A foliar pigment-based bioassay for interrogating chloroplast signalling revealed that carotenoid isomerisation regulates chlorophyll abundance

Abstract

Background Some plastid-derived metabolites can control nuclear gene expression, chloroplast biogenesis, and chlorophyll biosynthesis. For example, norflurazon (NFZ) induced inhibition of carotenoid biosynthesis in leaves elicits a protoporphyrin IX (Mg-ProtoIX) retrograde signal that controls chlorophyll biosynthesis and chloroplast development. Carotenoid cleavage products, known as apocarotenoids, also regulate plastid development. The key steps in carotenoid biosynthesis or catabolism that can regulate chlorophyll biosynthesis in leaf tissues remain unclear. Here, we established a foliar pigment-based bioassay using Arabidopsis rosette leaves to investigate plastid signalling processes in young expanding leaves comprising rapidly dividing and expanding cells containing active chloroplast biogenesis. Results We demonstrate that environmental treatments (extended darkness and cold exposure) as well as chemical (norflurazon; NFZ) inhibition of carotenoid biosynthesis, reduce chlorophyll levels in young, but not older leaves of Arabidopsis. Mutants with disrupted xanthophyll accumulation, apocarotenoid phytohormone biosynthesis (abscisic acid and strigolactone), or enzymatic carotenoid cleavage, did not alter chlorophyll levels in young or old leaves. However, 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 leaves of Arabidopsis plants. NFZ-induced inhibition of PHYTOENE DESATURASE (PDS) activity caused higher phytoene accumulation in younger crtiso leaves compared to WT indicating a continued substrate supply from the methylerythritol 4-phosphate (MEP) pathway. Conclusion The Arabidopsis foliar pigment-based bioassay can be used to differentiate signalling events elicited by environmental change, chemical treatment, and/or genetic perturbation, and determine how they control chloroplast biogenesis and chlorophyll biosynthesis. Genetic perturbations that impaired xanthophyll biosynthesis and/or carotenoid catabolism did not affect chlorophyll biosynthesis. The lack of CAROTENOID ISOMERISATION reduced chlorophyll accumulation, but not phytoene biosynthesis in young leaves of Arabidopsis plants growing under a long photoperiod. Findings generated using the newly customised foliar pigment-based bioassay implicate that carotenoid isomerase activity and NFZ-induced inhibition of PDS activity elicit different signalling pathways to control chlorophyll homeostasis in young leaves of Arabidopsis.

TheLYCOPENE EPSILON CYCLASEuntranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

“…NFZ treatment of seedlings blocks downstream carotenoid biosynthesis and elicits a retrograde signal that suppresses PhANG expression to thereby impair plastid biogenesis (Wu and Bock, 2021). ccr2 also blocks carotenoid accumulation in etiolated tissues, yet the lack of any PLB formation in ccr2 etioplasts and a unique transcriptomic profile indicated that ccr2 could trigger both independent and overlapping signalling pathways (Cazzonelli et al, 2020;Dhami et al, 2022). While εLCY has not been considered a traditional PhANG like LHCB1, our evidence reveals that both these genes are repressed in NFZ-treated etiolated seedlings.…”

“…The loss-of-function in crtiso (ccr2) triggers cis-carotene accumulation and suppresses εLCY expression in etiolated seedlings (Cuttriss et al, 2007). We recently reported a cis-ACS regulated plastid biogenesis, HY5, PIF3, and PhANG expression in etiolated and de-etiolated seedlings, as well as young emerging leaves of ccr2 that accumulate linear cis-carotenes (Cazzonelli et al, 2020;Dhami et al, 2022). We questioned if metabolic feedback regulation of εLCY expression was also linked to linear cis-carotene accumulation.…”

“…LHCB2.1) expression, and impairs carotenoid biosynthesis (MAYFIELD and TAYLOR, 1984;Brausemann et al, 2017;Dhami et al, 2022). NFZ blocked biosynthesis of cyclic carotenoids in etiolated seedlings, leaving only trace levels of lutein, zeaxanthin, and -carotene discernible from residual seed-derived pigments (Figure S3A).…”

Reducing PSY activity fine tunes threshold levels of acis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis

“…After prolycopene biosynthesis and prior to the branch of the epsilon/beta-branch in the pathway, CRTISO regulates isomerisation together with light forming a unidirectional photoswitch that rate-limits cis-carotene levels (Alagoz et al, 2018;Nayak et al, 2022). CRTISO appears to be the key rate-limiting step inducing threshold levels of acyclic linear cis-carotenes that impair signalling of plastid biogenesis (Dhami et al, 2022). During skotomorphogenesis, the etioplasts of seedling tissues develop prolamellar bodies (PLB), the characteristic paracrystalline membrane structure that defines etioplasts and accelerates photomorphogenesis upon illumination (Park et al, 2002;Rodriguez-Villalon et al, 2009).…”