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Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis.

ABSTRACT: Crocus sativus is a triploid sterile plant with long red stigmas which form commercial saffron. Saffron is the site for synthesis and accumulation of apocarotenoids like crocin, picrocrin and safranal which are responsible for its color, flavour and aroma making it world's most expensive spice. These compounds are formed by oxidative cleavage of zeaxanthin by carotenoid cleavage dioxygenases. Although the biosynthetic pathway of apocarotenoids is known to a considerable extent, the mechanism that regulates its tissue and developmental stage specific expression is not known.In the present work, we identified, cloned and characterized ultrapetala transcription factor called CsULT1 from Crocus. The gene contains an 80 amino acid long conserved SAND domain. The CsULT1 transcript was more abundant in stigma and showed increase in expression from pre anthesis stage till anthesis and decreased in post anthesis stage which corroborated with the accumulation pattern of crocin indicating its possible role in regulation of apocarotenoid biosynthesis. CsULT1 was found to be transcriptionally active and localized in nucleus. Its expression is induced in response to phytohormones like auxin, methyljasmonate and salicylic acid. Overexpression of CsULT1 in Crocus calli resulted in enhanced expression of key pathway genes like phytoene synthase (PSY), phytoene desaturase (PDS), beta carotene hydroxylase (BCH) and carotenoid cleavage dioxygenases (CCDs) indicating its role in regulation of apocarotenoid biosynthesis.This work presents first report on isolation and characterization of ultrapetala gene from Crocus. Our results suggest that CsULT1 is a novel regulator of Crocus apocarotenoid biosynthesis. We show for the first time involvement of plant SAND domain proteins in regulating secondary metabolic pathways.

SUBMITTER: Ashraf N

PROVIDER: S-EPMC4349709 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis.

Ashraf Nasheeman N Jain Deepti D Vishwakarma Ram A RA

BMC plant biology 20150201

<h4>Background</h4>Crocus sativus is a triploid sterile plant with long red stigmas which form commercial saffron. Saffron is the site for synthesis and accumulation of apocarotenoids like crocin, picrocrin and safranal which are responsible for its color, flavour and aroma making it world's most expensive spice. These compounds are formed by oxidative cleavage of zeaxanthin by carotenoid cleavage dioxygenases. Although the biosynthetic pathway of apocarotenoids is known to a considerable extent ...[more]

PMID: 25640597

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Project description:Crocus sativus stigmas form rich source of apocarotenoids like crocin, picrocrocin and saffranal which besides imparting color, flavour and aroma to saffron spice also have tremendous pharmacological properties. Inspite of their importance, the biosynthetic pathway of Crocus apocarotenoids is not fully elucidated. Moreover, the mechanism of their stigma specific accumulation remains unknown. Therefore, deep transcriptome sequencing of Crocus stigma and rest of the flower tissue was done to identify the genes and transcriptional regulators involved in the biosynthesis of these compounds.Transcriptome of stigma and rest of the flower tissue was sequenced using Illumina Genome Analyzer IIx platform which generated 64,604,402 flower and 51,350,714 stigma reads. Sequences were assembled de novo using trinity resulting in 64,438 transcripts which were classified into 32,204 unigenes comprising of 9853 clusters and 22,351 singletons. A comprehensive functional annotation and gene ontology (GO) analysis was carried out. 58.5 % of the transcripts showed similarity to sequences present in public databases while rest could be specific to Crocus. 5789 transcripts showed similarity to transcription factors representing 76 families out of which Myb family was most abundant. Many genes involved in carotenoid/apocarotenoid pathway were identified for the first time in this study which includes zeta-carotene isomerase and desaturase, carotenoid isomerase and lycopene epsilon-cyclase. GO analysis showed that the predominant classes in biological process category include metabolic process followed by cellular process and primary metabolic process. KEGG mapping analysis indicated that pathways involved in ribosome, carbon and starch and sucrose metabolism were highly represented. Differential expression analysis indicated that key carotenoid/apocarotenoid pathway genes including phytoene synthase, phytoene desaturase and carotenoid cleavage dioxygenase 2 are enriched in stigma thereby providing molecular proof for stigma to be the site of apocarotenoid biosynthesis.This data would provide a rich source for understanding the carotenoid/apocarotenoid metabolism in Crocus. The database would also help in investigating many questions related to saffron biology including flower development.

Project description:BACKGROUND: TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. RESULTS: We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. CONCLUSIONS: The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.

Project description: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.

Dataset Information

Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis.

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Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis.

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