Enzymatic Formation of β-Citraurin from β-Cryptoxanthin and Zeaxanthin by Carotenoid Cleavage Dioxygenase4 in the Flavedo of Citrus Fruit

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7′,8′ position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.Carotenoids, a diverse group of pigments widely distributed in nature, fulfill a variety of important functions in plants and play a critical role in human nutrition and health (Schwartz et al., 1997; Cunningham and Gantt, 1998; Havaux, 1998; Krinsky et al., 2003; Ledford and Niyogi, 2005). The pathway of carotenoid biosynthesis has been well documented in various plant species, including Arabidopsis (Arabidopsis thaliana; Park et al., 2002), tomato (Lycopersicon esculentum; Isaacson et al., 2002), pepper (Capsicum annuum; Bouvier et al., 1998), citrus (Citrus spp.; Kato et al., 2004, 2006; Rodrigo et al., 2004; Rodrigo and Zacarías, 2007; Kato, 2012; Zhang et al., 2012a), and apricot (Prunus armenaica; Kita et al., 2007). Genes encoding the enzymes in the carotenoid biosynthetic pathway have been cloned, and their expression profiles have also been characterized (Fig. 1). As carotenoids contain a series of conjugated double bonds in the central chain, they can be oxidatively cleaved in a site-specific manner (Mein et al., 2011). The oxidative cleavage of carotenoids not only regulates their accumulation but also produces a range of apocarotenoids (Walter et al., 2010). In higher plants, many different apocarotenoids derive from the cleavage of carotenoids and have important metabolic functions, such as plant hormones, pigments, aroma and scent compounds, as well as signaling compounds (Fig. 1). A well-known example is abscisic acid, which is a C₁₅ compound derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and 9′-cis-neoxanthin (Schwartz et al., 1997; Tan et al., 1997; Cutler and Krochko, 1999; Chernys and Zeevaart, 2000; Giuliano et al., 2003).Open in a separate windowFigure 1.Carotenoid and apocarotenoid metabolic pathway in plants. GGPP, Geranylgeranyl diphosphate. Enzymes, listed here from top to bottom, are named according to the designation of their genes: PSY, phytoene synthase; PDS, Phytoene desaturase; ZDS, ζ-carotene desaturase; ZISO, 15-cis-ζ-carotene isomerase; CRTISO, carotenoid isomerase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYe, ε-ring hydroxylase; HYb, β-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase.Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidative cleavage of carotenoids (Ryle and Hausinger, 2002). CCDs are nonheme iron enzymes present in plants, bacteria, and animals. In plants, CCDs belong to an ancient and highly heterogenous family (CCD1, CCD4, CCD7, CCD8, and 9-cis-epoxycarotenoid dioxygenases [NCEDs]). The similarity among the different members is very low apart from four strictly conserved His residues and a few Glu residues (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, the CCD family contains nine members (CCD1, NCED2, NCED3, CCD4, NCED5, NCED6, CCD7, CCD8, and NCED9), and orthologs in other plant species are typically named according to their homology with an Arabidopsis CCD (Huang et al., 2009). In our previous study, the functions of CitCCD1, CitNCED2, and CitNCED3 were investigated in citrus fruits (Kato et al., 2006). The recombinant CitCCD1 protein cleaved β-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9,10 and 9′,10′ positions and 9-cis-violaxanthin at the 9′,10′ position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11,12 position to form xanthoxin, a precursor of abscisic acid (Kato et al., 2006). To date, information on the functions of other CCDs in citrus fruits remains limited, while the functions of CCD7 and CCD8, as well as NCED5, NCED6, and NCED9, in Arabidopsis have been characterized (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, CCD7 cleaves all-trans-β-carotene at the 9′,10′ position to form all-trans-β-apo-10′-carotenal. All-trans-β-apo-10′-carotenal is further shortened by AtCCD8 at the 13,14 position to produce β-apo-13-carotenone (Alder et al., 2012). NCED5, NCED6, and NCED9 cleave 9-cis-violaxanthin at the 11,12 position to form xanthoxin (Tan et al., 2003). Compared with other CCDs, the function of CCD4 is poorly understood. In Chrysanthemum morifolium, CmCCD4a contributed to the white color formation by cleaving carotenoids into colorless compounds (Ohmiya et al., 2006). Recently, it has been reported that CsCCD4, CmCCD4a, and MdCCD4 could cleave β-carotene to yield β-ionone (Rubio et al., 2008; Huang et al., 2009).β-Citraurin, a C₃₀ apocarotenoid, is a color-imparting pigment responsible for the reddish color of citrus fruits (Farin et al., 1983). In 1936, it was first discovered in Sicilian oranges (Cual, 1965). In citrus fruits, the accumulation of β-citraurin is not a common event; it is only observed in the flavedos of some varieties during fruit ripening. The citrus varieties accumulating β-citraurin are considered more attractive because of their red-orange color (Ríos et al., 2010). Although more than 70 years have passed since β-citraurin was first identified, the pathway of its biosynthesis is still unknown. As its structure is similar to that of β-cryptoxanthin and zeaxanthin, β-citraurin was presumed to be a degradation product of β-cryptoxanthin or zeaxanthin (Oberholster et al., 2001; Rodrigo et al., 2004; Ríos et al., 2010; Fig. 1). To date, however, the specific cleavage reaction producing β-citraurin has not been elucidated. In this study, we found that the CitCCD4 gene was involved in the synthesis of β-citraurin, using two citrus varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. To confirm the role of the CitCCD4 gene further, functional analyses of the CitCCD4 enzyme were performed in vivo and in vitro. Additionally, the regulation of β-citraurin content and CitCCD4 gene expression in response to ethylene and red light-emitting diode (LED) light treatments was also examined. This study, to our knowledge, is the first to investigate the biosynthesis of β-citraurin in citrus fruits. The results might provide new strategies to enhance the nutritional and commercial qualities of citrus fruits.     

In Planta Assessment of the Role of Thioredoxin h Proteins in the Regulation of S-Locus Receptor Kinase Signaling in Transgenic Arabidopsis

The self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma.Many flowering plants possess self-incompatibility (SI), a genetic system that promotes outcrossing by preventing self-fertilization. In the Brassicaceae family, the SI response is controlled by haplotypes of the S locus, each of which contains two genes that encode highly polymorphic proteins, the S-locus receptor kinase (SRK), which is a plasma membrane resident single-pass transmembrane Ser/Thr receptor kinase displayed at the surface of stigma epidermal cells (Stein et al., 1991; Takasaki et al., 2000), and the S-locus Cys-rich protein (SCR), which is the pollen coat-localized ligand for SRK (Schopfer et al., 1999; Kachroo et al., 2001; Takayama et al., 2001). SRK and SCR exhibit allele-specific interactions, whereby only SRK and SCR encoded by the same S-locus haplotype interact. In a self-pollination, the binding of this “self” pollen-borne SCR to the extracellular domain of SRK activates the SRK kinase, thereby triggering a cellular response in stigma epidermal cells that causes inhibition of pollen germination and tube penetration into the stigma epidermal cell wall (for review, see Tantikanjana et al., 2010).Tight regulation of SRK kinase activity and its signaling cascade is critical for productive pollen-stigma interactions because constitutive (i.e. SCR-independent) activity of the receptor is expected to result in sterile stigmas that reject both compatible and incompatible pollen. In the classical view of ligand-activated receptor kinases, the receptor occurs as catalytically inactive monomers in the absence of ligand and only becomes activated upon ligand-induced dimerization (for review, see Lemmon and Schlessinger, 2010). However, some receptor kinases in both animals (Chan et al., 2000; Ehrlich et al., 2011) and plants (Giranton et al., 2000; Wang et al., 2005, 2008; Shimizu et al., 2010; Bücherl et al., 2013) form catalytically inactive dimers or oligomers in the absence of ligand, with receptor activation presumably resulting from ligand-induced higher order oligomerization or conformational changes (Lemmon and Schlessinger, 2010). Similar to the latter receptors, SRK forms oligomers in unpollinated stigmas, i.e. in the absence of SCR (Giranton et al., 2000), at least partly via ligand-independent dimerization domains located within the SRK extracellular domain (Naithani et al., 2007). It has been proposed that these ligand-independent SRK oligomers are maintained in an inactive state by thioredoxins, the ubiquitous small oxidoreductases that reduce disulfide bridges in proteins (Buchanan and Balmer, 2005). This hypothesis is supported by the following observations: (1) two Brassica napus thioredoxins, the Thioredoxin H-Like proteins THL1 and THL2, were identified as SRK interactors in a yeast (Saccharomyces cerevisiae) two-hybrid screen that used the B. napus SRK₉₁₀ kinase domain as bait (Bower et al., 1996); (2) when purified from pistils or insect cells, the Brassica oleracea SRK₃ variant was found to exhibit constitutive autophosphorylation activity in vitro, and this activity was inhibited by Escherichia coli-expressed THL1 proteins and was restored by addition of pollen coat proteins containing self but not of pollen coat proteins containing nonself SCR (Cabrillac et al., 2001); (3) the catalytic activity of THL1 was required for its inhibition of SRK₃ autophosphorylation activity in vitro (Cabrillac et al., 2001); and (4) antisense suppression of THL1/THL2 gene expression in the stigmas of a self-compatible B. napus strain reportedly produced a low-level constitutive incompatibility (Haffani et al., 2004), as might be expected if the THL1/THL2 proteins prevent the spontaneous activation of SRK-mediated signaling in stigmas.These observations notwithstanding, the in planta role of thioredoxin h proteins as negative regulators of SRK activity has not been conclusively demonstrated. To date, this proposed function has only been evaluated in a self-compatible strain of B. napus (Haffani et al., 2004). Consequently, it is not known if the proposed inhibitory effect of these thioredoxins on SRK catalytic activity is manifested in self-incompatible stigmas and if it applies to all SRK variants, be they derived from Brassica spp. or other self-incompatible species of the Brassicaceae such as Arabidopsis lyrata.In this study, we tested the in planta role of thioredoxin h proteins in the regulation of SI signaling using a transgenic self-incompatible Arabidopsis thaliana model that we generated by transforming A. thaliana with the SRKb-SCRb gene pair isolated from the Sb haplotype of self-incompatible A. lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). We had previously shown that the stigmas of A. thaliana SRKb-SCRb transformants can exhibit an SI response that is as robust as the SI response observed in naturally self-incompatible A. lyrata, demonstrating that A. thaliana, which harbors nonfunctional S-locus haplotypes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Shimizu et al., 2008; Boggs et al., 2009c), has nevertheless retained all other factors required for SI. In view of the availability in A. thaliana of a highly efficient transformation method and numerous genetic resources, the A. thaliana SRK-SCR transgenic model has enabled the use of experimental approaches that are difficult or impossible to implement in Brassica species and has thus proven to be an invaluable platform for in planta analysis of SRK and SI signaling (Liu et al., 2007; Boggs et al., 2009a, 2009b; Tantikanjana et al., 2009; Tantikanjana and Nasrallah, 2012).We therefore used this transgenic A. thaliana self-incompatible model to determine if abolishing the proposed SRK-thioredoxin h interaction or eliminating expression of the major thioredoxin h proteins expressed in stigmas would affect the outcome of self- or cross pollination. To this end, we expressed a mutant form of SRKb that lacked the Cys residue previously shown to be required for the interaction of SRK with THLs (Mazzurco et al., 2001), and we analyzed plants carrying knockout insertional mutations in thioredoxin h genes. Our results are inconsistent with the proposed role of thioredoxin h proteins as negative regulators of SRK catalytic activity and SI signaling.     

Priorities for selection and representation in natural tasks

Selecting and remembering visual information is an active and competitive process. In natural environments, representations are tightly coupled to task. Objects that are task-relevant are remembered better due to a combination of increased selection for fixation and strategic control of encoding and/or retaining viewed information. However, it is not understood how physically manipulating objects when performing a natural task influences priorities for selection and memory. In this study, we compare priorities for selection and memory when actively engaged in a natural task with first-person observation of the same object manipulations. Results suggest that active manipulation of a task-relevant object results in a specific prioritization for object position information compared with other properties and compared with action observation of the same manipulations. Experiment 2 confirms that this spatial prioritization is likely to arise from manipulation rather than differences in spatial representation in real environments and the movies used for action observation. Thus, our findings imply that physical manipulation of task relevant objects results in a specific prioritization of spatial information about task-relevant objects, possibly coupled with strategic de-prioritization of colour memory for irrelevant objects.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch

Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene.     

The Formation of Glucose 1,6-Diphosphate from Fructose 1,6-Diphosphate by Yeast Phosphoglucomutase

It was found that fructose 1,6-diphosphate, the main intermediate of glycolysis, was able to act as a coenzyme of yeast phosphoglucomutase reaction. The mechanism of the coenzymatic activity of fructose 1,6-diphosphate was studied. It was indicated in the fructose 1,6-diphosphate dependent reaction that glucose 1,6-diphosphate was formed by the phosphate-transfer of fructose 1,6-diphosphate to glucose 1-phosphate in the first step, and in the second step the conversion of glucose 1-phosphate to glucose 6-phosphate, the original mutase reaction, occurred in the presence of glucose 1,6-diphosphate. The kinetic constants in the reaction of the first step were determined from the time courses of the fructose 1,6-diphosphate dependent reaction.     

Carbon cycling and sequestration in a Japanese red pine (Pinus densiflora) forest on lava flow of Mt. Fuji

Biometric-based carbon flux measurements were conducted in a pine forest on lava flow of Mt. Fuji, Japan, in order to estimate carbon cycling and sequestration. The forest consists mainly of Japanese red pine (Pinus densiflora) in a canopy layer and Japanese holly (Ilex pedunculosa) in a subtree layer. The lava remains exposed on the ground surface, and the soil on the lava flow is still immature with no mineral soil layer. The results showed that the net primary production (NPP) of the forest was 7.3 ± 0.7 t C ha^?1 year^?1, of which 1.4 ± 0.4 t C ha^?1 year^?1 was partitioned to biomass increment, 3.2 ± 0.5 t C ha^?1 year^?1 to above-ground fine litter production, 1.9 t C ha^?1 year^?1 to fine root production, and 0.8 ± 0.2 t C ha^?1 year^?1 to coarse woody debris. The total amount of annual soil surface CO₂ efflux was estimated as 6.1 ± 2.9 t C ha^?1 year^?1, using a closed chamber method. The estimated decomposition rate of soil organic matter, which subtracted annual root respiration from soil respiration, was 4.2 ± 3.1 t C ha^?1 year^?1. Biometric-based net ecosystem production (NEP) in the pine forest was estimated at 2.9 ± 3.2 t C ha^?1 year^?1, with high uncertainty due mainly to the model estimation error of annual soil respiration and root respiration. The sequestered carbon being allocated in roughly equal amounts to living biomass (1.4 t C ha^?1 year^?1) and the non-living C pool (1.5 t C ha^?1 year^?1). Our estimate of biometric-based NEP was 25 % lower than the eddy covariance-based NEP in this pine forest, due partly to the underestimation of NPP and difficulty of estimation of soil and root respiration in the pine forest on lava flows that have large heterogeneity of soil depth. However, our results indicate that the mature pine forest acted as a significant carbon sink even when established on lava flow with low nutrient content in immature soils, and that sequestration strength, both in biomass and in soil organic matter, is large.     

Purification and Chemical Analysis of Microbial Cell Flocculant Produced by Aspergillus sojae AJ7002

A bio-flocculant was isolated from the culture broth of Asp. sojae AJ 7002. It was partially purified by acetone or ethanol precipitation, by ion-exchange and gel chromatography, and by dialysis. The isolated polymer possessed chemical characteristics of a poly-hexosamine and a protein. Glucosamine and galactosamine were not acetylated. The flocculant contained 2-ketogluconic acid, but sulfur or phosphorus was not detected. This flocculant was thermo-stable and its activity varied with pH. It was suggested that the hexosamine moiety in the polymer played a major role in bio-flocculation, assisted by protein portion in enlargement of the molecular weight of the flocculant, and by 2-ketogluconic acid in endowing it with amphoteric character.