Preharvest long-term exposure to UV-B radiation promotes fruit ripening and modifies stage-specific anthocyanin metabolism in highbush blueberry

Ultraviolet-B (UV-B) light (280–315 nm) is an important environmental signal that regulates plant development and photomorphogenesis, while also affecting the flavonoid pathway, including anthocyanin biosynthesis. Regarding the effects of UV-B radiation on fruits, the effects of a short-term or postharvest irradiation on fruit quality have been well-documented, but the effects of a long-term preharvest UV-B irradiation on fruit growth and coloration remain unclear. Thus, in this study, we investigated the effects of a long-term treatment involving an environmentally relevant UV-B dose on highbush blueberry (Vaccinium corymbosum) fruit. The preharvest UV-B treatment quickly promoted fruit growth and sugar accumulation, which is not commonly observed in other fruit tree species. The UV-B exposure also accelerated fruit ripening and coloration. The dual-luciferase assay proved that in blueberries, expression of VcUFGT encoding anthocyanin biosynthesis key enzyme, is positively and negatively regulated by VcMYBA1 and VcMYBC2, respectively. Throughout the fruit development stage, the UV-B treatment up-regulated VcMYBPA1 expression, which increased VcUFGT expression via VcMYBA1. In the green fruit stage, the UV-B treatment increased HY5 encoding UV receptor, which up-regulates VcMYBPA1 and down-regulates VcMYBC2, thereby promotes the accumulation of anthocyanins. On the other hand, excessive anthocyanin synthesis was inhibited by increased VcMYBC2 levels in mature fruits when exposed to UV-B light through HY5-independent pathway. In conclusion, anthocyanin-related MYB activators and repressor may coordinately balance the accumulation of anthocyanins in blueberry fruits, with UV-B treatments possibly influencing their effects in a stage-specific manner. The potential utility of preharvest UV-B treatments for improving blueberry fruit quality is discussed herein

Efficient Transient Expression for Functional Analysis in Fruit Using the Tsukuba System Vector

Evaluating the function of genes expressed in fruit tissues of fruit tree species using a genetic transformation approach is a long process because the trees are generally recalcitrant to genetic transformation and cannot bear fruit during their long juvenile phases. Transient gene expression in fruit enables the functional analysis of genes associated with fruit traits, which may accelerate the study of fruit physiology. Here, by using the recently developed “Tsukuba system”, we successfully established an efficient transient expression system in harvested fruit tissues. The “Tsukuba system” utilizes a combination of the geminiviral replication system and a double terminator, which ensures sufficient levels of transgene expression. We used blueberry fruit as a model to characterize the applicability of this system for transient expression in fruit tissue. The pTKB3-EGFP vector was introduced by agroinfiltration into the fruit tissues of several blueberry cultivars. We found that transient GFP fluorescence in fruit peaked 4–6 days after agroinfiltration. Agrobacterium suspensions were easily injected into soft, mature fruit, and GFP was strongly expressed; however, hard, immature fruit were not penetrable by Agrobacterium suspensions, and GFP was rarely detected. We then tested the applicability of the developed system to other fruit tree species: six families, 17 species, and 26 cultivars. GFP fluorescence was detected in all species, except for Japanese apricot. In blueberry, bilberry, sweet cherry, apricot, and satsuma mandarin, GFP was highly expressed and observed in a large proportion of the flesh. In kiwifruit, hardy kiwifruits, persimmon, peach, apple, European pear, and grape, GFP fluorescence was limited to certain parts of the fruits. Finally, transient VcMYBA1 overexpression in blueberry was tested as a model for gene functional analysis in fruit. Transient VcMYBA1 overexpression induced red pigmentation in the flesh, suggesting that VcMYBA1 expression caused anthocyanin accumulation. This study provides a technical basis for the rapid evaluation of genes expressed in fruit, which will be useful for gene function evaluation studies in fruit crops with long juvenile phases

Genomic insight into the developmental history of southern highbush blueberry populations

ハイブッシュブルーベリーに暖地適応性をもたらした遺伝要因を解明 --ゲノムに刻まれたブルーベリーの育種履歴--. 京都大学プレスリリース. 2020-09-07.Interspecific hybridization is a common breeding approach for introducing novel traits and genetic diversity to breeding populations. Southern highbush blueberry (SHB) is a blueberry cultivar group that has been intensively bred over the last 60 years. Specifically, it was developed by multiple interspecific crosses between northern highbush blueberry [NHB, Vaccinium corymbosum L. (2n = 4x = 48)] and low-chill Vaccinium species to expand the geographic limits of highbush blueberry production. In this study, we genotyped polyploid blueberries, including 105 SHB, 17 NHB, and 10 rabbiteye blueberry (RE) (Vaccinium virgatum Aiton), from the accessions planted at Poplarville, Mississippi, and accessions distributed in Japan, based on the double-digest restriction site-associated DNA sequencing. The genome-wide SNP data clearly indicated that RE cultivars were genetically distinct from SHB and NHB cultivars, whereas NHB and SHB were genetically indistinguishable. The population structure results appeared to reflect the differences in the allele selection strategies that breeders used for developing germplasm adapted to local climates. The genotype data implied that there are no or very few genomic segments that were commonly introgressed from low-chill Vaccinium species to the SHB genome. Principal component analysis-based outlier detection analysis found a few loci associated with a variable that could partially differentiate NHB and SHB. These SNP loci were detected in Mb-scale haplotype blocks and may be close to the functional genes related to SHB development. Collectively, the data generated in this study suggest a polygenic adaptation of SHB to the southern climate, and may be relevant for future population-scale genome-wide analyses of blueberry

Functional and expressional analyses of apple FLC-like in relation to dormancy progress and flower bud development.

We previously identified the FLOWERING LOCUS C (FLC)-like gene, a MADS-box transcription factor gene that belongs to Arabidopsis thaliana L. FLC clade, in apple (Malus ×domestica Borkh.), and its expression in dormant flower buds is positively correlated with cumulative cold exposure. To elucidate the role of the MdFLC-like in the dormancy process and flower development, we first characterized the phenotypes of MdFLC-like overexpressing lines with the Arabidopsis Columbia-0 background. The overexpression of MdFLC-like significantly delayed the bolting date and reduced the plant size, but it did not significantly affect the number of rosette leaves or flower organ formation. Thus, MdFLC-like may affect vegetative growth and development rather than flowering when expressed in Arabidopsis, which is not like Arabidopsis FLC that affects development of flowering. We compared seasonal expression patterns of MdFLC-like in low-chill ‘Anna’ and high-chill ‘Fuji’ and ‘Tsugaru’ apples collected from trees grown in a cold winter region in temperate zone and found an earlier upregulation in ‘Anna’ compared with ‘Fuji’ and ‘Tsugaru’. Expression patterns were also compared in relation to developmental changes in the flower primordia during the chilling accumulation period. Overall, MdFLC-like was progressively upregulated during flower primordia differentiation and development in autumn to early winter and reached a maximum expression level at around the same time as the genotype-dependent chilling requirements were fulfilled in high-chill cultivars. Thus, we hypothesize MdFLC-like may be upregulated in response to cold exposure and flower primordia development during the progress of endodormancy. Our study also suggests MdFLC-like may have a growth-inhibiting function during the end of endodormancy and ecodormancy when the temperature is low and unfavorable for rapid bud outgrowth

オウトウ ノ ジカ フワゴウセイ ニ カンスル ブンシ セイブツガクテキ セイカガクテキ ケンキュウ

京都大学0048新制・論文博士博士(農学)乙第11106号論農博第2454号新制||農||859(附属図書館)学位論文||H15||N3743(農学部図書室)UT51-2003-C676(主査)教授 米森 敬三, 教授 矢澤 進, 教授 谷坂 隆俊学位規則第4条第2項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Characterization of Auxin Metabolism in the Ovaries of the Lychee (Litchi chinensis) ‘Salathiel’

A part of this study was presented at the Autumn Annual Meeting of the Japanese Society for Horticultural Science, 2021.Seed size affects the edible portion rate of lychee fruit and is therefore an important trait for fruit quality in lychee (Litchi chinensis). Lychee fruits can be classified into four categories in terms of seed properties: normal, small, aborted seed and seedless. ‘Salathiel’ is known to bear a relatively high rate of aborted-seeded (pseudo-parthenocarpy, stenospermocarpy) and seedless (parthenocarpy) fruit regardless of environmental and cultivation conditions. In other horticultural fruit crops, such as tomato, auxin metabolism and signaling is critical for parthenocarpy, although auxin metabolism has not been characterized in relation to (pseudo) parthenocarpy in lychee. The purpose of this study was to characterize the physiological and morphological properties of maternal reproductive organs in ‘Salathiel’ female flowers, with a specific focus on the associated auxin metabolism. Microscopic observations of the internal structures of the ‘Salathiel’ embryo revealed that ‘Salathiel’ reproductive organ differentiation is similar to that of normal-seeded cultivars. However, the obturator was significantly smaller in ‘Salathiel’ than in other normal-seeded cultivars, suggesting that specific developmental characteristics may exist in the maternal reproductive organs of ‘Salathiel’. Our investigation of indole acetic acid (IAA) contents revealed that IAA levels were significantly higher in ‘Salathiel’ than in other cultivars. Moreover, the IAA metabolite contents also differed significantly between ‘Salathiel’ and the normal-seeded and small-seeded cultivars. Specifically, N-3-hydroxy-2-oxindole-3-acetyl glutamic acid contents were significantly higher in ‘Salathiel’ than in ‘Yu Her Pau’ and ‘Hei Ye’. 3-hydroxy-2-oxindole-3-acetic acid was lower in ‘Yu Her Pau’ and ‘Salathiel’ than in ‘Hei Ye’, while indole-acetylaspartic acid was significantly lower in ‘Salathiel’ compared to ‘Hei Ye’. Expression analyses of the genes related to auxin biosynthesis, catabolism, transport, and signaling indicated that the IAA influx-related and efflux-related gene expression levels were respectively higher and lower in ‘Salathiel’ than in the other cultivars, which is consistent with the increased IAA accumulation in the ‘Salathiel’ ovary. The possible involvement of auxin metabolism in the aborted-seeded and seedless fruit production in ‘Salathiel’ is discussed herein

マイクロアレイを用いた長期低温遭遇時のウメ休眠芽のトランスクリプトーム解析

Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season’s growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (P. persica) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (P < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included P. mume DORMANCY-ASSOCIATED MADS-box genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.永年性作物において越冬芽の休眠は, 環境に適応するために進化した成長制御機構のひとつであり, かつ翌年の成長を左右する重要な農業形質のひとつである. 芽の休眠には多くの遺伝的要因が関わるばかりでなく, 多岐にわたる環境要因や樹勢, 樹齢の影響もうけており, 多くの遺伝子の発現制御が休眠に関与していると考えられる. そこで本研究では, 休眠関与候補遺伝子の単離を目的に, カスタムマイクロアレイを用いたウメ休眠芽のトランスクリプトーム解析をおこなった. まず, 我々が先行研究で獲得したウメ芽 EST 配列情報をもとに, ゲノム全体をできるだけ偏りなく網羅するように選びだした 58539 ユニジーンに相当するプローブを搭載したマイクロアレイを構築した. 次いで, 休眠覚醒を誘導する長期の低温処理を施したウメ休眠芽の遺伝子発現変動を調査した. その結果, 有意に 2 倍以上発現が上方制御あるいは下方制御されるプローブが 2345 個あるいは 1059 個同定された. これらの変動遺伝子のなかには季節的な発現同調性がみられるものがあった. 下方制御される遺伝子のなかには, 先行研究で休眠への関与が示唆されている DORMANCY ASSOCIATED MADS-box が含まれており, 上方制御される遺伝子にはリポキシゲナーゼが含まれていた. Parametricanalysis of gene set enrichment 解析の結果, 長期の低温遭遇によってジャスモン酸やオキシリピン生合成・代謝の GO タームが有意に上昇し, 概日リズムの GO タームが有意に減少した. これらの GO タームに含まれる遺伝子について定量 RT-PCR を行った結果, マイクロアレイで検出された発現変動パターンとほぼ同様であった. 以上の結果より, 本研究で構築したマイクロアレイはウメ休眠芽の網羅的な遺伝子発現解析に有効であることが示された. 本実験では, ウメの休眠覚醒に関与する遺伝子ネットワークあるいは新規候補遺伝子の探索に活用した

Accumulation of Nonfunctional S-Haplotypes Results in the Breakdown of Gametophytic Self-Incompatibility in Tetraploid Prunus

The transition from self-incompatibility (SI) to self-compatibility (SC) is regarded as one of the most prevalent transitions in Angiosperm evolution, having profound impacts on the genetic structure of populations. Yet, the identity and function of mutations that result in the breakdown of SI in nature are not well understood. This work provides the first detailed genetic description of the breakdown of S-RNase-mediated gametophytic self-incompatibility (GSI) in a polyploid species that exhibits genotype-dependent loss of SI. Genetic analyses of six natural sour cherry (Rosaceae, Prunus cerasus) selections identified seven independent, nonfunctional S-haplotypes with disrupted pistil component (stylar-S) and/or pollen component (pollen-S) function. A genetic model demonstrating that the breakdown of SI in sour cherry is due to the accumulation of a minimum of two nonfunctional S-haplotypes within a single individual is developed and validated. Our finding that sour cherry is SI when only one nonfunctional S-haplotype is present has significant evolutionary implications since nonfunctional S-haplotypes would be maintained in the population without causing an abrupt shift to SC. Furthermore, we demonstrate that heteroallelic sour cherry pollen is self-incompatible, which is counter to the well-documented phenomenon in the Solanaceae where SC accompanying polyploidization is frequently due to the SC of heteroallelic pollen