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Background/Aim: Tears secreted from the lacrimal gland are essential for preserving the ocular surface. Thus, dysfunction of the lacrimal gland in Sjögren’s syndrome (SS) can lead to dry eye, resulting in a reduced quality of life. We previously reported that blueberry ‘leaf’ water extract prevents lacrimal hyposecretion in male non-obese diabetic (NOD) mice in a SS-like model. In this study, we investigated the effect of blueberry ‘stem’ water extract (BStEx) on lacrimal hyposecretion in NOD mice. Materials and Methods: Male NOD mice were fed 1% BStEx or control (AIN-93G) for 2, 4, or 6 weeks from 4 weeks of age. Pilocarpine-induced tear secretion was measured using a phenol red-impregnated thread. The lacrimal glands were histologically evaluated by HE staining. Inflammatory cytokine levels in the lacrimal glands were measured using ELISA. Immunostaining was performed to examine aquaporin 5 (AQP5) localization. The expression levels of autophagy-related proteins, AQP5, and phosphorylated AMPK were measured using western blotting. Results: After feeding BStEx to mice for 4 or 6 weeks, tear volume was observed to have increased in the BStEx group compared with that in the control group. There were no significant differences in inflammatory cell infiltration, autophagy-related protein expression, or the localization and expression of AQP5 in the lacrimal glands between the two groups. In contrast, AMPK phosphorylation increased in the BStEx group. Conclusion: BStEx prevented lacrimal hyposecretion in the SS-like model of male NOD mice, probably by opening tight junctions via the activation of AMPK in lacrimal acinar cells.

We have previously reported that the proanthocyanidin (PAC) fraction of blueberry leaf extract (BB-PAC) inhibits the proliferation of HTLV-1-infected adult T-cell leukemia (ATL) by inducing apoptosis. In the present study, we further analyzed the structure of BB-PAC and elucidated the molecular mechanism underlying the inhibitory function of HTLV-1-infected and ATL cells. After hot water extraction with fractionation with methanol-acetone, BB-PAC was found to be concentrated in fractions 4 to 7 (Fr7). The strongest inhibition of ATL cell growth was observed with Fr7, which contained the highest BB-PAC polymerization degree of 14. The basic structure of BB-PAC is mainly B-type bonds, with A-type bonds (7.1%) and cinchonain I units as the terminal unit (6.1%). The molecular mechanism of cytotoxicity observed around Fr7 against ATL cells was the degradation of JAK1 to 3 and the dephosphorylation of STAT3/5, which occurs by proteasome-dependent proteolysis, confirming that PAC directly binds to heat shock protein 90 (HSP90). JAK degradation was caused by proteasome-dependent proteolysis, and we identified the direct binding of PAC to HSP90. In addition, the binding of cochaperone ATPase homolog 1 (AHA1) to HSP90, which is required for activation of the cofactor HSP90, was inhibited by BB-PAC treatment. Therefore, BB-PAC inhibited the formation of the HSP90/AHA1 complex and promoted the degradation of JAK protein due to HSP90 dysfunction. These results suggest that the highly polymerized PAC component from blueberry leaves has great potential as a preventive and therapeutic agent against HTLV-1-infected and ATL cells.

BACKGROUND: In blueberries, the total polyphenol contents, total anthocyanin contents, and antioxidant activities of fruits in blueberries and their relatives are becoming important targets for breeders. Recently, intersectional hybrids between Japanese wild species Shashanbo and highbush blueberry were produced using polyploid breeding for the first time. However, the polyphenol contents, antioxidant activities, and anti-cancer cell proliferation properties of the fruits of these intersectional hybrids have not been studied sufficiently. OBJECTIVE: The objectives of this study were to determine the anthocyanin contents, polyphenol contents, and antioxidant activities at each stage of fruit development in intersectional hybrids, and to examine the effects of the fruit extracts on anti-cancer cell proliferation properties using human promyelocytic leukemia (HL-60) cells. METHODS: Freeze-dried fruit samples of three intersectional hybrids and their parents at five developmental stages were used to evaluate their polyphenol contents, anthocyanin contents, and levels of two types of antioxidant activities (DPPH and ORAC). The anti-cancer cell proliferation properties of each extract were also evaluated according to the survival rate of HL-60 cells. RESULTS: The total polyphenol contents in fruits of the intersectional hybrids gradually decreased as the fruits grew, but the contents increased slightly at the mature stage. Meanwhile, the total anthocyanin contents increased strongly to the mature stage. In addition, the fruit extract of intersectional hybrid JM1 was found to be more effective for inhibiting the growth of HL-60 human leukemia cells in vitro. Total polyphenol content correlated highly with antioxidant activities and anti-cancer cell proliferation properties. CONCLUSIONS: We clarified that the intersectional hybrids had produced have abundant bioactive compounds, high levels of antioxidant activities, and anti-cancer cell proliferation properties. The results proved the effectiveness of the use of Japanese wild Vaccinium species for the strategic breeding of blueberries with high levels of bioactive compounds.

Blue light causes retinal damage that can lead to ocular diseases such as age-related macular degeneration. In this study, we determined the protective effect of blueberry stem extract (BStEx) and active components on blue light-emitting diode (LED) light-induced retinal photoreceptor cell damage in vitro. Photoreceptor cells cultured in the presence of BStEx or components were exposed to blue light to induce cell damage. BStEx, fractions of BStEx containing proanthocyanidins, chlorogenic acid, catechin, and epicatechin prevented the cell damage and/or inhibited the generation of reactive oxygen species (ROS). Furthermore, BStEx reduced apoptosis and cell death, and inhibited the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase leading to cellular apoptosis induced by blue light exposure. These findings suggest that BStEx and components exert a protective effect against blue light-induced photoreceptor cell damage through the inhibition of MAPK phosphorylation and ROS production.

Background/Aim: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren’s syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. Materials and Methods: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1β and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. Results: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. Conclusion: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

With the current worldwide pandemic of COVID-19, there is an urgent need to develop effective treatment and prevention methods against SARS-CoV-2 infection. We have previously reported that the proanthocyanidin (PAC) fraction in blueberry (BB) leaves has strong antiviral activity against hepatitis C virus (HCV) and human T-lymphocytic leukemia virus type 1 (HTLV-1). In this study, we used Kunisato 35 Gou (K35) derived from the rabbit eye blueberry (Vaccinium virgatum Aiton), which has a high PAC content in the leaves and stems. The mean of polymerization (mDP) of PAC in K35 was the highest of 7.88 in Fraction 8 (Fr8) from the stems and 12.28 of Fraction 7 (Fr7) in the leaves. The composition of BB-PAC in K35 is that most are B-type bonds with a small number of A-type bonds and cinchonain I as extension units. A strong antiviral effect was observed in Fr7, with a high polymerized PAC content in both the leaves and stems. Furthermore, when we examined the difference in the action of BB-PAC before and after SARS-CoV-2 infection, we found a stronger inhibitory effect in the pre-infection period. Moreover, BB-PAC Fr7 inhibited the activity of angiotensin II converting enzyme (ACE2), although no effect was observed in a neutralization test of pseudotyped SARS-CoV-2. The viral chymotrypsin-like cysteine protease (3CLpro) of SARS-CoV-2 was also inhibited by BB-PAC Fr7 in leaves and stems. These results indicate that BB-PAC has at least two different inhibitory effects, and that it is effective in suppressing SARS-CoV-2 infection regardless of the time of infection.

We previously reported that rabbit-eye blueberry (

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Prevention of postprandial hypertriglyceridemia is an important consideration for reducing the risk of developing cardiovascular disease. While blueberry fruits have been reported to ameliorate lipid metabolism in humans, there are only few research reports on the effects of blueberry leaves (BL). Here, we investigated the efficacy of BL on postprandial hyperlipidemia in subjects with high fasting triacylglycerol (TG) concentrations. Randomized, double-blind, cross-over design study was conducted. The subjects consumed a BL containing beverage or a placebo beverage before a fat-enriched test meal. Blood samples were collected prior to and 1, 2, 3, 4, and 5 hours after consuming the test beverage. The postprandial serum TG and remnant-like particle cholesterol (RLP-C) concentrations were significantly lower in the BL beverage compared with those in the placebo beverage. Additionally, BL was more effective in subjects with high fasting ghrelin with gastric emptying function. In current study, fasting ghrelin correlated with the increase in postprandial serum TG, suggesting that BL ameliorates hypertriglyceridemia through delayed gastric emptying. In conclusion, this pilot study suggests that BL may be useful as an early dietary therapy for treating postprandial hyperlipidemia.

Blueberry leaf is currently a popular dietary supplement. Effects of dietary blueberry leaf and its active components on body fat accumulation were examined. C57BL/6J mice were fed high-fat, high-sucrose diet with or without 3% blueberry leaf extract (BLEx) or 3% concentrated-polyphenolic BLEx (CP BLEx) for 8 weeks. Compared to mice fed a high-fat, high-sucrose diet without blueberry leaf, BLEx and CP BLEx significantly reduced body weight and adipose tissue weight gain. Adipocytes were also smaller and and liver lipid accumulatioin was significantly inhibited in mice fed either BLEx or CP BLEx. These effects tended to be more pronounced in mice fed CP BLEx compared to in mice fed BLEx. Together, results suggest that blueberry leaf inhibits body fat accumulation typically observed in mice fed a high-fat, high-sucrose diet, and that inhibition is attributable to polyphenolic components in leaf extracts.

Three Fortunella (kumquat) species, the Meiwa kumquat (F. crassifolia Swingle), the Round kumquat [F. japonica (Thunb.) Swingle] and the Changshou kumquat (F. obovata hort. ex Tanaka), showing tetraploid, which had been induced by treating the seeds with colchicine, were examined for origin and horticultural characteristics (e. g. morphology of leaves, flowers, pollen and fruits). Additionally, these tetraploid kumquats were crossed to produce triploid kumquats. All of the tetraploids were confirmed to be derived from the nucellar embryo of each original kumquat by examining their chromosomal composition with chromomycin A3 staining. All of the tetraploid kumquats had the typical morphological characteristics of tetraploid Citrus plants, such as round and thick leaves, and large flowers and pollen grains. On the other hand, the fruits of these tetraploid plants showed desirable traits for kumquats such as thicker pericarp and higher soluble solids content. Furthermore, when these tetraploids were crossed with some diploid cultivars, triploid progenies were obtained from almost all of the cross combinations.

Metabolic profiling is often used to identify possible correlations between a compoundʼs metabolic profile and biological activity. Direct-injection electron ionization-mass spectrometry “fingerprinting” is useful for characterizing biological materials. We demonstrate the utility of direct-injection electron ionization-mass spectrometry for metabolic profiling using 100 different extracts of leaves from 20 blueberry cultivars collected at 5 time points from April to December 2008. A qualitative direct-injection electron ionization-mass spectrometry method was used to profile the major and/or minor constituents in the blueberry leaf extracts. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Correlated m/z values indicating the collection month were determined in the same analysis, and air temperature variance factors were extracted from score plots by principal component analysis. We previously reported that blueberry extracts inhibit the proliferation of adult T-cell leukemia cells. Leaves of Vaccinium virgatum collected in December of 2008 exhibited significantly greater inhibition of adult T-cell leukemia cell proliferation than other species. Highly bioactive cultivars or species were identified by direct-injection electron ionization-mass spectrometry metabolomics analysis of blueberry leaf extracts. The components extracted based on our direct-injection electron ionization-mass spectrometry analyses could be used to construct a model to predict anti-adult T-cell leukemia bioactivity. This is the first study to report a relationship between seasonal variation and bioactivity of natural products using a direct-injection electron ionization-mass spectrometry metabolomics method.

Background: Blueberry stems, a by-product of blueberry leaf tea production, are typically discarded. We evaluated the effects of hot-water extracts of rabbiteye blueberry ( Vaccinium virgatum Aiton; RB species) leaves and stems on hepatic lipid levels in rats consuming a high-sucrose diet. Methods: Male Sprague-Dawley rats were divided into groups that received a control high-sucrose diet alone or supplementation with 2.0% blueberry leaf extract or 0.5% or 2.0% blueberry stem extract. Blood and hepatic lipid levels, hepatic lipogenic enzyme activity, and hepatic quercetin metabolites were evaluated after 28 days of ad libitum consumption. Results: Supplementation with the extracts did not affect body weight gain, food intake, liver and white adipose tissue weights, or serum lipid levels. Hepatic triglyceride and total cholesterol levels were reduced in the groups that received 2.0% supplementation of either extract. Hepatic malic enzyme activity was also reduced in those groups. Quercetin and its glycosides, the major polyphenols identified in the extracts, accumulated in the liver as quercetin aglycone and quercetin metabolites. Conclusion: We demonstrated how daily consumption of blueberry leaf and stem extracts can decrease hepatic lipid levels, potentially downregulating malic enzyme activity. These effects were intensive in leaf extracts. The active compounds existed in both extracts may be quercetin and its glycosides. Therefore, blueberry stems and leaves may be an attractive candidate novel functional food. Keywords: Blueberry leaf; blueberry stem; quercetin; hepatic lipid; rat; functional food

The breeding of raspberries (Rubus idaeus L.) and blackberries (R. fruticosus Agg.) has shifted in focus toward traits associated with the sensory qualities and potential health benefits of the fruit, and Rubus wild species have attracted a great deal of attention as new sources of genetic diversity. In this study, the anthocyanin and polyphenol contents and compositions, and three types of antioxidant capacities were compared in mature fruits including raspberries, blackberries, and Japanese wild Rubus species. ‘Blackcap’ black raspberry showed the significantly highest values of total anthocyanin content and total polyphenol content among 12 Rubus species or cultivars. Cyanidin-3-xylosyl-rutinoside was contained only in black raspberries, and was characteristic anthocyanin in Rubus species. R. croceacanthus H. Lev., a Japanese wild species that is adapted to warm regions, had a deep red fruit color, and had the highest total anthocyanin and total polyphenol contents among the four Japanese wild species examined. As for the antioxidant capacities, the highest values were observed in ‘Blackcap’ among 12 Rubus species. Furthermore, a high positive correlation was shown between total anthocyanin or total polyphenol content and antioxidant capacities. In particular, black raspberries and blackberries contained high concentrations of rutin. Thus, rutin might contribute to the high antioxidant capacities in these cultivars.

Anthocyanins and polyphenols in the sweetpotato (Ipomoea batatas L.) have attracted attention due to their multiple physiological functions. Therefore, sweetpotato breeders have focused on increasing the anthocyanin and polyphenol contents. In this study, we investigated the diversity of anthocyanins, polyphenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activities in sweetpotato genotypes. We also assessed how the anthocyanin and polyphenol contents and compositions were related to DPPH radical-scavenging activities during tuber growth in several cultivars of sweetpotatoes. In six purple-fleshed cultivars, the percentage of acylated anthocyanins that were evaluated was high (92.0–98.5 %), whereas in ‘Fukumurasaki’ this percentage was particularly low (63.6 %). Moreover, the percentages of anthocyanins that were acylated in the purple-fleshed cultivars ‘Tanegashimamurasaki’, ‘Purple Sweet Lord’, and ‘Akemurasaki’ had already reached high levels in the early stage of tuber development. On the other hand, these three cultivars with high anthocyanin contents had high DPPH radical-scavenging activities, and the activities during tuber growth showed a high correlation with total anthocyanin content. Elucidating the changes in acylated anthocyanin content and in antioxidant activities during tuber growth can help promote high-quality sweetpotato cultivation and breeding.

Cryopreservation is an important tool for long-term storage of plant germplasm that is currently used for plant germplasm storage at many institutes worldwide. Recently, novel cryogenic procedures (V and D cryo-plate methods) have been developed. In this study, the most suitable conditions for preserving blueberry shoot tips derived from in vitro and current shoots using the D cryo-plate method were investigated. The D cryo-plate method has advantages such as higher regrowth after cryopreservation and a more user-friendly process compared with conventional cryogenic methods. The optimum duration of desiccation for regrowth of shoot tips from each shoot type was 1 h. To induce dehydration tolerance for the shoot tips, the effects of two cryoprotection treatments (sucrose preculture and loading solution [LS] treatment) on shoot regrowth after cryopreservation were investigated. The combined effect of both treatments significantly increased percentage regrowth (approximately 90%). No regrowth of shoot tips was attained without the two treatments. Thus, preculture and LS treatment were effective to induce dehydration tolerance for cryopreservation of blueberry shoot tips. The optimized conditions for blueberry shoot tips using the D cryo-plate technique were: preculture with 0.3 M sucrose for 1 day, LS treatment (2 M glycerol +0.4–1.0 M sucrose) for 30 min, and air dehydration for 1 h. This optimized procedure was applied to additional blueberry cultivars shoot tips derived from in vitro shoots (regrowth 46.7–100%) and current shoots (regrowth 17.2–62.7%). Furthermore, in vitro shoot tips were suitable material for the D cryo-plate method in blueberry.

The genetic analysis of five haploid plants (BX1-BX5) of ‘Banpeiyu’ pummelo (BP), [Citrus maxima (Burm.) Merr.] using 32 simple sequence repeat (SSR) markers carried out for the purpose of confirming their gynogenetic origin and genetic differences. As the results, all the haploid plants possessed only either one of the BP alleles, confirming that they are gynogenetic origin. We also confirmed that BX1 and its doubled haploid (BX1-DH) have exactly the same SSR alleles. Interestingly, all the five haploids had common alleles on the linkage group (LG) 6 in the case of three markers. Then, we performed self-pollination of DH plants and reciprocal crosses between DH and BP, which has strong self-incompatibility. The seeds obtained by these crosses germinated in vitro or ex vitro normally and developed into diploid seedlings, except for one tetraploid seedling. Genetic analysis of these seedlings with 32 SSR markers revealed that segregation ratios of the 32 loci in the 30 BP inbred, 30 BP x DH and 30 DH x BP progenies mostly fitted to the expected ratios. However, significantly distorted segregation ratios were found in one, five and three SSR markers for BP inbred, BP x DH and DH x BP progenies, respectively. All of the 30 DH inbred progenies showed exactly the same SSR alleles as the original DH. This is the first example in which self and back cross progenies of DH in fruit crops were genetically analyzed using SSR markers.

Rubus L. subgenus Idaeobatus is one of the largest groups comprising mostly diploid species within the genus. Idaeobatus species is distributed throughout Japan, showing a wide range of trait variation and climatic distribution. We constructed phylogenetic trees of Idaeobatus that are naturally distributed in Japan. The Bayesian and ML phylogenetic tree found that this subgenus was polyphyletic with at least three genetic groups. The phylogenetic tree of Japanese Idaeobatus also reflects the trait variations for flower colors, fruit colors, and leaf shape (simple vs compound). The species that occurs in subtropical climates was found to be closely related to their sister temperate species in Japanese Idaeobatus. Crossability generally reflected the phylogenetic distances between species. Trait diversity and its phylogenetic relationship among Japanese Idaeobatus can provide additional insights for future breeding and ecological and evolutionary studies.

The genus Rubus comprises more than 740 species divided into 12 subgenera and contains the raspberries, blackberries, arctic fruits, and flowering raspberries, all of which have been utilized in breeding programs. The objective of this study was to evaluate the phylogenetic relationships among wild and cultivated Rubus species mainly collected in Japan. To evaluate genetic resources in Rubus, 81 accessions were analyzed with three amplified fragment length polymorphism (AFLP) primer pairs and data were analyzed with the neighbor-joining and unweighted pair group methods with arithmetic mean. Two of the generated phylogenetic trees grouped subgenera Anoplobatus, Eubatus, Idaeobatus, and Malachobatus into different clusters. Accessions of Rubus idaeus L. var. aculeatissimus collected from four regions in Hokkaido formed distinct clusters reflecting sampling sites. Four hybrid accessions between raspberry cultivars (R. idaeus L.) and R. idaeus L. var. aculeatissimus, and one hybrid accession between a raspberry cultivar and Rubus spectabilis Pursh were clearly distinguished from parental accessions. These results indicated that AFLP markers are a reliable technique for assessing genetic diversity and studying phylogenetic relationships in Rubus. Data from diversity and phylogenetic studies revealed valuable information on the availability of unique fragments in different accessions that would be useful for the development of improved genotypes through conventional breeding and marker-assisted selection.

Self-incompatibility (SI) is an important mechanism in higher plants that promotes outcrossing and prevents self-fertilization. ‘Banpeiyu’ (Citrus maxima) and ‘Hyuganatsu’ (Citrus tamurana), two of the Citrus cultivars distributed in Kyusyu, Japan, show gametophytic SI. In this study, we used the Citrus mature pollen culture system and stylar crude protein extracts to simulate compatible (C) and SI responses in ‘Banpeiyu’ pollen tubes. We analyzed the protein changes in pollen tubes with the C- and SI-like treatments by nano-liquid chromatography–mass spectrometry (nano-LC-MS); 14 and 27 proteins were identified in C- and SI-like treatments, respectively. We picked up some candidate genes that were particularly prevalent in SI-like treatment and analyzed their expression level changes during C- and SI-like treatments in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes. The expression levels of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese SOD (Mn SOD), catalase (CAT), and cysteine protease (CYP) increased after SI-like treatment. We used a fluorescent probe to visualize reactive oxygen species (ROS) level changes in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes after C- and SI-like treatments and found that 2-hour SI-like treatment induced ROS levels to increase in the pollen tubes of both cultivars. These results suggest that an ROS increase could be one of the key phenomena in the SI response of Citrus and that gene expression changes were responses to ROS generation.

Citrus is a genus with gametophytic self-incompatibility, which prevents fertilization by self-pollination. Polyamines (PAs), as a class of active small molecules, widely participate in various cellular activities. To investigate the effects of the changes in PA contents [putrescine, spermidine (SPD), and spermine] on pollen tube growth in self-incompatible (SI) plants, we cultured pollen of Citrus maxima ‘Banpeiyu’ and C. tamurana ‘Hyuganatsu’ in a Citrus mature pollen culture system and detected the PA content changes in pollen by high-performance liquid chromatography (HPLC) analysis during germination and after treatment with crude protein extracts of compatible or incompatible styles. We profiled the PA content changes during the germination course in pollen of ‘Banpeiyu’ and ‘Hyuganatsu’ by HPLC, and identified increases of free and perchloric-acid-insoluble SPD contents in pollen tubes in the incompatible treatment. Exogenous SPD treatment obviously increased the SPD contents in the pollen tube, inhibited pollen tube elongation, and resulted in some abnormal morphological alterations, such as tip inflation, tube content leakage and tip twist, which were observed in both ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes exposed to incompatible treatment. This finding implied that SPD might participate in the SI response in Citrus.

To analyze the protein expression of pollen grains and tubes having self-incompatible reactions in Hyuganatsu (Citrus tamurana Hort. ex Tanaka), we cultured mature pollen and treated it with crude extracts from the styles using a mass or single liquid culture system, and we collected pollen grains and tubes for proteome analysis. The relative expression of each identified protein was quantified by an imager and revealed as a value relative to the pollen grain. Eleven identified proteins were remarkably up-regulated (above 1.2-fold) or down-regulated (under 0.8-fold) in treatment with self-incompatible reactions. Eight of these proteins were predicted to be self-incompatible-related proteins with the reported functions. In this study, F-box protein was identified as showing minimum expression in the treatment with crude extracts from styles of Hyuganatsu (self-incompatible reactions). The S-locus gene product expressed in pollen has been identified as an F-box protein in Solanaceae and Rosaceae. It is not clear whether a mechanism similar to that of Solanaceae and Rosaceae exists in the self-incompatible reaction in Hyuganatsu; however, the reduced expression of F-box protein may induce a self-incompatible reaction.

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.