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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 c

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, tran

Bud dormancy helps woody perennials survive winter and activate robust plant development in the spring. For apple (Malus × domestica), short-term chilling induces bud dormancy in autumn, then prolonged chilling leads to dormancy release and a shift to a quiescent state in winter, with subsequent warm periods promoting bud break in spring. Epigenetic regulation contributes to seasonal responses such as vernalization. However, how histone modifications integrate seasonal cues and internal signals during bud dormancy in woody perennials remains largely unknown. Here, we show that H3K4me3 plays a key role in establishing permissive chromatin states during bud dormancy and bud break in apple. The global changes in gene expression strongly correlated with changes in H3K4me3, but not H3K27me3. High expression of DORMANCY-ASSOCIATED MADS-box (DAM) genes, key regulators of dormancy, in autumn was associated with high H3K4me3 levels. In addition, known DAM/SHORT VEGETATIVE PHASE (SVP) target genes significantly overlapped with H3K4me3-modified genes as bud dormancy progressed. These data suggest that H3K4me3 contributes to the central dormancy circuit, consisting of DAM/SVP and abscisic acid (ABA), in autumn. In winter, the lower expression and H3K4me3 levels at DAMs and gibberellin metabolism genes control chilling-induced release of dormancy. Warming conditions in spring facilitate the expression of genes related to phytohormones, the cell cycle, and cell wall modification by increasing H3K4me3 toward bud break. Our study also revealed that activation of auxin and repression of ABA sensitivity in spring are conditioned at least partly through temperature-mediated epigenetic regulation in winter.

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.

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.

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.

Litchi (Litchi chinensis Sonn.) originated from China and many of its cultivars have been produced in China so far during the long history of cultivation. One problem in litchi production and research is the worldwide confusion regarding litchi cultivar nomenclature. Because litchi cultivars can be described in terms of cultivar-dependent fruit appearance, it should be possible to discriminate cultivars of postharvest fruits. In this study, we explored this possibility using recently developed deep learning technology for four common Taiwanese cultivars 'Gui Wei', 'Hei Ye', 'No Mai Tsz', and 'Yu Her Pau'. First, we quantitatively evaluated litchi fruit shapes using elliptic Fourier descriptors and characterized the relationship between cultivars and fruit shapes. Results suggest that 'Yu Her Pau' can be clearly discriminated from others mainly based on its higher length-to-diameter ratio. We then fine-tuned a pre-trained VGG16 to construct a cultivar discrimination model. Relatively few images were sufficient to train the model to classify fruit images with 98.33% accuracy. We evaluated our model using images of fruits collected in different seasons and locations and found the model could identify 'Yu Her Pau' fruits with 100% accuracy and 'Hei Ye' fruits with 84% accuracy. A Grad-CAM visualization reveals that this model uses different cultivar-dependent regions for cultivar recognition. Overall, this study suggests that deep learning can be used to discriminate litchi cultivars from images of the fruit.