Naoki Yamauchi

Tomatidine and lycotetraose, hydrolysis products of α‐tomatine by Fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells

Many fungal pathogens of tomato produce extracellular enzymes, collectively known as tomatinases, that detoxify the preformed antifungal steroidal glycoalkaloid α‐tomatine. Tomatinase from the vascular wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici cleaves α‐tomatine into the aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt). Although modes of action of α‐tomatine have been extensively studied, those of Td and Lt are poorly understood. Here, we show that both Td and Lt inhibit the oxidative burst and hypersensitive cell death in suspension‐cultured tomato cells. A tomatinase‐negative F. oxysporum strain inherently non‐pathogenic on tomato was able to infect tomato cuttings when either Td or Lt was present. These results suggest that tomatinase from F. oxysporum is required not only for detoxification of α‐tomatine but also for suppression of induced defense responses of host.

Peroxidase-mediated chlorophyll degradation in horticultural crops

One of the symptoms of senescence in harvested horticultural crops is the loss of greenness that comes with the degradation of chlorophyll (Chl). With senescence, peroxidase, which is involved in Chl degradation, increased greatly in stored horticultural crops. C132-hydroxychlorophyll a, an oxidized form of Chl a, is formed in vitro through Chl oxidation by peroxidase. Peroxidase mediates Chl degradation in the presence of phenolic compounds such as p-coumaric acid and apigenin, which have a hydroxyl group at the p-position. Apparently, not all phenolic compounds are able to degrade Chl in this system, and their effectiveness appears to depend on their molecular configuration. In peroxidase-mediated Chl degradation, peroxidase oxidizes the phenolic compounds with hydrogen peroxide and forms phenoxy radical; then, the phenoxy radical oxidizes Chl and its derivatives to colorless low molecular weight compounds through the formation of C132-hydroxychlorophyll a,a fluorescent Chl catabolite and a bilirubin-like compound as an intermediate. In addition to the phenoxy radical, superoxide anion, which is formed in the peroxidase-catalyzed reaction, might be involved in Chl oxidation. Moreover, Chl degradation by peroxidase seems to occur in the chloroplast and/or the vacuole. The involvement of peroxidase in Chl degradation in senescing horticultural crops is also discussed.

Chlorophyll degradation in Wase satsuma mandarin (Citrus unshiu Marc.) fruit with on-tree maturation and ethylene treatment

Abstract Changes in chlorophylls content and their derivatives were determined to elucidate the differences in chlorophyll degradation of Wase satsuma mandarin (Citrus unshiu Marc. cv. Miyagawa-Wase) fruit with on-tree maturation or ethylene treatment. Chlorophylls content in the ethylene-treated fruit decreased markedly after 2 days of storage at 20 °C. 132-hydroxychlorophyll a and pheophorbide a also decreased during storage, whereas chlorophyllide a did not decrease for the first 5 days of storage. Chlorophylls, 132-hydroxychlorophyll a, chlorophyllide a and pheophorbide a in on-tree matured fruit decreased with degreening. A small amount of pyropheophorbide a was detected in on-tree matured fruit, but the content showed almost no change during on-tree maturation. These results suggest that the acceleration of chlorophyll degradation by ethylene treatment could be due to the enhancement of chlorophyllide a formation by chlorophyllase.

Effects of sugars and abscisic acid on somatic embryogenesis from melon (Cucumis melo L.) expanded cotyledon

Effects of sugars and abscisic acid (ABA) on somatic embryogenesis from melon (Cucumis melo L.) expanded cotyledon and changes in ABA content on cultures on the various sucrose treatment media were investigated. Sucrose induced somatic embryogenesis, but mannitol did not. Two hundred millimolar sucrose treatments were optimal for somatic embryogenesis; the formation of somatic embryos was suppressed at lower or higher sucrose concentrations. The frequency of somatic embryogenesis, however, was increased by the addition of mannitol to the initial medium, even if the sucrose concentration was less than 200 mM. Somatic embryogenesis was enhanced by the addition of 0.5 μM ABA to 200 mM sucrose treatment. In the 200 mM sucrose treatment, in which somatic embryos were easily formed, endogenous levels of ABA in callus increased greatly after 2 weeks of culture. ABA content of embryogenic callus was markedly higher than that of non-embryonic callus at any given period. These results indicate that ABA formation, which was controlled by osmotic conditions induced by the sugars, could be involved in somatic embryogenesis.

In vitro chlorophyll degradation in stored broccoli (Brassica oleracea L. var. italica Plen.) florets

Abstract In vitro chlorophyll (Chl) degradation in broccoli (Brassica oleracea L. var. italica Plen.) florets was studied by HPLC analysis of the degradative products. With the degradation of Chl, chlorophyllide a (Chlide a) and phaeophorbide a (Phb a) increased gradually in the reaction mixture containing the broccoli extract. This was followed by the formation of pyrophaeophorbide a (Pyro-Phb a) after 2 h of reaction time. 132-hydroxychlorophyll a (Chl a-1) showed minimal change during the reaction. Chl a degradation and Chlide a formation decreased significantly in an extract of yellow broccoli florets during 4 days storage at 15°C. Pyro-Phb a formation also decreased gradually, but Chl a-1 formation did not decrease during storage. Thus, in an in vitro system of extracted broccoli florets, Chl a was degraded initially to Chlide a or Chl a-1. Chlide a subsequently was degraded to Pyro-Phb a through Phb a. Chl a-1 and Pyro-Phb a could be finally degraded to colourless low molecular weight compounds.

Involvement of peroxidase in chlorophyll degradation in stored broccoli (Brassica oleracea L.) and inhibition of the activity by heat treatment

Cell localization of chlorophyll (Chl)-degrading peroxidase and the effects of heat treatment on the activity were determined to elucidate the involvement of the enzyme with yellowing in stored broccoli. Subcellular fractions and intact chloroplasts were prepared by differential and Percoll gradient centrifugation, respectively. In fresh broccoli, only low levels of Chl-degrading peroxidase activity were located in the intact chloroplast, and the highest activity was present in the cytosolic fraction. The Chl content of broccoli without heat treatment decreased greatly after 4 days storage at 15 °C, whereas the content in broccoli treated at 50 °C for 2 h showed almost no change during storage. In microsomal and cytosolic fractions, the activity of C2 (Rf=0.47) isoperoxidase, which is involved in Chl degradation, increased greatly with floret yellowing. The Chl-degrading peroxidase activities in microsomal and cytosolic fractions also increased strongly after 4 days of storage. In addition, the Chl-degrading peroxidase activity as well as C2 isoperoxidase activity was suppressed by heat treatment. These results indicate that heat treatments may be effective in inhibiting senescence in part by suppressing the enhancement of Chl-degrading peroxidase activity in the microsomes and the cytosol.

Direct comparison between genomic constitution and flavonoid contents in Allium multiple alien addition lines reveals chromosomal locations of genes related to biosynthesis from dihydrokaempferol to quercetin glucosides in scaly leaf of shallot (Allium cepa L.)

The extrachromosome 5A of shallot (Allium cepa L., genomes AA) has an important role in flavonoid biosynthesis in the scaly leaf of Allium fistulosum–shallot monosomic addition lines (FF+nA). This study deals with the production and biochemical characterisation of A. fistulosum–shallot multiple alien addition lines carrying at least 5A to determine the chromosomal locations of genes for quercetin formation. The multiple alien additions were selected from the crossing between allotriploid FFA (♀) and A. fistulosum (♂). The 113 plants obtained from this cross were analysed by a chromosome 5A-specific PGI isozyme marker of shallot. Thirty plants were preliminarily selected for an alien addition carrying 5A. The chromosome numbers of the 30 plants varied from 18 to 23. The other extrachromosomes in 19 plants were completely identified by using seven other chromosome markers of shallot. High-performance liquid chromatography analyses of the 19 multiple additions were conducted to identify the flavonoid compounds produced in the scaly leaves. Direct comparisons between the chromosomal constitution and the flavonoid contents of the multiple alien additions revealed that a flavonoid 3′-hydroxylase (F3′H) gene for the synthesis of quercetin from kaempferol was located on 7A and that an anonymous gene involved in the glucosidation of quercetin was on 3A or 4A. As a result of supplemental SCAR analyses by using genomic DNAs from two complete sets of A. fistulosum–shallot monosomic additions, we have assigned F3′H to 7A and flavonol synthase to 4A.

Development of microsatellite markers in cultivated and wild species of sections Cepa and Phyllodolon in Allium

The potential of microsatellite markers for use in genetic studies has been evaluated in Allium cultivated species (Allium cepa, A. fistulosum) and its allied species (A. altaicum, A. galanthum, A. roylei, A. vavilovii). A total of 77 polymerase chain reaction (PCR) primer pairs were employed, 76 of which amplified a single product or several products in either of the species. The 29 AMS primer pairs derived from A. cepa and 46 microsatellites primer pairs from A. fistulosum revealed a lot of polymorphic amplicons between seven Allium species. Some of the microsatellite markers were effective not only for identifying an intraspecific F1 hybrid between shallot and bulb onion but also for applying to segregation analyses in its F2 population. All of the microsatellite markers can be used for interspecific taxonomic analyses among two cultivated and four wild species of sections Cepa and Phyllodolon in Allium. Generally, our data support the results obtained from recently performed analyses using molecular and morphological markers. However, the phylogeny of A. roylei, a threatened species with several favorable genes, was still ambiguous due to its different positions in each dendrogram generated from the two primer sets originated from A. cepa and A. fistulosum.

Integrating transcriptome and target metabolome variability in doubled haploids of Allium cepa for abiotic stress protection

Environmental stress conditions such as drought, heat, salinity, or pathogen infection can have a devastating impact on plant growth and yield, resulting in a need for stress-tolerant crop varieties. Crossbreeding tropical and cultivated onion species produced a hybrid F1 generation possessing genetic and metabolic parental properties that aided abiotic stress tolerance. Targeted metabolite profiling using liquid chromatography–tandem mass spectrometry integrated with transcriptional analysis of their relevant genes provided insights into the metabolic and genomic architecture of the onion doubled haploid (Allium cepa L., DHC), shallot doubled haploid (A. cepa L. Aggregatum group, DHA), and F1 hybrid. Out of a complete set of 113 targeted metabolites, 49 metabolites were found to be statistically significantly different between genotypes: 11 metabolites were characteristic for DHC, 10 for DHA, 14 for F1, and 14 metabolites were shared among the three genotypes. Several key genes and metabolites introgressed in abiotic stress response were up-regulated in DHA and F1 genotypes as compared to DHC. Principal component analysis and Volcano plot analysis revealed that metabolic traits and their relevant genes (namely, amino acid, carbohydrate, flavonoid, and phospholipid biosynthesis) were strongly linked with DHA and F1, reflecting the adaptability of DHA and F1 toward abiotic stress as compared to DHC.

Chromosomal Organization and Sequence Diversity of Genes Encoding Lachrymatory Factor Synthase in Allium cepa L.

Lachrymatory factor synthase (LFS) catalyzes the formation of lachrymatory factor, one of the most distinctive traits of bulb onion (Allium cepa L.). Therefore, we used LFS as a model for a functional gene in a huge genome, and we examined the chromosomal organization of LFS in A. cepa by multiple approaches. The first-level analysis completed the chromosomal assignment of LFS gene to chromosome 5 of A. cepa via the use of a complete set of A. fistulosum–shallot (A. cepa L. Aggregatum group) monosomic addition lines. Subsequent use of an F2 mapping population from the interspecific cross A. cepa × A. roylei confirmed the assignment of an LFS locus to this chromosome. Sequence comparison of two BAC clones bearing LFS genes, LFS amplicons from diverse germplasm, and expressed sequences from a doubled haploid line revealed variation consistent with duplicated LFS genes. Furthermore, the BAC-FISH study using the two BAC clones as a probe showed that LFS genes are localized in the proximal region of the long arm of the chromosome. These results suggested that LFS in A. cepa is transcribed from at least two loci and that they are localized on chromosome 5.