Deregulation of ζ-carotene desaturase in Arabidopsis and tomato exposes a unique carotenoid-derived redundant regulation of floral meristem identity and function.

A level of redundancy and interplay among the transcriptional regulators of floral development safeguards a plant's reproductive success and ensures crop production. In the present study, an additional layer of complexity in the regulation of floral meristem (FM) identity and flower development is elucidated linking carotenoid biosynthesis and metabolism to the regulation of determinate flowering. The accumulation and subsequent cleavage of a diverse array of ζ-carotenes in the chloroplast biogenesis 5 (clb5) mutant of Arabidopsis results in the reprogramming of meristematic gene regulatory networks establishing FM identity mirroring that of the FM identity master regulator, APETALA1 (AP1). The immediate transition to floral development in clb5 requires long photoperiods in a GIGANTEA-independent manner, whereas AP1 is essential for the floral organ development of clb5. The elucidation of this link between carotenoid metabolism and floral development translates to tomato exposing a regulation of FM identity redundant to and initiated by AP1 and proposed to be dependent on the E class floral initiation and organ identity regulator, SEPALLATA3 (SEP3).

The role of carotenoids as a source of retrograde signals: impact on plant development and stress responses.

Communication from plastids to the nucleus via retrograde signal cascades is essential to modulate nuclear gene expression, impacting plant development and environmental responses. Recently, a new class of plastid retrograde signals has emerged, consisting of acyclic and cyclic carotenoids and/or their degradation products, apocarotenoids. Although the biochemical identity of many of the apocarotenoid signals is still under current investigation, the examples described herein demonstrate the central roles that these carotenoid-derived signals play in ensuring plant development and survival. We present recent advances in the discovery of apocarotenoid signals and their role in various plant developmental transitions and environmental stress responses. Moreover, we highlight the emerging data exposing the highly complex signal transduction pathways underlying plastid to nucleus apocarotenoid retrograde signaling cascades. Altogether, this review summarizes the central role of the carotenoid pathway as a major source of retrograde signals in plants.

Detection and analysis of novel and known plant volatile apocarotenoids.

As the climate becomes increasingly unpredictable due to global warming, plants will encounter a greater challenge to adapt to their hostile environment (e.g., drought, heat, pollution). Volatile apocarotenoids (VAs) are an integral part of this necessary adaptation. VAs are involved in diverse plant life processes such as defense against biotic or abiotic stresses and regulate various aspects of plant development. The discovery of new VAs will help enhance abiotic and biotic stress tolerance, optimize biomass and crop yield, improve root development to better search for nutrients and promote symbiotic associations. This chapter describes an optimized method, HeadSpace Solid-Phase MicroExtraction (HS-SPME) coupled to Gas Chromatography-Mass Spectrometry (GC/MS), for the sensitive, reproducible, accurate, and high-throughput detection and quantification of novel and known VAs. Further optimization of this method can be performed by (1) adapting optimal growth conditions for your plants, (2) identifying the correct SPME fiber coating chemistry for the VAs of interest, (3) adapting optimal sample HS-SPME extraction temperature and time, and the desorption time in the GC inlet, (4) identifying the correct GC column and applying the optimal GC/MS parameters for good chromatographic baseline separation of the VAs, mass spectral matching and retention index (RI) validation, and (5) performing suitable quantification and statistical analyses. With this optimized and validated analytical technique, we detected and quantified 28 VAs; 20 of these were identified for the first time in Arabidopsis.

Deconvoluting apocarotenoid-mediated retrograde signaling networks regulating plastid translation and leaf development.

Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1-dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development.

Manipulation of ZDS in tomato exposes carotenoid- and ABA-specific effects on fruit development and ripening.

Spontaneous mutations in fruit-specific carotenoid biosynthetic genes of tomato (Solanum lycopersicum) have led to improved understanding of ripening-associated carotenogenesis. Here, we confirm that ZDS is encoded by a single gene in tomato transcriptionally regulated by ripening transcription factors RIN, NOR and ethylene. Manipulation of ZDS was achieved through transgenic repression and heterologous over-expression in tomato. CaMV 35S-driven RNAi repression inhibited carotenoid biosynthesis in all aerial tissues examined resulting in elevated levels of ζ-carotene isomers and upstream carotenoids, while downstream all trans-lycopene and subsequent photoprotective carotenes and xanthophylls were diminished. Consequently, immature fruit displayed photo-bleaching consistent with reduced levels of the photoprotective carotenes and developmental phenotypes related to a reduction in the carotenoid-derived phytohormone abscisic acid (ABA). ZDS-repressed ripe fruit was devoid of the characteristic red carotenoid, all trans-lycopene and displayed brilliant yellow pigmentation due to elevated 9,9' di-cis-ζ-carotene. Over-expression of the Arabidopsis thaliana ZDS (AtZDS) gene bypassed endogenous co-suppression and revealed ZDS as an additional bottleneck in ripening-associated carotenogenesis of tomato. Quantitation of carotenoids in addition to multiple ripening parameters in ZDS-altered lines and ABA-deficient fruit-specific carotenoid mutants was used to separate phenotypic consequences of ABA from other effects of ZDS manipulation and reveal a unique and dynamic ζ-carotene isomer profile in ripe fruit.

Genetic and metabolic effects of ripening mutations and vine detachment on tomato fruit quality.

Tomato (Solanum lycopersicum) fruit ripening is regulated co-operatively by the action of ethylene and a hierarchy of transcription factors, including RIPENING INHIBITOR (RIN) and NON-RIPENING (NOR). Mutations in these two genes have been adopted commercially to delay ripening, and accompanying textural deterioration, as a means to prolong shelf life. However, these mutations also affect desirable traits associated with colour and nutritional value, although the extent of this trade-off has not been assessed in detail. Here, we evaluated changes in tomato fruit pericarp primary metabolite and carotenoid pigment profiles, as well as the dynamics of specific associated transcripts, in the rin and nor mutants during late development and postharvest storage, as well of those of the partially ripening delayed fruit ripening (dfd) tomato genotype. These profiles were compared with those of the wild-type tomato cultivars Ailsa Craig (AC) and M82. We also evaluated the metabolic composition of M82 fruit ripened on or off the vine over a similar period. In general, the dfd mutation resulted in prolonged firmness and maintenance of quality traits without compromising key metabolites (sucrose, glucose/fructose and glucose) and sectors of intermediary metabolism, including tricarboxylic acid cycle intermediates. Our analysis also provided insights into the regulation of carotenoid formation and highlighted the importance of the polyamine, putrescine, in extending fruit shelf life. Finally, the metabolic composition analysis of M82 fruit ripened on or off the vine provided insights into the import into fruit of compounds, such as sucrose, during ripening.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

Volatile apocarotenoid discovery and quantification in Arabidopsis thaliana: optimized sensitive analysis via HS-SPME-GC/MS.

INTRODUCTION: In the field of carotenoid metabolism researchers' focus has been directed recently toward the discovery and quantification of carotenoid cleavage products (i.e. apocarotenoids, excluding the well-studied carotenoid-derived hormones abscisic acid and strigolactones), due to their emerging roles as putative signaling molecules. Gas chromatography mass spectrometry (GC/MS) and sample preparation via headspace solid phase micro-extraction (HS-SPME) are widely used analytical techniques for broad untargeted metabolomics studies and until now, no optimized quantitative targeted HS-SPME-GC/MS method has been developed specifically for volatile apocarotenoids (VAs) in planta. OBJECTIVES: Optimization and subsequent validation of the HS-SPME technique for extracting and quantifying volatile apocarotenoids in planta. METHODS: Factors considered during method optimization were HS-SPME parameters; vial storage conditions; different adsorbent SPME fibre coating chemistries; plant tissue matrix effects; and fresh tissues to be analyzed. RESULTS: Mean linear regression in planta calibration correlation coefficients (R2) for VAs was 0.974. The resultant method mean limits of detection (LOD) and lower limits of quantification (LLOQ) for VAs using in planta standard additions were 0.384 ± 0.139 and 0.640 ± 0.231 µg/L, respectively. VAs remained stable at elevated SPME incubation temperatures, with no observable effects of thermal and photo-stereoisomerisation and oxidation. The bipolar 50/30 µm divinylbenzene/carboxen on polydimethylsiloxane (PDMS/DVB/CAR) was identified as the optimal fibre for broad molecular weight range VA analysis. CONCLUSIONS: An optimized HS-SPME-GC/MS method for VA detection and quantification was validated in vitro and in planta: based on biological replicates and stringent QA/QC approaches, thereby providing robust detection and quantification of VAs across a broad range of Arabidopsis tissues, fifteen of which were identified for the first time in Arabidopsis.

AtPDS overexpression in tomato: exposing unique patterns of carotenoid self-regulation and an alternative strategy for the enhancement of fruit carotenoid content.

The regulation of plant carotenogenesis is an active research area for both biological discovery and practical implementation. In tomato (Solanum lycopersicum), we demonstrate additional bottlenecks exist in the poly-cis-transformation of phytoene to lycopene in the context of ripening-induced PSY1 expression and activity and reveal phytoene desaturase (PDS), as a target for manipulation towards elevated lycopene content in maturing tomato fruit. Overexpression of Arabidopsis PDS, AtPDS, elevated PDS transcript abundance in all aerial tissues resulting in both altered carotenoid accumulation and associated pathway gene expression in a tissue-specific manner. Significant increases in downstream carotenoids (all-trans-lycopene and ß-carotene) and minimal changes in carotenogenic gene expression (carotenoid isomerase-like 1, CRTIL1) suggest overexpression of heterologous AtPDS in tomato circumvents endogenous regulatory mechanism observed with previous strategies. In transgenic leaves, depletion of the PDS substrate, phytoene, was accompanied by minor, but significant increases in xanthophyll production. Alterations in the leaf carotenogenic transcript profile, including the upstream MEP pathway, were observed revealing unique feedback and feedforward regulatory mechanisms in response to AtPDS overexpression. AtPDS overexpression in the background of the tangerine (carotenoid isomerase, CRTISO) mutant exposes its potential in elevating downstream cis-lycopene accumulation in ripe tomato fruit, as cis-lycopene is more bioavailable yet less abundant than all-trans-lycopene in the wild-type control. In summary, we demonstrate the limitation of PDS in ripening fruit, its utility in modifying carotenoid profiles towards improved quality, and reveal novel carotenoid pathway feedback regulation.

Synthesis and Function of Apocarotenoid Signals in Plants.

In plants, carotenoids are essential for photosynthesis and photoprotection. However, carotenoids are not the end products of the pathway; apocarotenoids are produced by carotenoid cleavage dioxygenases (CCDs) or non-enzymatic processes. Apocarotenoids are more soluble or volatile than carotenoids but they are not simply breakdown products, as there can be modifications post-cleavage and their functions include hormones, volatiles, and signals. Evidence is emerging for a class of apocarotenoids, here referred to as apocarotenoid signals (ACSs), that have regulatory roles throughout plant development beyond those ascribed to abscisic acid (ABA) and strigolactone (SL). In this context we review studies of carotenoid feedback regulation, chloroplast biogenesis, stress signaling, and leaf and root development providing evidence that apocarotenoids may fine-tune plant development and responses to environmental stimuli.

Fruit carotenoid-deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis.

Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2-C-methyl-d-erythritol-4-phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map-based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.

Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond.

The chloroplast can act as an environmental sensor, communicating with the cell during biogenesis and operation to change the expression of thousands of proteins. This process, termed retrograde signaling, regulates expression in response to developmental cues and stresses that affect photosynthesis and yield. Recent advances have identified many signals and pathways-including carotenoid derivatives, isoprenes, phosphoadenosines, tetrapyrroles, and heme, together with reactive oxygen species and proteins-that build a communication network to regulate gene expression, RNA turnover, and splicing. However, retrograde signaling pathways have been viewed largely as a means of bilateral communication between organelles and nuclei, ignoring their potential to interact with hormone signaling and the cell as a whole to regulate plant form and function. Here, we discuss new findings on the processes by which organelle communication is initiated, transmitted, and perceived, not only to regulate chloroplastic processes but also to intersect with cellular signaling and alter physiological responses.

More than meets the eye: from carotenoid biosynthesis, to new insights into apocarotenoid signaling.

Carotenoids are a class of isoprenoids synthesized almost exclusively in plants involved in a myriad of roles including the provision of flower and fruit pigmentation for the attraction of pollinators and seed dispersing organisms. While carotenoids are essential throughout plant development, they are also extremely important in human diets providing necessary nutrition and aiding in the prevention of various cancers, age-related diseases and macular degeneration. Utilization of multiple plant models systems (i.e. Arabidopsis; maize; and tomato) has provided a comprehensive framework detailing the regulation of carotenogenesis throughout plant development covering all levels of genetic regulation from epigenetic to post-translational modifications. That said, the understanding of how carotenoids self-regulate remains fragmented. Recent reports demonstrate the potential influence of carotenoid-cleavage products (apocarotenoids) as signaling molecules regulating carotenoid biosynthesis in addition to various aspects of plants development (i.e. leaf and root development). This review highlights recent advances in carotenogenic regulation and insights into potential roles of novel apocarotenoids in plants.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid ß-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.

Molecular and genetic regulation of fruit ripening.

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During this maturation process, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organisms. In addition, advanced or over-ripening and senescence, especially through tissue softening and eventual decay, render fruit susceptible to invasion by opportunistic pathogens. While ripening and senescence are often used interchangeably, the specific metabolic activities of each would suggest that ripening is a distinct process of fleshy fruits that precedes and may predispose the fruit to subsequent senescence.

Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening.

Ripening of tomato fruits is triggered by the plant hormone ethylene, but its effect is restricted by an unknown developmental cue to mature fruits containing viable seeds. To determine whether this cue involves epigenetic remodeling, we expose tomatoes to the methyltransferase inhibitor 5-azacytidine and find that they ripen prematurely. We performed whole-genome bisulfite sequencing on fruit in four stages of development, from immature to ripe. We identified 52,095 differentially methylated regions (representing 1% of the genome) in the 90% of the genome covered by our analysis. Furthermore, binding sites for RIN, one of the main ripening transcription factors, are frequently localized in the demethylated regions of the promoters of numerous ripening genes, and binding occurs in concert with demethylation. Our data show that the epigenome is not static during development and may have been selected to ensure the fidelity of developmental processes such as ripening. Crop-improvement strategies could benefit by taking into account not only DNA sequence variation among plant lines, but also the information encoded in the epigenome.

Tissue specificity and differential expression of transcription factors in tomato provide hints of unique regulatory networks during fruit ripening.

Tissue specificity or dramatically different expression levels of transcription factors in different tissue types allows differential regulation of tissue development as well as alternate modes of metabolic regulation. Recently we reported (Rohrmann et al., 2011) the development of a quantitative real-time PCR platform (qRT-PCR) that allows accurate quantification of the expression level of approximately 1000 tomato transcription factors. Application of this platform to samples collected during a ripening time course of wild type tomato and the high pigment mutant hp1 allowed us to identify transcription factors of importance both to ripening per se and to the metabolic shifts that occur during this critical biological process. Here we extend the quantitative real-time PCR analyses to include samples from flower, leaf, stem and root of wild type tomato. Co-expression network analysis to identify both conserved and unique regulatory networks both between individual tissues of tomato and also in cross-species comparisons of specific tissues, suggested some key TF genes which are involved in photosynthesis and fruit development.

Altered chloroplast development and delayed fruit ripening caused by mutations in a zinc metalloprotease at the lutescent2 locus of tomato.

The chloroplast is the site of photosynthesis in higher plants but also functions as the center of synthesis for primary and specialized metabolites including amino acids, fatty acids, starch, and diverse isoprenoids. Mutants that disrupt aspects of chloroplast function represent valuable tools for defining structural and biochemical regulation of the chloroplast and its interplay with whole-plant structure and function. The lutescent1 (l1) and l2 mutants of tomato (Solanum lycopersicum) possess a range of chlorophyll-deficient phenotypes including reduced rates of chlorophyll synthesis during deetiolation and enhanced rates of chlorophyll loss in leaves and fruits as they age, particularly in response to high-light stress and darkness. In addition, the onset of fruit ripening is delayed in lutescent mutants by approximately 1 week although once ripening is initiated they ripen at a normal rate and accumulation of carotenoids is not impaired. The l2 locus was mapped to the long arm of chromosome 10 and positional cloning revealed the existence of a premature stop codon in a chloroplast-targeted zinc metalloprotease of the M50 family that is homologous to the Arabidopsis (Arabidopsis thaliana) gene ETHYLENE-DEPENDENT GRAVITROPISM DEFICIENT AND YELLOW-GREEN1. Screening of tomato germplasm identified two additional l2 mutant alleles. This study suggests a role for the chloroplast in mediating the onset of fruit ripening in tomato and indicates that chromoplast development in fruit does not depend on functional chloroplasts.

Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation.

Solanum lycopersicum (tomato) and its wild relatives harbor genetic diversity that yields heritable variation in fruit chemistry that could be exploited to identify genes regulating their synthesis and accumulation. Carotenoids, for example, are essential in plant and animal nutrition, and are the visual indicators of ripening for many fruits, including tomato. Whereas carotenoid synthesis is well characterized, factors regulating flux through the pathway are poorly understood at the molecular level. To exploit the impact of tomato genetic diversity on carotenoids, Solanum pennellii introgression lines were used as a source of defined natural variation and as a resource for the identification of candidate regulatory genes. Ripe fruits were analyzed for numerous fruit metabolites and transcriptome profiles generated using a 12,000 unigene oligoarray. Correlation analysis between carotenoid content and gene expression profiles revealed 953 carotenoid-correlated genes. To narrow the pool, subnetwork analysis of carotenoid-correlated transcription revealed 38 candidates. One candidate for impact on trans-lycopene and ß-carotene accumulation was functionally charaterized, SlERF6, revealing that it indeed influences carotenoid biosynthesis and additional ripening phenotypes. Reduced expression of SlERF6 by RNAi enhanced both carotenoid and ethylene levels during fruit ripening, demonstrating an important role for SlERF6 in ripening, integrating the ethylene and carotenoid synthesis pathways.