Phosphorus starvation induces post-transcriptional CHS gene silencing in Petunia corolla.

KEY MESSAGE: The corolla of Petunia 'Magic Samba' exhibits unstable anthocyanin expression depending on its phosphorus content. Phosphorus deficiency enhanced post-transcriptional gene silencing of chalcone synthase - A in the corolla. Petunia (Petunia hybrida) 'Magic Samba' has unstable red-white bicolored corollas that respond to nutrient deficiency. We grew this cultivar hydroponically using solutions that lacked one or several nutrients to identify the specific nutrient related to anthocyanin expression in corolla. The white area of the corolla widened under phosphorus (P)-deficient conditions. When the P content of the corolla grown under P-deficient conditions dropped to <2,000 ppm, completely white corollas continued to develop in >40 corollas until the plants died. Other elemental deficiencies had no clear effects on anthocyanin suppression in the corolla. After phosphate was resupplied to the P-deficient plants, anthocyanin was restored in the corollas. The expression of chalcone synthase-A (CHS-A) was suppressed in the white area that widened under P-suppressed conditions, whereas the expression of several other genes related to anthocyanin biosynthesis was enhanced more in the white area than in the red area. Reddish leaves and sepals developed under the P-deficient condition, which is a typical P-deficiency symptom. Two genes related to anthocyanin biosynthesis were enhanced in the reddish organs. Small interfering RNA analysis of CHS-A showed that the suppression resulted from post-transcriptional gene silencing (PTGS). Thus, it was hypothesized that the enhancement of anthocyanin biosynthetic gene expression due to P-deficiency triggered PTGS of CHS-A, which resulted in white corolla development.

Defense response of a pepper cultivar cv. Sy-2 is induced at temperatures below 24°C.

Temperature is one of the most important environmental factors that influence plant growth and development. Recent studies imply that plants show various responses to non-extreme ambient temperatures. Previously, we have found that a pepper cultivar cv. Sy-2 (Capsicum chinense) shows developmental defects at temperatures below 24°C. In this study, to gain new insights into the temperature sensitivity of cv. Sy-2, temperature-sensitive genes were screened using microarray techniques. At restrictive temperature of 20°C, almost one-fourth of the 411 up-regulated genes were defense related or predicted to be defense related. Further expression analyses of several defense-related genes showed that defense-related genes in cv. Sy-2 were constitutively expressed at temperatures below 24°C. Moreover, accumulation of high level of salicylic acid (SA) in cv. Sy-2 grown at 20°C suggests that the defense response is activated in the absence of pathogens. To confirm that the defense response is induced in cv. Sy-2 below 24°C, we evaluated the resistance to biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria and necrotrophic fungal pathogen Cercospora capsici. Cv. Sy-2 showed enhanced resistance to X. campestris pv. vesicatoria, but not to C. capsici.

Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis).

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Capsinoids, non-pungent capsaicin analogs, reduce body fat accumulation without weight rebound unlike dietary restriction in mice.

Enhancing energy expenditure and reducing energy intake are both crucial for weight control. Capsinoids, which are non-pungent capsaicin analogs, are known to suppress body fat accumulation and reduce body weight by enhancing energy expenditure in both mice and humans. However, it is poorly understood whether the suppression of body fat accumulation by capsinoids has an advantage over dietary restriction. This study shows that the oxygen consumption was increased in mice administered with capsinoids but not in dietary-restricted mice, although there was a similar suppression of body fat accumulation in both groups. The weight rebound was more notable in the dietary-restricted mice than in the mice administered with capsinoids. These results indicate that suppressing body fat accumulation by capsinoids was more beneficial than a restricted diet for maintaining body weight.

Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 Sweet.

Capsaicinoids are responsible for the spicy flavor of pungent peppers (Capsicum). The cultivar CH-19 Sweet is a non-pungent pepper mutant derived from a pungent pepper strain, Capsicum annuum CH-19. CH-19 Sweet biosynthesizes capsaicinoid analogs, capsinoids. We determined the genetic and metabolic mechanisms of capsinoid biosynthesis in this cultivar. We analyzed the putative aminotransferase (pAMT) that is thought to catalyze the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. Enzyme assays revealed that pAMT activity catalyzing vanillylamine formation was completely lost in CH-19 Sweet placenta tissue. RT-PCR analysis showed normal mRNA transcription of the pAMT gene; however, SNP analysis of the cDNA sequence showed a T nucleotide insertion at 1291 bp in the pAMT gene of CH-19 Sweet. This insertion formed a new stop codon, TGA, that prevented normal translation of the gene, and the pAMT protein did not accumulate in CH-19 Sweet as determined using Western blot analysis. We developed a dCAPS marker based on the T insertion in the pAMT gene of CH-19 Sweet, and showed that the pAMT genotype co-segregated with the capsinoid or capsaicinoid fruit phenotype in the F(2) population. The T insertion was not found in other pungent and non-pungent Capsicum lines, suggesting that it is specific to CH-19 Sweet. CH-19 Sweet's pAMT gene mutation is an example of a nonsense mutation in a single gene that alters a secondary metabolite biosynthetic pathway, resulting in the biosynthesis of analogs. The dCAPS marker will be useful in selecting lines with capsinoid-containing fruits in pepper-breeding programs.

Dramatic changes in leaf development of the native Capsicum chinense from the Seychelles at temperatures below 24 degrees C.

When a pepper cultivar (Capsicum chinense cv. Seychelles-2, Sy-2) native to the Seychelles was grown in Japan, all seedlings showed seasonal developmental abnormalities such as development of abnormally shaped leaves. Other pepper cultivars grew well in all seasons while the growth of cv. Sy-2 was stunted. In this study, we first examined the effects of various changes in temperature and photoperiod on the cv. Sy-2 phenotype. The results showed that temperatures lower than 24 degrees C led to the formation of abnormal leaves. Second, morphological and anatomical analyses of cotyledons and true leaves developed at 28 and 20 degrees C were conducted. The narrower and thicker cotyledons developed at 20 degrees C had fewer palisade cells in the leaf-length direction, and more cells in the leaf-thickness direction. True leaves developed at 20 degrees C were irregularly shaped, thicker and had smaller leaf area. In addition, true leaves developed at 20 degrees C had fewer palisade cells in the leaf-length and leaf-width directions and had more cells in the leaf-thickness direction. Furthermore, abnormal periclinal cell divisions in the mesophyll and/or epidermal cell layers were observed during leaf blade development at 20 degrees C. These results suggest that the observed changes in cell proliferation and abnormal periclinal cell divisions were related, at least in part, to abnormal leaf development of cv. Sy-2 at temperatures below 24 degrees C.

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum.

BACKGROUND AND AIMS: Helichrysum bracteatum is called an 'eternal flower' and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. METHODS: DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. KEY RESULTS: Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. CONCLUSIONS: Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.

Isolation of coniferyl esters from Capsicum baccatum L., and their enzymatic preparation and agonist activity for TRPV1.

Coniferyl esters--capsiconiate and dihydrocapsiconiate--were isolated from the fruits of the pepper, Capsicum baccatum L. var. praetermissum. Their structures were determined by spectroscopic methods to be coniferyl (E)-8-methyl-6-nonenoate (capsiconiate) and coniferyl 8-methylnonanoate (dihydrocapsiconiate). This finding was further confirmed by the lipase-catalyzed condensation of coniferyl alcohol with its corresponding fatty acid derivative. The agonist activity of the esters for transient receptor potential vanilloid 1 (TRPV1) was evaluated by conducting an analysis of the intracellular calcium concentrations in TRPV1-expressing HEK293 cells. The EC50 values of capsiconiate and dihydrocapsiconiate were 3.2 and 4.2 microM, respectively.

Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1.

Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca2+]i). These compounds would be expected to activate TRPV1 via different mechanism(s), depending on their properties. We synthesized several capsaicinoids and capsinoids that have variable lengths of acyl moiety. The activities of these compounds towards TRPV1 heterologously expressed in HEK293 cells were determined by measuring [Ca2+]i. When an extracellular or intracellular Ca2+ source was removed, some agonists such as capsaicin could increase [Ca2+]i. However, a highly lipophilic capsaicinoid containing C18:0 and capsinoids containing C14:0, C18:0, or C18:1 (the latter was named olvanilate) could not elicit a large increase in [Ca2+]i in the absence of an extracellular or intracellular Ca2+ source. These results suggest that highly lipophilic compounds cause only a slight Ca2+ influx, via TRPV1 in the plasma membrane, and are not able to activate TRPV1 in the endoplasmic reticulum.

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.

Potent production of capsaicinoids and capsinoids by Capsicum peppers.

The fundamental structure of capsinoids is a fatty acid ester with vanillyl alcohol, whereas in capsaicinoids, a fatty acid amide is linked to vanillylamine. To clarify the relationship between their biosynthesis in Capsicum plants, we carried out an in vivo tracer experiment using stable isotopically labeled putative precursors. Liquid chromatography-tandem mass spectrometry was used to measure the uptake of isotopes into metabolites after injection of the labeled precursors into intact fruits of a pungent cultivar, Peru, and a non-pungent cultivar, CH-19 Sweet. Labeled vanillylamine was incorporated into capsaicinoids in both cultivars. While labeled vanillyl alcohol was incorporated into capsinoids in both cultivars, the accumulation of intact capsaicinoids in Peru was suppressed by over 60% after administration of vanillyl alcohol. In Peru, labeled vanillin was converted to both vanillylamine and, in 5-fold excess, vanillyl alcohol. Moreover, labeled vanillin was converted exclusively to vanillyl alcohol in CH-19 Sweet. These data are consistent with the incorporation of labeled vanillin into capsaicinoids and capsinoids in both cultivars. We conclude that pungent cultivars are highly potent producers of vanillyl alcohol that is incorporated into capsinoids and that biosynthesis of capsinoids is catalyzed by capsaicin synthase.

Activation of transient receptor potential A1 by a non-pungent capsaicin-like compound, capsiate.

BACKGROUND AND PURPOSE: Capsiate is produced by 'CH-19 Sweet' (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels. EXPERIMENTAL APPROACH: Using Ca(2+) imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons. KEY RESULTS: We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC(50) for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid. CONCLUSIONS AND IMPLICATIONS: Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.

Capsiate, a non-pungent capsaicin analog, reduces body fat without weight rebound like swimming exercise in mice.

Enhancement of energy expenditure and reducing energy intake are crucial for weight control. Capsiate, a non-pungent capsaicin analog, is known to suppress body fat accumulation and reduce body weight by enhancing of energy expenditure in both mice and humans. However, it is poorly understood whether suppressing body fat accumulation by capsiate administration is equal to exercise or not. The aim of this study is to compare the effects of repeated administration of capsiate and exercise and to investigate the weight rebound after repeated capsiate administration and/or exercise. In the present study, we report that 2 weeks treatment of capsiate and exercise increased energy metabolism and suppressed body fat accumulation during 4 more weeks of ad libitum feeding. The body weight in capsiate and exercise groups was significantly lower than that of control group. The oxygen consumption was significanlty increased in capsiate and exercise groups than in the vehicle administered mice. In addition, the abdominal adipose tissue weight in capsiate and exercise groups was significantly lower than that of control group. These results indicate that suppressing body fat accumulation by capsiate intake is beneficial for maintaining an ideal body weight as exercise.

Newly mutated putative-aminotransferase in nonpungent pepper (Capsicum annuum) results in biosynthesis of capsinoids, capsaicinoid analogues.

Capsinoids make up a group of nonpungent capsaicinoid analogues produced in Capsicum fruits. They have bioactivities similar to those of capsaicinoids such as suppression of fat accumulation and antioxidant activity. Because of their low pungency, they are more palatable ingredients in dietary supplements than capsaicinoids. We recently reported that capsinoid biosynthesis is caused by nonsense mutation in a putative aminotransferase gene (p-AMT) in a nonpungent cultivar CH-19 Sweet. Here we report on the screening of nonpungent germplasm that revealed a nonpungent cultivar Himo, which contains high levels of capsinoids. We have shown that Himo has a recessive allele of p-amt, which contains a mutation different from that of CH-19 Sweet. Sequence analysis of p-amt in Himo revealed that a single-nucleotide substitution results in one amino acid substitution from cysteine to arginine in the pyridoxal 5-phosphate binding domain. Genetic analysis using a cleaved amplified polymorphic sequence marker confirmed that the p-AMT genotype was precisely cosegregated with capsinoid biosynthesis and nonpungency. Himo will provide a new natural source of capsinoids.

Novel loss-of-function putative aminotransferase alleles cause biosynthesis of capsinoids, nonpungent capsaicinoid analogues, in mildly pungent chili peppers (Capsicum chinense).

Capsinoids are a group of nonpungent capsaicinoid analogues produced in Capsicum fruits. They have similar bioactivities to capsaicinoids such as suppression of fat accumulation and antioxidant activity. They are more palatable ingredients in dietary supplements than capsaicinoids because of their low pungency. Previous studies on nonpungent Capsicum annuum cultivars showed that capsinoid biosynthesis is caused by loss-of-function putative aminotransferase (p-amt) alleles. This study showed that three mildly pungent cultivars of Capsicum chinense (Zavory Hot, Aji Dulce strain 2, and Belize Sweet) contain high levels of capsinoid. It was shown that these cultivars have novel p-amt alleles, which contain mutations that differ from those of C. annuum. Sequence analysis of p-amt in Belize Sweet revealed that a 5 bp insertion (TGGGC) results in a frameshift mutation. A transposable element (Tcc) was found in the p-amt of Zavory Hot and Aji Dulce strain 2. Tcc has features similar to those of the hAT transposon family. This was inserted in the fifth intron of Zavory Hot and in third intron of Aji Dulce strain 2. The p-amt alleles harboring Tcc cannot produce an active p-AMT. These mildly pungent cultivars will provide a new natural source of capsinoids.

Assessment of capsiconinoid composition, nonpungent capsaicinoid analogues, in capsicum cultivars.

Capsiconinoid is a group of nonpungent capsaicinoid analogues produced in Capsicum fruits, which we recently identified. Capsiconinoids have agonist activity for transient receptor potential vanilloid type 1 (TRPV1), which is reported to be a receptor for capsaicin. It is, therefore, important to screen cultivars containing high levels of capsiconinoid for their use as a vegetable or dietary supplement. This study describes the quantitative analysis of capsiconinoid content in fruits of 35 Capsicum cultivars: 18 cultivars of C. annuum, 7 of C. baccatum, 5 of C. chinense, 4 of C. frutescens, and 1 of C. pubescens. Using high-performance liquid chromatography (HPLC), we found that 10 cultivars contained capsiconinoids. Capsiconinoid Baccatum (CCB) (C. baccatum var. praetermissum) showed the highest capsiconinoid content (3314 microg/g DW) and Charapita (C. chinense) had the second highest content. The other 8 cultivars had much lower capsiconinoid content than these two cultivars (<300 microg/g DW). Time-course analysis during fruit development clarified that capsiconinoid content in CCB fruits increased until 30 days after flowering (DAF) and then decreased rapidly until 40 DAF.

Effects of CH-19 Sweet, a non-pungent cultivar of red pepper, on sympathetic nervous activity, body temperature, heart rate, and blood pressure in humans.

We investigated the changes in autonomic nervous activity, body temperature, blood pressure (BP), and heart rate (HR) after intake of the non-pungent pepper CH-19 Sweet and of hot red pepper in humans to elucidate the mechanisms of diet-induced thermogenesis (DIT) due to CH-19 Sweet. We found that CH-19 Sweet activates the sympathetic nervous system (SNS) and enhances thermogenesis as effectively as hot red pepper, ant that the heat loss effect due to CH-19 Sweet is weaker than that due to hot red pepper. Furthermore, we found that intake of CH-19 Sweet does not affect systolic BP or HR, while hot red pepper transiently elevates them. These results indicate that DIT due to CH-19 Sweet can be induced via the activation of SNS as well as hot red pepper, but that the changes in BP, HR, and heat loss effect are different between these peppers.

Capsinoid is biosynthesized from phenylalanine and valine in a non-pungent pepper, Capsicum annuum L. cv. CH-19 sweet.

The biosynthetic pathway of capsinoid in 'CH-19 Sweet' was investigated. [(3)H]Valine and [(14)C]phenylalanine were injected into the fruits of the intact plant. Both of radioactivities were detected in capsinoid fractions. (14)C radioactivity was observed in phenylpropanoid compounds, and in vanillin, vanillylamine, vanillyl alcohol, and vanillic acid. We confirmed that capsinoid is biosynthesized from phenylalanine and valine.

Capsiate, a nonpungent capsaicin analog, increases endurance swimming capacity of mice by stimulation of vanilloid receptors.

We investigated the effect of capsiate, a nonpungent natural capsaicin analog, on the swimming capacity of mice in an adjustable-current water pool. Male BALB/c mice orally given capsiate (10 mg/kg) were able to keep swimming longer before exhaustion than the control mice. After 30 min of swimming, the residual glycogen in the gastrocnemius muscle was higher, the serum free fatty acid concentration tended to be higher, and the serum lactic acid concentration was significantly lower in the capsiate-administered mice. The value for the respiratory exchange ratio of the capsiate group was significantly lower during both resting and treadmill running. These physiological differences were abolished by administering the vanilloid receptor antagonist, capsazepin (0.17 mmol/kg, i.p.). The mice were not averse to the capsiate solution during a 4-h two-bottle choice test. These results suggest that the oral administration of capsiate enhanced fat oxidation and spared carbohydrate utilization, and consequently increased the endurance swimming capacity of the mice via stimulation of their vanilloid receptors. Practical application of capsiate is expected.