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Takaya Moriguchi and Roger J. Romani

A strong association is implicit between mitochondrial function and the energy demands of cells responding to stress. Yet, the dynamics of this organelle-cellular dependency have been difficult to resolve. This study examines a new diagnostic parameter namely, mitochondrial maintenance and self-restoration as exhibited by the course of respiratory functions (states 3 and 4 respiratory rates, respiratory control) of mitochoudria extracted during and after exposure of intact `Hass' avocado (Persea americana) fruit to different stress atmospheres: anoxia (100% N2) or high (25% and 75%) CO2 for varying durations. Comparisons are made with direct exposure of the mitochondria themselves to similar atmospheres. In general, exposure of the fruit to CO2 rich atmospheres enhanced the capacity of their mitochondria to restore energy-linked functions whereas anoxia caused irreparable damage. The physiological (climacteric) state of the fruit also affected the stress capacity of the mitochondria contained therein, anaerobiosis being more harmful to mitochondria in riper fruit. In contrast to their effects in vivo, in vitro anoxia appeared to sustain mitochondrial energy-linked functions, whereas high CO2 was clearly harmful. These and other observations are discussed in the context of mitochondrial self-restoration or homeostasis and its relevance to postharvest stress-atmosphere storage for purposes such as pathogen suppression or insect control.

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Takaya Moriguchi, Tetsuro Sanada, and Shohei Yamaki

Sugar levels and composition were determined in developing `Hakuto' peach (Prunus persica Batsch var. vulgaris Maxim.) fruit. Glucose and fructose in nearly equal amounts were the predominant sugars detected during the early stage of development. Sucrose subsequently began to accumulate and was the predominant sugar in mature fruit. Sorbitol remained at a low level throughout development. The large increase in the amount of sucrose was accompanied by a rapid increase in sucrose synthase (EC activity. Sucrose phosphate synthase (EC was also detected in flesh extracts, but the activities were low throughout development. Acid invertase (EC activity was highest in young fruit and declined with development. Activity, however, increased again at a later stage of development. Peach fruit contained appreciable sorbitol oxidase activity, while other sorbitol-related enzymes were barely detectable, suggesting that transported sorbitol was predominantly converted to glucose. These results suggest that the supply of glucose and fructose depends on acid invertase and sorbitol oxidase, and that accumulation of sucrose depends on-sucrose synthase.

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Sunao Hisada, Tomoya Akihama, Tomoko Endo, Takaya Moriguchi, and Mitsuo Omura

A cDNA library was constructed from satsuma mandarin (Citrus unshiu Marc.) fruit tissues during the rapid cell enlargement phase. A total of 950 individual cDNA clones was partially sequenced and compared with GenBank databases for characterizing the gene repertoire expressed during this developmental phase. Among these, 426 cDNA clones (44.8%) showed sequence identity with previously characterized genes with optimized (OPT) scores of ≥200, while 524 clones (55.2%) resulted in low OPT scores (<200) and did not show any significant sequence identity with previously published genes. Based on nucleotide sequence, most clones with OPT scores of ≥200 were assumed to be transcription-, translation-, cell-wall-metabolism-, and stress-response-related genes. Other clones showed homology with published sequences related to housekeeping and stress-response-related genes, including metallothionein-like proteins, late-embryogenesis-abundant (LEA) proteins, and heat-shock proteins. These results suggested that Citrus fruit during rapid cell enlargement were metabolically active and expanding in response to biotic and abiotic stress. For clones with low nucleotide sequence homology, the recurrence was evaluated by aligning nucleotide sequences of each clone and generating contig maps. Expressed sequence tags (ESTs) of 162 clones with OPT scores <200 have not been reported for any other organism. Collectively, randomly sequenced cDNA clones described in this study provided information on types of genes expressed during the rapid cell enlargement phase in Citrus fruit. These genes should be used as candidates for Citrus genome mapping projects.

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Takaya Moriguchi, Kazuyuki Abe, Tetsuro Sanada, and Shohei Yamaki

Soluble sugar content and activities of the sucrose-metabolizing enzymes sucrose synthase (SS) (EC, sucrose-phosphate synthase (SPS) (EC, and acid invertase (EC were analyzed in the pericarp of fruit from pear cultivars that differed in their potential to accumulate sucrose to identify key enzymes involved in sucrose accumulation in Asian pears. The Japanese pear `Chojuro' [Pyrus pyrifolia (Burro. f.) Nakai] was characterized as a high-sucrose-accumulating type based on the analysis of mature fruit, while the Chinese pear `Yali' (P. bretschneideri Rehd.) was a low-sucrose-accumulating type throughout all developmental stages. The activity of SS and SPS in `Chojuro' increased during maturation concomitant with sucrose accumulation, whereas the activity of these enzymes in `Yali' did not increase during maturation. The activity of SS and SPS in the former were seven and four times, respectively, higher than those in the latter at the mature stage. Further, among 23 pear cultivars, SS activity was closely correlated with sucrose content, while SPS activity was weakly correlated. Soluble acid invertase activity in `Chojuro' and `Yali' decreased with fruit maturation, but the relationships between soluble invertase activity and sucrose content were not significant. The results indicate that SS and SPS are important determinants of sucrose accumulation in Asian pear fruit and that a decrease of soluble acid invertase activity is not absolutely required for sucrose accumulation.

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Takaya Moriguchi, Tetsushi Hidaka, Mitsuo Omura, Toshiaki Motomura, and Tomoya Akihama

Interspecific hybridizations by electrofusion of embryogenic callus cells from `Seminole' tangelo (Citrus reticulata Blanco × C. paradisi Macf.), `Hazzara (Abohar)', or `Ohta' ponkan (C. reticulata Blanco) and leaf cells from `Lisbon' lemon [C. limon (L.) Burm. f.] or rough lemon (C. jambhili Lush.), respectively, were performed. Electrofusion of `Seminole' tangelo and `Lisbon' lemon, `Hazzara (Abohar)' and rough lemon, and `Ohta' ponkan and rough lemon resulted in 33, 43, and 36 plants, respectively. Seven to 10 plants in each combination were selected randomly and used to investigate nuclear and cytoplasmic genomes. Regenerated plants derived from electrofusion of `Seminole' tangelo and `Lisbon' lemon, and `Hazzara (Abohar)' and rough lemon possessed the same restriction fragment pattern for nuclear rDNA as that of the mesophyll parents and the same mitochondrial DNA (mtDNA) restriction pattern as that of the respective suspension parents, indicating that they were cybrids. In contrast, all the plants resulting from a combination between `Ohta' ponkan and rough lemon were confirmed by nuclear rDNA and mtDNA analysis to be somatic hybrids. The analysis of chromosome number supported the results of Southern blot hybridization. The results suggest that specific cell lines, parental combinations, or both can increase the efficiency of inducing cybrids in Citrus by electrofusion.

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Kuniaki Sugawara, Atsushi Oowada, Takaya Moriguchi, and Mitsuo Omura

Random amplified polymorphic DNA (RAPD) markers were used to detect chimerism of citrus cultivars. Polymerase chain reaction conditions suitable for discriminating citrus chimeras were determined. Primers that produced consistent and repeatable bands that differed between the parental cultivars were chosen to create discriminating band patterns. Our results show that selected 12-mer primers can be useful for identifying the four citrus chimeras tested using RAPD technology.

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Kuniaki Sugawara, Takumi Wakizuka, Atsushi Oowada, Takaya Moriguchi, and Mitsuo Omura

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the histogenic structure of leaf and fruit tissues in four graft chimeras, two intentional chimeras that were produced in combination with `Hamlin' orange [Citrus sinensis (L.) Osbeck] and `Satsuma' mandarin (C. unshiu Marc.), and two naturally occurring periclinal chimera cultivars, Kobayashi Mikan (a graft chimera of C. unshiu and C. natsudaidai Hayata), and Kinkoji Unshu (a graft chimera of C. unshiu and C. obovoidea hort. ex Takahashi). RAPD profiles of the lamina epidermis and the mesophyll cells of specific individuals indicated that the four graft chimeras were interspecific monekto chimeras, whose outermost layer (histogenic layer L-1) of the shoot apical meristem consisted of a species that was different from that in the inner layers (histogenic layers L-2 and L-3). Moreover, juice vesicles, which develop from the inside cells of the pericarp and become the main edible parts of Citrus fruit, were a mixture of the cells from both parental source cultivars. Therefore, the vesicles were at least composed of L-1 and subepidermal inner L-2 cells. This determination of interspecific chimeral construction (which was made possible by molecular techniques) is a valuable finding, in terms of improving Citrus through intentional use of periclinal chimerism.

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Tomomi Tsuda, Masami Yamaguchi, Chikako Honda, and Takaya Moriguchi

We used RNA blot analysis to examine the expression of six genes of the anthocyanin biosynthesis pathway in the flowers and fruit skins at three developmental stages of white and red peaches and a deep-red nectarine [Prunus persica (L.) Batch]. In the red peach `Akatsuki' and the deep-red nectarine `Flavortop', expression levels of anthocyanin biosynthesis genes were related to anthocyanin accumulation in the fruit skin; expression of all six genes dramatically increased at Stage III of fruit development, and anthocyanin concentration also increased at this stage. In the white peach `Mochizuki', however, expression of the chalcone synthase gene (CHS) and the dihydroflavonol 4-reductase gene (DFR) was undetectable in Stage III, although the chalcone isomerase gene (CHI), the flavanone 3-hydroxylase gene (F3H), the anthocyanidin synthase gene (ANS), and the UDP-glucose-flavonoid 3-O-glucosyltransferase gene (UFGT) were expressed. We occasionally found red pigment in the skin of `Mochizuki' peach. In these red skin areas, both CHS and DFR were clearly expressed in Stage III. These results suggest that CHS and DFR are the key regulatory genes in the process of anthocyanin biosynthesis in mature red peach and nectarine.

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Daisuke Sakamoto, Yuri Nakamura, Hiroyoshi Sugiura, Toshihiko Sugiura, Toshikazu Asakura, Mineyuki Yokoyama, and Takaya Moriguchi

The effect of 9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid (KODA) on endodormancy breaking was studied in flower buds of japanese pear (Pyrus pyrifolia Nakai). The optimal concentration of KODA (100 μM) for endodormancy breaking was established over a 2-year period during the endodormancy stage of 2006 and 2007 in japanese pear cultivars Kosui, Natsushizuku, and Hosui. The effect of KODA on endodormancy breaking in flower buds was similar between ‘Natsushizuku’ and ‘Kosui’ but somewhat lower in ‘Hosui’. These results indicate that KODA can be an effective agent for promoting endodormancy breaking of flower buds in ‘Natsushizuku’ and ‘Kosui’. Although not as effective as hydrogen cyanamide, KODA may be preferable at late endodormancy stages because it has no apparent phytotoxicity.

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Hiroyasu Kitashiba*, Yu-Jin Hao, Chikako Honda, Masayuki Kita, and Takaya Moriguchi

Polyamines [putrescine (put), spermidine (spd), and spermine (spm)] are aliphatic amines that are implicated in the regulation of many basic physiological processes such as cell growth, proliferation and stress responses in organisms including plants (Walden et al. 1997). Put is metabolized to spd and spm through the successive enzymatic reactions of spd synthase (SPDS) and spm synthase (SPMS) with the use of decarboxylated S-adenosylmethionine (dcSAM) as an aminopropyl donor, which is generated by SAM decarboxylase (SAMDC). So far, two MdSAMDC (MdSAMDC1 and MdSAMDC2) homologous to SAMDC and two MdACL5 (MdACL5-1 and MdACL5-2) homologous to ACL5 encoding SPMS in Arabidopsis (Hanzawa et al. 2000) were isolated from `Orin' apple. To investigate the function of these genes, complementation analyses were carried out using yeast mutants. Each of the MdSAMDCs consists of three ORFs; tiny- and small-ORFs in the 5' regions, and main ORF like other plant SAMDC genes. Both constructs for MdSAMDC containing all ORFs (SAM-DCall) or containing only main ORF (SAMDCorf) were capable of recovering the growth of yeast SAMDC-deficient mutants (delta spe2) without supplement of spd, although the SAMDCall constructs always showed the lower growth speed than the SAMDCorf constructs. On the other hand, yeast SPMS-deficient mutant (delta spe4) introduced by MdACL5 cDNA produced significantly higher amount of spm than the delta spe4 with control vector by HPLC. Collectively, these results suggest that both MdSAMDCs are functional as a SAMDC and the tiny- and small-ORFs are negative-regulatory factor for the translation efficiency of SAMDC, and also that MdACL5 encodes a functional SPMS like as ACL5 in Arabidopsis. The first and second authors contributed equally to this work.