, E, F). Whereas warmer temperatures only slightly decreased chlorophyll levels and had no signi cant impact on carotenoid content (Figure 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.
The impact of environmental change on pigment levels in aging leaf types was further investigated in combination with NFZ, to determine what might be perturbing chloroplast biogenesis. Young leaves from the NFZ-treated plants accumulated phytoene under warm, cold, and illuminated conditions, that was signi cantly higher than older leaves ( Figure 2G-I). Warmer conditions signi cantly increased phytoene content in both leaf types, whereas cold exposure substantially reduced phytoene levels compared to the standard 22 o C growth temperature ( Figure 2G-I). Intriguingly, dark-exposed young and old leaves did not accumulate phytoene upon simultaneous NFZ treatment ( Figure 2I). Compared to their respective control without NFZ, total chlorophylls and carotenoids were signi cantly reduced in young, but not old leaves from plants treated with NFZ and exposed to 32 o C, 7 o C, and darkness ( Figure 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 ( Figure 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.
Mutants that block the production of lutein (lut2), violaxanthin and neoxanthin (npq2), lutein, violaxanthin and neoxanthin (npq2 lut2), lutein and neoxanthin (npq1 lut2 aba4), or hyperaccumulate zeaxanthin (npq2, npq2 lut2), did not affect the higher chlorophyll levels in younger relative to older leaves ( Figure 3C-D). There were differences in the total carotenoid content among the different mutant 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. 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) (Maass et al., 2009) ( Figure 4A-J). There were no signi cant differences in pigment levels between PSY-OE and WT in both leaf types, with the exception for a subtle reduction in neoxanthin in older leaves from PSY-OE. Therefore, overexpression of PSY did impact chlorophyll levels in young or old leaves.
Next, we investigated if the loss-of-function in z-iso or crtiso mutants, that accumulate acyclic ciscarotenes 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 ( Figure 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 ( Figure 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 ( Figure 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 ( Figure 4A-B). Total carotenoid content was signi cantly lower in young leaves from crtiso relative to WT, yet carotenoid levels were similar in older leaves ( Figure 4J). Transgene 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 ( Figure 4C, J).
Unlike WT, the level of lutein and β-carotene were similar in young and old leaves from crtiso ( Figure 4D-E). Whereas vioaxanthin, antheraxanthin, zeaxanthin and neoaxanthin were all signi cantly 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 quanti ed 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 ( Figure 5A). Curiously, phytoene content was signi cantly 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 ( Figure  5B-D). Similarly, total carotenoid content in young leaves from crtiso plants treated with NFZ were signi cantly lower than older leaves, while young leaves form NFZ treated WT plants showed carotenoid levels similar to older leaves ( Figure 5K). Hence, NFZ and crtiso might affect chlorophyll levels and perhaps chloroplast biogenesis by independent signalling pathways.
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 βcarotene levels in young leaves from both WT and crtiso to levels below that observed in older leaves ( Figure 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 ( Figure 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 ( Figure 5G). NFZ treated younger leaves contained more antheraxanthin and zeaxanthin compared to older leaves, which was further elevated almost 3-fold in the crtiso mutant, evidence of continued carotenoid biosynthesis or reduced catabolism ( Figure 5H-I). The levels of neoxanthin were similar in young and older leaves from WT and crtiso plants treated with NFZ ( Figure 5J). The impact of NFZ on individual carotenoid levels in young leaves from WT and crtiso are complex and highlight a continued supply of isoprenoid substrates for carotenoid biosynthesis. The additive effect of NFZ treatment on accumulation of some chlorophylls and carotenoids in crtiso relative to WT, reveal 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 (Dhami et al., 2018, Gugel and Soll, 2017, Gonzalez et al., 2012. We demonstrate that extended darkness and prolonged cold for 24 hrs can reduce chlorophyll by 20-50% to match levels 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 unidenti ed acyclic ciscarotene 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 (Cazzonelli et al., 2020). 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 unidenti ed 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.
Nor urazon and environmental factors impede pigmentation in young emerging leaves Our pigment-based signalling bioassay showed that NFZ caused a 2-3-fold higher accumulation of phytoene in young compared to old leaves in agreeance with previous reports (Beisel et al., 2011). Despite a presumable impairment in plastid biogenesis in young leaves containing dividing cells and developing plastids, phytoene biosynthesis continued revealing a su cient substrate availability from the MEP pathway. Inhibition of carotenoid biosynthesis by NFZ was previously shown to initially enhance pathway ux, presumably compensating for the short supply of β-carotene (Beisel et al., 2011). 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) ( 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 (Stettler et al., 2009). 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 (Possingham et al., 1975). 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 (Gonzalez et al., 2012, Gugel and Soll, 2017, Jarvis and Lopez-Juez, 2013, Pyke, 2010). 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 14 CO 2 in Arabidopsis (Beisel et al., 2010). However, dark exposure of pepper leaves downregulated the expression of PSY and PDS thereby stalling carotenoid biosynthesis (Simkin et al., 2003). 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 rst 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 (Liu et al., 2018, Gan et al., 2019. Cold stress can cause irreversible damage to chloroplast structure and photosynthetic capacity and trigger ABA biosynthesis in order to enhance cold acclimation in maize (Guo et al., 2021). We showed that colder exposure reduced chlorophyll and carotenoid accumulation in younger leaves. It was previously shown that spinach leaf discs grown at lower temperatures (12°C continuous) contained small cells and fewer chloroplasts (Possingham and Smith, 1972). Cold exposure at 4 o C was also shown to inhibit cell division and arrest growth as evident in Arabidopsis roots and maize leaves (Ashraf and Rahman, 2019, Rymen et al., 2007). Hence, exposure of young leaves to cooler temperatures could suppress cell cycle progression, thereby limiting chloroplast biogenesis and/or division during leaf cell division and/or expansion. The reduction of phytoene and total carotenoid levels in both young and old leaves from the cold exposed plants support an overall reduction in cellular growth processes. This contrasts to higher temperatures that enhanced phytoene accumulation in both young and old leaves yet had no effect on total carotenoid levels and only slightly reduced chlorophyll levels. The reduction in chlorophyll a could be a cellular strategy to reduce heat-induced oxidative stress as was shown in ag leaves at the grain-lling stage of different heat-resistant winter wheat varieties (Wang et al., 2018, Feng et al., 2014. The increased level of phytoene in response to higher temperature could compensate for the higher rate of carotenoid degradation and higher demand of xanthophylls, particularly zeaxanthin which is crucial to maintain functional integrity of chloroplasts (Grudzinski et al., 2017) and the biosynthesis of apocarotenoids that signal stress events (Havaux, 2014). Low temperature and NFZ exposure have similar effects on chlorophyll and carotenoid biosynthesis in young Arabidopsis leaves, contrasting a different signalling mechanism to that induced by darkness which blocks phytoene biosynthesis.
Chlorophyll levels in young leaves are not altered in mutants that disrupt abscisic acid, strigolactone, and/or aporcarotenoid biosynthesis Strigolactone and β-apocarotenoid signalling metabolites regulate stress acclimation and plant development (Hou et al., 2016, Beltran and Stange, 2016. CCD1 cleaves various carotenoid bonds to generate multiple apocarotenoid products (Vogel et al., 2008). CCD4 cleaves βcarotene (Gonzalez-Jorge et al., 2013). CCD7 catalyses the production of strigolactone from β-carotene (Jia et al., 2018). The mature leaves we used to quantify pigment accumulation were unlikely to have transitioned into a phase of senescence and likely harbor mature chloroplasts (Gugel and Soll, 2017). It was not surprising that a single ccd mutant (ccd1, ccd4, or ccd7) or double mutant combination (ccd1 ccd4, ccd1 ccd7, ccd4 ccd7), were unable to signi cantly alter chlorophyll or carotenoid levels in older leaves containing mature chloroplasts. The pigment levels reached a threshold in older leaves that remained unchanged in the absence of SL or CCD-derived β-apocarotenoids. The signi cantly higher level of carotenoid content in the young leaves from Arabidopsis single and double mutants indicates that lower carotenoid content in older leaves could not be attributed to CCD catalysed carotenoid degradation.
The lower pigment levels of older leaves are more likely due to a combination of lower cell density, less cell division, and a low rate of chloroplast turnover ( Violaxanthin and neoxanthin are precursors of abscisic acid that regulates guard cell closure in stomata, mediates stress acclimation and plant development (Du et al., 2013). Mutants such as npq2 (ZEP; also known as aba1-3, aba de cient 1) and aba4 (NXS) alter xanthophyll accumulation and block ABA biosynthesis in Arabidopsis (Ware et al., 2016). The consistently higher level of chlorophyll pigments in younger compared to older leaves in lut2, npq2, lut npq2, and aba4 npq1 lut2 mutants, revealed that altering xanthophyll composition and/or their derived oxidation products did not alter chlorophyll levels and therefore may not have affected chloroplast biogenesis. In addition, the lack of ABA biosynthesis in npq2, lut npq2, and aba4 npq1 lut2 mutants revealed that ABA may not regulate chlorophyll levels in young expanding Arabidopsis leaves in plants grown under a long photoperiod. Taken together, chlorophyll levels and hence chloroplast biogenesis in young leaves of Arabidopsis were not impacted by perturbations in ABA biosynthesis.
A cis-carotene derived ACS could regulate chloroplast development and chlorophyll accumulation in young leaves The higher carotenoid and chlorophyll levels in younger compared to older leaves of the PSY-OE plants showed that substrate supply was not rate-limiting. The substantially higher level of phytoene accumulation in response to NFZ treated leaves, further supported a continual supply of substrates for carotenoid biosynthesis in young leaves, even when NFZ inhibited chloroplast biogenesis. Therefore, substrate supply into the carotenoid pathway does not necessarily have to be affected for a plastid derived signal to mediate a change in chloroplast biogenesis. cis-carotenes have been recently proposed to act as substrates in generating an ACS that controls plastid development (e.g. etioplast, chromoplast, Phytoene and phyto uene accumulation in response to NFZ treatment are unlikely to be signals themselves, although a burst in their production was shown to elicit arti cial chloroplast-to-chromoplast differentiation in leaves (Llorente et al., 2020). Etiolated cotyledons of z-iso accumulate cis-carotenes; 15cis-phytoene, 15, 9' di-cis-phyto uene, and 9,15,9'-tri-cis-zeta-carotene . The z-iso mutant was shown to generate a subtle yellow leaf phenotype when plants were grown under a shorter photoperiod, and there was a slight effect on plastid development as de-etiolated seedlings showed a subtle reduction in chlorophyll accumulation in cotyledons after light exposure . However, the younger leaves from z-iso mutants grown under a longer photoperiod displayed chlorophyll levels similar to that of WT. Therefore, cis-carotenes that accumulate when isomerisation is impaired by z-iso are not provide the right precursors for the biosynthesis of a cis-ACS that regulates chlorophyll accumulation and plastid development in photosynthetic tissues. ζ-carotene, neurosporene and/or tetra-cis-lycopene have been direct linked to the generation of a yet to be identi ed cis-ACS in crtiso mutant tissues that regulate PLB formation in etioplasts and chloroplast biogenesis in plants grown under a shorter photoperiod causing young leaves to display a virescent phenotype (Park et al., 2002. Su cient light exposure facilitates photoisomerisation of tetra-cis-lycopene into all-trans-lycopene, thereby reducing cis-carotene accumulation, restoring plastid development, and greening in older leaves of the Arabidopsis crtiso as well as tomato tangerine mutants , Isaacson et al., 2002. Indeed, we observed similar levels of chlorophyll in older leaves of the crtiso mutant relative to WT plants growing under a longer photoperiod. Surprisingly, the level of total chlorophyll, lutein, and β-carotene were similar between young and old leaves of crtiso when plants are grown under a longer photoperiod, revealing that a ciscarotene derived ACS could be produced under longer photoperiods in order to control plastid development. However, the levels of violaxanthin, zeaxanthin and antheraxanthin were considerably higher in younger relative to older leaves revealing that such a cis-ACS regulates chlorophyll pigmentation and individual carotenoid biosynthesis by different mechanisms. We conclude that isomerisation mediated by CRTISO, and perhaps photoisomerisation, are major rate-limiting steps in regulating a plastid-derived signal that controls chloroplast development during cell division and expansion in young emerging leaves. Given there were similarities (e.g. reduced chlorophyll accumulation) and differences (differential carotenoid biosynthesis) in how crtiso and NFZ impact upon pigment levels in young leaves, this prompted us to explore if they perturb similar signalling pathways. We previously proposed that the crtiso-mediated cis-ACS regulated gene expression was independent of gun-mediated NFZ retrograde signalling . Here, we demonstrated that NFZ heightened the accumulation of phytoene and in crtiso revealing that neither NFZ nor crtiso, or both in combination, impaired substrate supply for carotenogenesis. However, NFZ should have blocked the production of the downstream cis-ACS, as it was able to restore PLB formation in crtiso etiolated seedlings (Cuttriss et al., 2007). The treatment of crtiso with NFZ further decreased chlorophylls, lutein and total carotenoids below that of NFZ treated WT older leaves, and collectively reduced violaxanthin and antheraxanthin to levels below that of crtiso older leaves. This additive effect indicated that NFZ and crtiso generate different signals, neither of which affected the level of the stress pigment zeaxanthin that remained higher in younger leaves, irrespective of the treatment or genotype. It seems likely that the cis-ACS produced by crtiso and retrograde signal (e.g. Mg-ProtoIX) generated by NFZ inhibition of PDS activity, act in different manners to control chloroplast development and maintain pigment homeostasis in young leaves.

Conclusion
The Arabidopsis foliar pigment-based signalling bioassay has utility to combine chemical (e.g NFZ), environmental (darkness and temperature), and genetic (e.g. crtiso or gun signalling mutants) perturbations to decipher how plastid-generated signalling metabolites operate mechanistically in planta to control chlorophyll biosynthesis and chloroplast development. NFZ treatment of whole rosettes likely triggered a chlorophyll-derived signal that affected chloroplast biogenesis during early leaf development leading to a reduction in chlorophyll levels. The Arabidopsis foliar pigment-based signalling bioassay allowed us to demonstrate that carotenoid isomerization was the rate-limiting step in the carotenoid pathway that regulates an unidenti ed apocarotenoid signal controlling chlorophyll biosynthesis.
Photoperiod and light quality are factors affecting photoisomerization of cis-carotenes in a tissuespeci c manner in this bioassay can differentiate between signalling processes by growing plants under different light conditions. Here, we utilised the Arabidopsis foliar pigment-based signalling bioassay to demonstrate that plants grown under a longer photoperiod and lacking carotenoid isomerisation trigger the production of a signal that is likely to control chloroplast biogenesis.

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