Yoshiyuki Tanaka

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 Sweets 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.

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.

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.

Multiple loss-of-function putative aminotransferase alleles contribute to low pungency and capsinoid biosynthesis in Capsicum chinense

Capsicum chinense is a domesticated hot pepper species in the Capsicum genus that originated in the Amazon and is consumed in USA, the Caribbean and South America. Although a characteristic of this species is high pungency, some non-pungent or low-pungent strains, called “Aji Dulce” (sweet pepper in Spanish), exist in the Caribbean region. In the present study, low-pungent C. chinense accessions were analyzed in order to elucidate the genetic mechanisms responsible for low pungency. All low-pungent C. chinense accessions in this study carried non-functional alleles of putative aminotransferase (pAMT), which catalyzes the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. These low-pungent accessions produced capsinoids, low-pungent capsaicinoid analogs. The pamt mutation in each strain was characterized using allele-specific markers, and one novel pamt allele (pamt7) was identified. The pamt7 had a new hAT family transposon insertion in the second exon region, which caused the loss of pAMT expression. pamt7 is apparently an ancestral allele for pamt6 because the 7-bp insertion in pamt6 can be regarded as a footprint of the transposon. A phylogenetic analysis of pamt alleles was performed to examine their relationships. Combined with previously reported pamt alleles, the Tcc family transposon insertion and its excision were involved in the generation of various pamt alleles in C. chinense. A phylogenetic analysis of pamt alleles showed that at least five occurred within C. chinense after speciation of the Capsicum genus. In conclusion, the results of the present study identified pamt as the main and most frequent gene controlling low pungency in C. chinense. Allelic variations in loss-of function pamt and their wide distribution demonstrated the potential of C. chinense bioresources for genetic improvements to pungency and metabolic profiles in hot pepper breeding programs.

Complete mitochondrial genome sequence of black mustard (Brassica nigra; BB) and comparison with Brassica oleracea (CC) and Brassica carinata (BBCC).

Crop species of Brassica (Brassicaceae) consist of three monogenomic species and three amphidiploid species resulting from interspecific hybridizations among them. Until now, mitochondrial genome sequences were available for only five of these species. We sequenced the mitochondrial genome of the sixth species, Brassica nigra (nuclear genome constitution BB), and compared it with those of Brassica oleracea (CC) and Brassica carinata (BBCC). The genome was assembled into a 232 145 bp circular sequence that is slightly larger than that of B. oleracea (219 952 bp). The genome of B. nigra contained 33 protein-coding genes, 3 rRNA genes, and 17 tRNA genes. The cox2-2 gene present in B. oleracea was absent in B. nigra. Although the nucleotide sequences of 52 genes were identical between B. nigra and B. carinata, the second exon of rps3 showed differences including an insertion/deletion (indel) and nucleotide substitutions. A PCR test to detect the indel revealed intraspecific variation in rps3, and in one line of B. nigra it amplified a DNA fragment of the size expected for B. carinata. In addition, the B. carinata lines tested here produced DNA fragments of the size expected for B. nigra. The results indicate that at least two mitotypes of B. nigra were present in the maternal parents of B. carinata.

High Pressure Phases in the Ge-P System

ゲルマニウムとリンの反応系について Bridgman 型ピストンシリンダーおよび正六面体型アンピル装置を用いて温度 600～1500℃, 圧力 10～50 kb の高温高圧下で反応性が検討された。X 線解析の結果, 実験条件の範囲内で約 8 種類の結晶性の高圧相がえられることが明らかになった。これらの高圧相の中には乱れセン亜鉛鉱型構造で格子定数 α= 5. 581Å のGePと, ケイ素 - リソ系での SiP に対応するセン亜鉛鉱型構造で格子定数 = 5.463Å の GeP が含まれている。黄鉄鉱型構造の SiP2に対応する高圧相は認められないが, 生成過程において対応関係にある立方晶系で格子定数 α = 5.619Å のGeP2と考えられる相が確認された。X線回折により高温高圧におけるゲルマニウム - リン系の主要な反応過程が推定され, この反応過程に対する温度, 圧力, 反応時間および試料中のゲルマニウムとリンの混合比の効果が調べられ, ケイ素 - リン系との異同が比較検討された。ケイ素 - リン系とゲルマニウムニ - リン系では1300℃, 40kb以下の領域では完全に対応関係にある高圧相は存在しないが, 1400℃, 40kb以上の高温高圧下において対応関係にあるセン亜鉛鉱型構造の SiP,GePを生じる。