Volatile Apocarotenoids Research Articles

Conserved carotenoid pigmentation in reproductive organs of Charophyceae.

Sexual reproduction in Charophyceae abounds in complex traits. Their gametangia develop as intricate structures, with oogonia spirally surrounded by envelope cells and richly pigmented antheridia. The red-probably protectant-pigmentation of antheridia is conserved across Charophyceae. Chara tomentosa is, however, unique in exhibiting this pigmentation and also in vegetative tissue. Here, we investigated the two sympatric species, C. tomentosa and Chara baltica, and compared their molecular chassis for pigmentation. Using reversed phase C30 high performance liquid chromatography (RP-C30-HPLC), we uncover that the major pigments are β-carotene, δ-carotene and γ-carotene; using headspace solid-phase microextraction coupled to gas chromatography equipped with a mass spectrometer (HS-SPME-GC-MS), we pinpoint that the unusually large carotenoid pool in C. tomentosa gives rise to diverse volatile apocarotenoids, including abundant 6-methyl-5-hepten-2-one. Based on transcriptome analyses, we uncover signatures of the unique biology of Charophycaee and genes for pigment production, including monocyclized carotenoids. The rich carotenoid pool probably serves as a substrate for diverse carotenoid-derived metabolites, signified not only by (i) the volatile apocarotenoids we detected but (ii) the high expression of a gene coding for a cytochrome P450 enzyme related to land plant proteins involved in the biosynthesis of carotenoid-derived hormones. Overall, our data shed light on a key protection strategy of sexual reproduction in the widespread group of macroalgae. The genetic underpinnings of this are shared across hundreds of millions of years of plant and algal evolution. This article is part of the theme issue 'The evolution of plant metabolism'.

Molecular cloning, expression, and biochemical characterization of carotenoid cleavage dioxygenase 1 (LbCCD1) from Lycium barbarum

Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, which have significant industrial applications due to their aromatic and bioactive properties. This study focuses on the molecular cloning and biochemical characterization of the LbCCD1 from Lycium barbarum. The LbCCD1 gene was successfully cloned and heterologously expressed in Escherichia coli and several carotenoids were using as substrates for investigate its specificity by in vitro and in vivo experiment. The LbCCD1 protein was able to cleave a variety of carotenoids including β-carotene, zeaxanthin, astaxanthin, and β-apo-8′-carotenal, at the 9, 10 (9′, 10′) double bond to produce β-ionone, 3‑hydroxy-4-oxo-β-ionone, and 3‑hydroxy-β-ionone, respectively in vitro. LbCCD1 could also cleave zeaxanthin and β-carotene at the 9, 10 (9′, 10′) double bond to produce β-ionone, respectively, in E. coli accumulating carotenoids. Interestingly, LbCCD1 did not exhibit cleavage activity on lycopene either in vivo or in vitro unlike other CCD1 family enzymes.In the previous experiment, it was confirmed that LbCCD1 exhibits cleavage activity towards β-apo-8′-carotenal in vitro, so we used β-apo-8′-carotenal as the substrate for characterizing the enzymatic properties. The expression of LbCCD1 was optimized at such conditions (temperature 24 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of LbCCD1 revealed the optimal activities were at pH 9 and 55 °C. The addition of 10 % ethanol could increase enzyme activity to above 15 %. However, the concentration of Fe2+ has a minimal effect on enzyme activity. The Vmax for β-apo-8′-carotenal was 8.6 U/mg, while the Km was 0.27 mM. To preliminarily verify the potential of LbCCD1 as a biological component for β-ionone production. By introducing LbCCD1 into the β-carotene-high-producing chassis cell and optimizing the conditions (temperature 30 °C, IPTG 0.01 mM, Fe2+ concentration 0.05 mM), the β-ionone yield reached 21.45 mg/L. This study focused on one of the CCDs derived from woody plants, which have been relatively underexplored. It lays the groundwork for expanding the CCD enzyme library, identifying suitable CCDs, and investigating the structure-function relationship of CCDs. Furthermore, it sets the stage for engineering novel CCD genes and developing advanced applications of CCDs as biocatalysts and platforms for synthetic biology. These advancements will enable the efficient production of volatile aroma compounds from carotenoids.

Characterization of Carotenoid Cleavage Oxygenase Genes in Cerasus humilis and Functional Analysis of ChCCD1.

Carotenoid cleavage oxygenases (CCOs) are key enzymes that function in degrading carotenoids into a variety of apocarotenoids and some other compounds. In this study, we performed genome-wide identification and characterization analysis of CCO genes in Cerasus humilis. Totally, nine CCO genes could be classified into six subfamilies, including carotenoid cleavage dioxygenase 1 (CCD1), CCD4, CCD7, CCD8, CCD-like and nine-cis-epoxycarotenoid dioxygenase (NCED), were identified. Results of gene expression analysis showed that ChCCOs exhibited diverse expression patterns in different organs and in fruits at different ripening stages. To investigate the roles of ChCCOs in carotenoids degradation, enzyme assays of the ChCCD1 and ChCCD4 were performed in Escerichia coli BL21(DE3) that can accumulate lycopene, β-carotene and zeaxanthin. The prokaryotic expressed ChCCD1 resulted in obvious degradation of lycopene, β-carotene and zeaxanthin, but ChCCD4 did not show similar functions. To further determine the cleaved volatile apocarotenoids of these two proteins, headspace gas chromatography/mass spectrometer analysis was performed. Results showed that ChCCD1 could cleave lycopene at 5, 6 and 5', 6' positions to produce 6-methy-5-hepten-2-one and could catalyze β-carotene at 9, 10 and 9', 10' positions to generate β-ionone. Our study will be helpful for clarifying the roles of CCO genes especially ChCCD1 in regulating carotenoid degradation and apocarotenoid production in C. humilis.

Metabolism of Carotenoids and β-Ionone Are Mediated by Carotenogenic Genes and PpCCD4 Under Ultraviolet B Irradiation and During Fruit Ripening.

Carotenoids are essential pigments widely distributed in tissues and organs of higher plants, contributing to color, photosynthesis, photoprotection, nutrition, and flavor in plants. White- or yellow-fleshed colors in peach were determined by expression of carotenoids cleavage dioxygenase (PpCCD) genes, catalyzing the degradation of carotenoids. The cracked volatile apocarotenoids are the main contributors to peach aroma and flavor with low sensory threshold concentration. However, the detailed regulatory roles of carotenoids metabolism genes remained unclear under UV-B irradiation. In our study, metabolic balance between carotenoids and apocarotenoids was regulated by the expression of phytoene synthase (PSY), β-cyclase (LCY-B), ε-cyclase (LCY-E), and PpCCD4 under UV-B irradiation. The transcript levels of PpPSY, PpLCY-B, PpLCY-E, and PpCHY-B were elevated 2- to 10-fold compared with control, corresponding to a nearly 30% increase of carotenoids content after 6 h UV-B irradiation. Interestingly, the total carotenoids content decreased by nearly 60% after 48 h of storage, while UV-B delayed the decline of lutein and β-carotene. The transcript level of PpLCY-E increased 17.83-fold compared to control, partially slowing the decline rate of lutein under UV-B irradiation. In addition, the transcript level of PpCCD4 decreased to 30% of control after 48 h UV-B irradiation, in accordance with the dramatic reduction of apocarotenoid volatiles and the delayed decrease of β-carotene. Besides, β-ionone content was elevated by ethylene treatment, and accumulation dramatically accelerated at full ripeness. Taken together, UV-B radiation mediated the metabolic balance of carotenoid biosynthesis and catabolism by controlling the transcript levels of PpPSY, PpLCY-B, PpLCY-E, and PpCCD4 in peach, and the transcript level of PpCCD4 showed a positive relationship with the accumulation of β-ionone during the ripening process. However, the detailed catalytic activity of PpCCD4 with various carotenoid substrates needs to be studied further, and the key transcript factors involved in the regulation of metabolism between carotenoids and apocarotenoids need to be clarified.

The biochemical and molecular investigation of flower color and scent sheds lights on further genetic modification of ornamental traits in Clivia miniata.

Clivia miniata is renowned for its evergreen and strap-like leaves, whereas its floral color and scent are lacking diversity. Here, anthocyanin, volatile terpene, and carotenoid metabolisms were integrally investigated in C. miniata flowers. The results showed that pelargonidins and lutein might cooperate to confer orange or yellow color to C. miniata flowers, but only a trace amount of (+)-limonene was detected. The expression levels of CmF3′H and CmDFR appeared to be responsible for the ratio of cyanidin and pelargonidin derivatives in C. miniata, and the low expression of CmF3′H was responsible for the lack of cyanidins in flowers. Moreover, the CmF3′H promoter could not be activated by CmMYBAs, suggesting that it was controlled by novel regulators. Only two CmTPSs were functional, with CmTPS2 responsible for (+)-limonene synthesis, contributing to the monotonous flower volatile terpenes of C. miniata. CmCCD1a and CmCCD1b were able to cleave carotenoids at the 5,6 (5′,6′), and 9,10 (9′,10′) positions to generate volatile apocarotenoids, whereas the substrates found in low-quantities or specific subcellular localizations of CmCCD1s might constrain volatile apocarotenoid release. Consequently, activating F3′H and introducing novel F3′5′H or versatile TPS may be effective ways to modify the floral color and scent, respectively. Alternatively, modifying the carotenoid flux or CCD1 localization might affect floral color and scent simultaneously. Taking these results together, the present study provides a preliminary deciphering of the genetic constraints underlying flower color and scent development, and proposes possible schemes for further genetic modification of ornamental traits in C. miniata and other 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.

The Genes of CYP, ZEP, and CCD1/4 Play an Important Role in Controlling Carotenoid and Aroma Volatile Apocarotenoid Accumulation of Apricot Fruit.

Carotenoids are important coloration molecules and indispensable component of the human diet. And these compounds confer most of the apricot fruit yellow or orange color. In China, fruit of some apricot cultivar present light-yellow color but strong flowery flavor, however, the chemical mechanism remains unknown. Here, carotenoids and aroma volatile apocarotenoids (AVAs) in three skin types of apricot cultivars (orange, yellow, and light-yellow skinned) were determined by HPLC and GC-MS, respectively. And the transcript levels of carotenogenic genes were analyzed by qRT-PCR. The orange-skinned cultivars “Hongyu” and “Danxing” fruit presented the most abundant total carotenoid, β-carotene and specific α-carotene contents, and β-carotene (52–77%) increased to become the dominant carotenoid during fruit ripening. The transcript levels of lycopene β-cyclase (LCYb) and β-carotene hydroxylase (CHYb) sharply increased during ripening. The yellow-skinned cultivars “Sulian No. 2” and “Akeyaleke” fruit contained lower levels of total carotenoids and β-carotene but were rich in phytoene. The light-yellow coloration of “Baixing” and “Luntaixiaobaixing” fruit was attributed to low amounts of total carotenoids, lutein, and neoxanthin and an absence of β-cryptoxanthin, but high level of aroma volatile apocarotenoids (AVAs) such as β-ionone were detected in these cultivars fruit, accompanied by low transcript levels of carotene hydroxylase (CYP) and zeaxanthin epoxidase (ZEP) but high levels of carotenoid cleavage dioxygenase 1 (CCD1) and CCD4. Correlation analysis showed that the expression level of CCD1 negatively correlated with carotenoid accumulation but positively with AVAs production. These collected results suggest that both carotenoid biosynthesis and degradation are important for apricot coloration and aroma formation. CYP, ZEP, CCD1, and CCD4 may be the key regulation points for carotenoid and AVAs accumulation in apricot fruit, which provide important targets for quality-oriented molecular breeding.

The honeysuckle genome provides insight into the molecular mechanism of carotenoid metabolism underlying dynamic flower coloration.

Lonicera japonica is a widespread member of the Caprifoliaceae (honeysuckle) family utilized in traditional medical practices. This twining vine honeysuckle also is a much-sought ornamental, in part due to its dynamic flower coloration, which changes from white to gold during development. The molecular mechanism underlying dynamic flower coloration in L.japonica was elucidated by integrating whole genome sequencing, transcriptomic analysis and biochemical assays. Here, we report a chromosome-level genome assembly of L.japonica, comprising nine pseudochromosomes with a total size of 843.2Mb. We also provide evidence for a whole-genome duplication event in the lineage leading to L.japonica, which occurred after its divergence from Dipsacales and Asterales. Moreover, gene expression analysis not only revealed correlated expression of the relevant biosynthetic genes with carotenoid accumulation, but also suggested a role for carotenoid degradation in L.japonica's dynamic flower coloration. The variation of flower color is consistent with not only the observed carotenoid accumulation pattern, but also with the release of volatile apocarotenoids that presumably serve as pollinator attractants. Beyond novel insights into the evolution and dynamics of flower coloration, the high-quality L.japonica genome sequence also provides a foundation for molecular breeding to improve desired characteristics.

Determination of Plant Volatile Apocarotenoids.

Volatile compounds derived from carotenoid cleavage are biologically relevant molecules in plants, as they are involved in different processes of plant communication and also have a significant effect on our perception of food flavor.Here we describe two methods for the determination of volatile apocarotenoids in fruit based on extraction by means of headspace solid-phase microextraction (HS-SPME) and separation and detection by means of gas chromatography coupled to mass spectrometry (GC-MS). The first method is optimised for the detection of the complete volatile profile in Citrus fruit, including those compounds derived from carotenoids. The second is a shorter method focused to the detection of volatile apocarotenoids in the tomato fruit.

Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes

In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including β-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and β-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9′,10′) double bond position of cyclic carotenoids to generate α-ionone and β-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.

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

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. Optimization and subsequent validation of the HS-SPME technique for extracting and quantifying volatile apocarotenoids in planta. 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. 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 ± 0.231µg/L, respectively. VAs remained stable at elevated SPME incubation temperatures, with no observable effects of thermal and photo-stereoisomerisation and oxidation. The bipolar 50/30µm divinylbenzene/carboxen on polydimethylsiloxane (PDMS/DVB/CAR) was identified as the optimal fibre for broad molecular weight range VA analysis. 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.

Scent Chemicals of the Tail Gland of the Red Fox,Vulpes vulpes

Like all animals, the red fox uses chemical signals for social communication. The supracaudal or tail gland smells of violets, attributed to the presence of carotenoid degradation products, or apocarotenoids, which commonly occur as aromatics in flowers. We have more fully characterized the scent chemistry of the fox tail gland. Volatile chemicals were analyzed by gas chromatography-mass spectrometry (GC-MS) and identified from their electron ionization mass spectra and Kovats retention indices. The 3 previously reported apocarotenoids were confirmed, and many additional compounds found. These include the apocarotenoids β-cyclocitral, β-homocitral, β-ionone, cyclic β-ionone, β-ionone-5,6-epoxide, α-ionene, α-ionone, 2,6,6-trimethylcyclohexanone (IUPAC 2,2,6-), 2,6,6-trimethyl-2-cyclohexen-1-one, 6-methyl-5-hepten-2-one (sulcatone), and geranyl acetone. Notably, sulcatone is a semiochemical in several species. 3,3-Dimethyl-2,7-octanedione was identified as a probable apocarotenoid which is likely to be a significant fox scent chemical. The γ-lactone of 4-hydroxyhexadecanoic acid (hexadecan-4-olide) was also found, one of a group of known mammalian signaling compounds. This rich mixture of volatile apocarotenoids implies an adequate consumption of plant carotenoids, which are known to be necessary for optimal health. Dietary carotenoids color the skin and feathers of some birds, used as a visual signal to conspecifics, and the floral aroma of the fox tail gland may provide an olfactory signal to other foxes.

Repellent and Attractive Effects of α-, β-, and Dihydro-β- Ionone to Generalist and Specialist Herbivores.

In plants, the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including α-ionone, β-ionone, and dihydro-β-ionone, compounds that are important in herbivore-plant communication. For example, β-ionone is part of an induced defense in canola, Brassica napus, and is released following wounding by herbivores. The objectives of the research were to evaluate whether these volatile compounds would: 1) be released in higher quantities from plants through the over-expression of the carotenoid cleavage dioxygenase1 (CCD1) gene and 2) cause herbivores to be repelled or attracted to over-expressing plants relative to the wild-type. In vivo dynamic headspace collection of volatiles coupled with gas chromatography-mass spectrometry was used to determine volatile organic compounds (VOC) in the headspace of the Arabidopsis thaliana ecotype Columbia-0 (L.) over-expressing the AtCCD1 gene. The analytical method allowed the detection of β-ionone in the Arabidopsis headspace where emission rates ranged between 2 and 5-fold higher compared to the wild type, thus corroborating the in vivo enhancement of gene expression. A two chamber choice test between wild type and AtCCD1 plants revealed that crucifer flea beetle Phyllotreta cruciferae (Goeze) adults were repelled by the AtCCD1 plants with the highest transcription and β-ionone levels. α-Ionone and dihydro-β-ionone were not found in the headspace analysis, but solutions of the three compounds were tested in the concentration range of β-ionone found in the Arabidopsis headspace (0.05 to 0.5ng/μl) in order to assess their biological activity with crucifer flea beetle, two spotted spider mite Tetranychus urticae (Koch), and silverleaf whiteflies Bemisia tabaci (Gennadius). Choice bioassays demonstrated that β-ionone has a strong repellent effect toward both the flea beetle and the spider mite, and significant oviposition deterrence to whiteflies. In contrast, dihydro-β-ionone had attractant properties, especially to the crucifer flea beetle, while α-ionone did not show any significant activity. These findings demonstrate how regulating genes of the carotenoid pathway can increase herbivore deterrent volatiles, a novel tool for insect pest management.

Carotenoid content, antioxidant capacity and volatile compounds of the aroma during tomato ripening

During ripening, tomato fruit accumulates high levels of carotenoids. These compounds, besides their function as pigments, nutrients and antioxidants, are precursors of some important volatile compounds for the development of the characteristic aroma of these fruits. In the present work, the antioxidant capacity, lycopene and β-carotene levels, and the production of aromatic substances derived from the carotenoid biosynthetic pathway were analyzed in the tomato cultivar 7705 at different ripening stages. Results showed a significant (p<0.05) increase of lycopene and β-carotene (22.1 and 2.9 fold, respectively) from the mature green to the breaker stage. Lycopene levels increased and those of β-carotene decreased from the light red to the red ripening stages. There was a 1.6 fold increase of the antioxidant capacity from the mature green to the breaker stage, with no changes between the light red to the red stage. The highest antioxidant capacity observed in the last stages of ripening correlated with the accumulation of lycopene and β-carotene pigments which participate in several antioxidant mechanisms. On the other hand, levels of the volatile compounds of the tomato aroma derived from the carotenoid metabolism significantly increased (p<0.05) in red tomatoes compared to mature green fruits (4.7 fold for 6-methyl-5-hepten-2-one, 3.8 fold for damascenone and approximately twice fold for β-ionone and geranyl acetone). The increase in the volatile compoundss might be explained by the lycopene and β-carotene accumulation in the last ripening stages (light red and red), which are the precursors of the volatile apocarotenoids.

The Carotenoid Cleavage Dioxygenase 1 Enzyme Has Broad Substrate Specificity, Cleaving Multiple Carotenoids at Two Different Bond Positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').