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SUMMARY: The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all‐trans‐ into 9‐cis‐β‐carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27‐homologs, D27‐like1 and ‐like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27‐like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13‐cis/15‐cis/9‐cis‐ and a 9‐cis/all‐trans‐β‐carotene isomerization. Although disruption of AtD27‐like1 did not cause SL deficiency phenotypes, overexpression of AtD27‐like1 in the d27 mutant restored the more‐branching phenotype, indicating a contribution of AtD27‐like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27‐like1 to SL biosynthesis is likely a result of its formation of 9‐cis‐β‐carotene that was present at higher levels in AtD27‐like1 overexpressing lines. By contrast, AtD27‐like1 expression correlated negatively with the content of 9‐cis‐violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone‐treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis‐β‐carotene isomerase that affects the content of both cis‐carotenoid‐derived plant hormones, ABA and SLs. Significance Statement: AtD27LIKE1 is involved in the biosynthesis of the two carotenoid‐derived plant hormones ABA and strigolactones (SLs), by feeding SL biosynthesis at the same time as negatively impacting ABA content. [ABSTRACT FROM AUTHOR]

Summary: Carotenoid‐derived regulatory metabolites and hormones are generally known to arise through the oxidative cleavage of a single double bond in the carotenoid backbone, which yields mono‐carbonyl products called apocarotenoids. However, the extended conjugated double bond system of these pigments predestines them also to repeated cleavage forming dialdehyde products, diapocarotenoids, which have been less investigated due to their instability and low abundance. Recently, we reported on the short diapocarotenoid anchorene as an endogenous Arabidopsis metabolite and specific signaling molecule that promotes anchor root formation. In this work, we investigated the biological activity of a synthetic isomer of anchorene, iso‐anchorene, which can be derived from repeated carotenoid cleavage. We show that iso‐anchorene is a growth inhibitor that specifically inhibits primary root growth by reducing cell division rates in the root apical meristem. Using auxin efflux transporter marker lines, we also show that the effect of iso‐anchorene on primary root growth involves the modulation of auxin homeostasis. Moreover, by using liquid chromatography–mass spectrometry analysis, we demonstrate that iso‐anchorene is a natural Arabidopsis metabolite. Chemical inhibition of carotenoid biosynthesis led to a significant decrease in the iso‐anchorene level, indicating that it originates from this metabolic pathway. Taken together, our results reveal a novel carotenoid‐derived regulatory metabolite with a specific biological function that affects root growth, manifesting the biological importance of diapocarotenoids. [ABSTRACT FROM AUTHOR]

Carotenoids are ubiquitous precursors of important metabolites including hormones, such as strigolactones (SLs) and abscisic acid (ABA), and signaling and regulatory molecules, such as the recently discovered zaxinone. Strigolactones and ABA are key regulators of plant growth and development, adaptation to environmental changes and response to biotic and abiotic stress. Previously, we have shown that zaxinone, an apocarotenoid produced in rice by the enzyme zaxinone synthase (ZAS) that is common in mycorrhizal plants, is required for normal rice growth and development, and a negative regulator of SL biosynthesis. Zaxinone is also formed in Arabidopsis, which lacks ZAS, via an unknown route. In the present study, we investigated the biological activity of zaxinone in Arabidopsis, focusing on its effect on SL and ABA biosynthesis. For this purpose, we quantified the content of both hormones and determined the levels of related transcripts in Arabidopsis (Arabidopsis thaliana), roots upon zaxinone treatment. For SL quantification, we also employed Striga seed germination bioassay. Results obtained show that zaxinone application to hydroponically grown Arabidopsis seedlings enhanced transcript levels of key biosynthetic genes of both hormones, led to higher root ABA and SL (methyl carlactonoate, MeCLA) content, and increased SL release, even under sufficient phosphate supply. Using the SL insensitive (max2-1) and the ABA deficient (aba1-6, aba2-1 , and nced3) mutants, we also show that zaxinone application reduced hypocotyl growth and that this effect is caused by increasing ABA content. Our results suggest that zaxinone is a regulatory metabolite also in Arabidopsis, which triggers the biosynthesis of both carotenoid-derived hormones, SLs and ABA, in roots. In the non-mycotrophic plant Arabidopsis, zaxinone does not increase growth and may be perceived as a stress signal, while it acts as a growth-promoting metabolite and suppressor of SL biosynthesis in rice. [ABSTRACT FROM AUTHOR]

INDETERMINATE DOMAIN (IDD)/BIRD proteins belong to a highly conserved plant-specific group of transcription factors with dedicated functions in plant physiology and development. Here, we took advantage of the chimeric repressor gene-silencing technology (CRES-T, SRDX) to widen our view on the role of IDD4 / IMPERIAL EAGLE and IDD family members in plant immunity. The hypomorphic idd4SRDX lines are compromised in growth and show a robust autoimmune phenotype. Hormonal measurements revealed the concomitant accumulation of salicylic acid and jasmonic acid suggesting that IDDs are involved in regulating the metabolism of these biotic stress hormones. The analysis of immunity-pathways showed enhanced activation of immune MAP kinase-signaling pathways, the accumulation of hydrogen peroxide and spontaneous programmed cell death. The transcriptome of nonelicited idd4SRDX lines can be aligned to approximately 40% of differentially expressed genes (DEGs) in flg22-treated wild-type plants. The pattern of DEGs implies IDDs as pivotal repressors of flg22-dependent gene induction. Infection experiments showed the increased resistance of idd4SRDX lines to Pseudomonas syringae and Botrytis cinerea implying a function of IDD s in defense adaptation to hemibiotrophs and necrotrophs. Genome-wide IDD4 DNA-binding studies (DAP-SEQ) combined with DEG analysis of idd4SRDX lines identified IDD4-regulated functional gene clusters that contribute to plant growth and development. In summary, we discovered that the expression of idd4SRDX activates a wide range of defense-related traits opening up the possibility to apply idd4SRDX as a powerful tool to stimulate innate immunity in engineered crops. [ABSTRACT FROM AUTHOR]

Apocarotenoid glycosylation serves as a valve regulating carotenoid homeostasis in plants and may contribute to their response to photo-oxidative stress. However, an analytical method that allows comprehensive and sensitive profiling of glycosylated apocarotenoids (GAPOs) is still missing. We developed an efficient ultra-high performance liquid chromatography-high resolution-mass spectrometry (UHPLC-HR-MS) method to analyze 25 GAPOs present in carotenoid-accumulating E. coli cells and plant tissues. Optimized HR-heated-electrospray ionization (HESI)-MS parameters enabled, based on HR MS and tandem mass spectrometry (MS/MS) data, the identification of yet undescribed GAPOs from Arabidopsis, which include Glc-apo-11-carotenal (GAPO11), Glc-apo-13-carotenone (GAPO13), and their isomers. The identity of these compounds was confirmed by the transformation of deuterium-labelled non-hydroxylated carotene cleavage products into the corresponding GAPOs in planta. Quantitative analysis of GAPOs in Arabidopsis showed that the levels of Glc-cyclocitral (GAPO7), Glc-cyclocitral isomer I (GAPO7I), Glc-ionone (GAPO9), Glc-ionone isomer I (GAPO9I), Glc-apo-11-carotenal isomer I (GAPO11I), Glc-apo-13-carotenone (GAPO13), and Glc-apo-13-carotenone isomers (GAPO13I, GAPO13II, and GAPO13III) significantly increase after high light (HL) treatment. This treatment also led to an obvious increase in the levels of most carotene- and all xanthophyll-derived apocarotenoids detected in our system. Our work demonstrates for the first time that HL stress induces apocarotenoid glycosylation in Arabidopsis and unravels a novel plant metabolic pathway that leads from carotene cleavage products to GAPOs that are identical to xanthophyll derived GAPOs. Thus, our new approach allows sensitive and reliable profiling of GAPOs, which is crucial for understanding the function of apocarotenoid glycosylation in plants and its role in the acclimation to HL stress. [ABSTRACT FROM AUTHOR]

Highlights • Arabidopsis AtD27 catalyzes the reverse isomerization of all- trans -/9- cis -β-carotene and carotenes with unmodified β-ionone ring. • Using promoter- GUS lines and qRT-PCR assays, we show that AtD27 expression is regulated by auxin, ABA and phosphate availability. • We provide evidence for the isomerization activity in planta and for a role of AtD27 in determining shoots ABA content. Abstract Strigolactones (SLs) are carotenoid-derived plant hormones that influence various aspects of plant growth and development in response to environmental conditions, especially nutrients deficiency. SLs are synthesized via a strict stereo-specific core pathway that leads to the intermediate carlactone, requiring the iron-containing polypeptide DWARF27 (D27) and the carotenoid cleavage dioxygenases 7 (CCD7) and 8 (CCD8). It has been shown that the rice OsD27 is a β-carotene isomerase catalyzing the interconversion of all- trans - into 9- cis -β -carotene. However, data about the enzymatic activity of D27 from other species are missing. Here, we investigated the activity and substrate specificity of the Arabidopsis AtD27 by testing a broad range of carotenoid substrates. Both in vivo and in vitro assays show that AtD27 catalyzes the reverse isomerization of all- trans -/9- cis -β-carotene. AtD27 did not isomerize 13- cis - or 15- cis -β-carotene, indicating high specificity for the C9-C10 double bond. The isomerization reaction was inhibited in the presence of silver acetate, pointing to the involvement of an iron-sulfur cluster. We further investigated the expression of AtD27 , using Arabidopsis transgenic lines expressing β-glucuronidase (GUS) under the control of AtD27 native promoter. AtD27 is ubiquitously expressed throughout the plant with the highest expression in immature flowers. In lateral roots, AtD27 expression was induced by treatment with auxin and ABA, while the application of SL analogs did not show an effect. Lower ABA levels in atd27 mutant indicated an interference with the ABA pathway. Quantitative real-time RT-PCR showed that transcript levels of AtD27 and other SL biosynthetic genes in roots are induced upon phosphate starvation. Taken together, our study on AtD27 confirms the postulated enzymatic function of this enzyme, shows its strict substrate- and regio-specificity and indicates an important role in response to multiple plant hormones and phosphate deficiency. [ABSTRACT FROM AUTHOR]