'Hongmian miyou' (Citrus grandis L. Osbeck) is mutated from 'Guanxi miyou', with a different spongy layer coloration. Trifoliate orange (Poncirus trifoliata) is widely used as rootstocks in 'Guanxi miyou' grafting, whereas 'Hongmian miyou' is incompatible with available trifoliate orange rootstocks. To explore the reasons for the etiolation of leaves of 'Hongmian miyou'/trifoliate orange, anatomical differences among different graft unions, gene expression profiles, and auxin levels of scion were investigated in this study. A histological assay indicated that there was no significant difference in anatomical structure between the compatible and incompatible combinations. A total of 1950 significant differentially-expressed genes (DEGs) were identified and analyzed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that genes involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction were significantly enriched. Moreover, the expression of nine genes in the auxin pathway were upregulated and three were downregulated in compatible combinations compared with those in the incompatible group. Further experiments verified that indole-3-acetic acid (IAA) content increases in the compatible graft combination, which suggests that IAA might promote graft compatibility.

Flowering connects vegetative and generative developmental phases and plays a significant role in strawberry production. The mechanisms that regulate strawberry flowering time are unclear. B-box transcription factors (BBXs) play important roles in the flowering time regulation of plants. Nevertheless, BBXs in octoploid cultivated strawberry (Fragaria ananassa) and their functions in flowering time regulation have not been identified. Here, we identified 51 FaBBXs from cultivated strawberry and 16 FvBBXs from diploid wild strawberry (Fragaria vesca), which were classified into five groups according to phylogenetic analysis. Further evolutionary analysis showed that whole-genome duplication or segmental duplication is a crucial factor that leads to the expansion of the BBX gene family in two strawberry species. Moreover, some loss and acquisition events of FaBBX genes were identified in the genome of cultivated strawberry that could have affected traits of agronomic interest, such as fruit quality. The promoters of FaBBX genes showed an enrichment in light-responsive, cis-regulatory elements, with 16 of these genes showing changes in their transcriptional activity in response to blue light treatment. On the other hand, FaBBX28c1, whose transcriptional activity is reduced in response to blue light, showed a delay in flowering time in Arabidopsis transgenic lines, suggesting its role in the regulation of flowering time in cultivated strawberry. Our results provide new evolutionary insight into the BBX gene family in cultivated strawberry and clues regarding their function in flowering time regulation in plants.

A precise, rapid and straightforward approach to chromosome identification is fundamental for cytogenetics studies. However, the identification of individual chromosomes was not previously possible for Chinese cherry or other Prunus species due to the small size and similar morphology of their chromosomes. To address this issue, we designed a pool of oligonucleotides distributed across specific pseudochromosome regions of Chinese cherry. This oligonucleotide pool was amplified through multiplex PCR with specific internal primers to produce probes that could recognize specific chromosomes. External primers modified with red and green fluorescence tags could produce unique signal barcoding patterns to identify each chromosome concomitantly. The same oligonucleotide pool could also discriminate all chromosomes in other Prunus species. Additionally, the 5S/45S rDNA probes and the oligo pool were applied in two sequential rounds of fluorescence in situ hybridization (FISH) localized to chromosomes and showed different distribution patterns among Prunus species. At the same time, comparative karyotype analysis revealed high conservation among P. pseudocerasus, P. avium, and P. persica. Together, these findings establish this oligonucleotide pool as the most effective tool for chromosome identification and the analysis of genome organization and evolution in the genus Prunus.

Melatonin (MT) is crucial in plant growth, development, and response to stress. Celery is a vegetable that grows in a cool climate, and a hot climate can deteriorate its growth, yield, and quality. This study investigates the effect of exogenous melatonin on celery physiology. Transcriptional levels were analyzed by spraying celery with exogenous MT before exposing it to high temperatures. The regulatory mechanism of exogenous MT-mediated heat tolerance was examined. The results show that the exogenous MT reduced the thermal damage state of celery seedlings, as well as the malondialdehyde (MDA) content and relative conductivity (REC), increasing the oxidase activity, the osmotic regulatory substances, and chlorophyll, enhancing the leaf transpiration and the light energy utilization efficiency. We examined the mechanism of exogenous MT in mitigating high-temperature damage using the transcriptome sequencing method. A total of 134 genes were expressed differently at high temperature in the celery treated with MT compared with the untreated celery. Functional annotation analysis revealed that the differentially expressed genes were abundant in the "pyruvate metabolism" pathway and the "peroxidase activity" pathway. According to the pathway-based gene expression analysis, exogenous MT can inhibit the upregulation of pyruvate synthesis genes and the downregulation of pyruvate consumption genes, preventing the accumulated pyruvate from rapidly upregulating the expression of peroxidase genes, and thereby enhancing peroxidase activity. RT-qPCR verification showed a rising encoding peroxidase gene expression under MT treatment. The gene expression pattern involved in pyruvate anabolism and metabolism agreed with the abundant transcriptome expression, validating the physiological index results. These results indicate that the application of exogenous MT to celery significantly enhances the ability of plant to remove reactive oxygen species (ROS) in response to heat stress, thereby improving the ability of plant to resist heat stress. The results of this study provide a theoretical basis for the use of MT to alleviate the damage caused by heat stress in plant growth and development.

Celery (Apium graveolens L.) is an important vegetable crop cultivated worldwide for its medicinal properties and distinctive flavor. Volatile organic compound (VOC) analysis is a valuable tool for the identification and classification of species. Currently, less research has been conducted on aroma compounds in different celery varieties and colors. In this study, five different colored celery were quantitatively analyzed for VOCs using HS-SPME, GC-MS determination, and stoichiometry methods. The result revealed that γ-terpinene, d-limonene, 2-hexenal,-(E)-, and β-myrcene contributed primarily to the celery aroma. The composition of compounds in celery exhibited a correlation not only with the color of the variety, with green celery displaying a higher concentration compared with other varieties, but also with the specific organ, whereby the content and distribution of volatile compounds were primarily influenced by the leaf rather than the petiole. Seven key genes influencing terpenoid synthesis were screened to detect expression levels. Most of the genes exhibited higher expression in leaves than petioles. In addition, some genes, particularly AgDXS and AgIDI, have higher expression levels in celery than other genes, thereby influencing the regulation of terpenoid synthesis through the MEP and MVA pathways, such as hydrocarbon monoterpenes. This study identified the characteristics of flavor compounds and key aroma components in different colored celery varieties and explored key genes involved in the regulation of terpenoid synthesis, laying a theoretical foundation for understanding flavor chemistry and improving its quality.

Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.

The negative association between psychological stress and male fertility has been known for many years. This study was aimed at (i) identifying spermatogenesis impairment induced by psychological stress in rats and (ii) exploring the role of glucocorticoid receptor (GR) signaling in these adverse effects (if they exist). Male Sprague Dawley rats were exposed to a six-week period of unpredictable chronic mild stress (uCMS) along with cotreatment of GR antagonist RU486 (1 mg/kg/day). Testicular damage was assessed by testicular pathological evaluation, epididymal sperm concentration, serum testosterone levels, testicular apoptotic cell measurements, and cell cycle progression analyses. Rats in the uCMS group had decreased levels of serum testosterone and decreased epididymal sperm concentration. The uCMS-treated rats also had decreased numbers of spermatids and increased levels of apoptotic seminiferous tubules; additionally, cell cycle progression of spermatogonia was arrested at the G0/G1 phase. Furthermore, uCMS exposure caused an increase in serum corticosterone level and activated GR signaling in the testes including upregulated GR expression. RU486 treatment suppressed GR signaling and alleviated the damaging effects of stress, resulting in an increased epididymal sperm concentration. Overall, this work demonstrated for the first time that the activation of GR signaling mediates stress-induced spermatogenesis impairment and that this outcome is related to cell apoptosis and cell cycle arrest in germ cells.

Chinese cherry [Cerasus pseudocerasus (Lindl.) G. Don] is an important fruit tree from China that has excellent ornamental, economic, and nutritional values with various colors. The dark-red or red coloration of fruit, an attractive trait for consumers, is determined by anthocyanin pigmentation. In this study, the coloring patterns during fruit development in dark-red and yellow Chinese cherry fruits were firstly illustrated by integrated transcriptome and widely-targeted metabolome analyses. Anthocyanin accumulation in dark-red fruits was significantly higher compared with yellow fruits from the color conversion period, being positively correlated to the color ratio. Based on transcriptome analysis, eight structural genes (CpCHS, CpCHI, CpF3H, CpF3'H, CpDFR, CpANS, CpUFGT, and CpGST) were significantly upregulated in dark-red fruits from the color conversion period, especially CpANS, CpUFGT, and CpGST. On contrary, the expression level of CpLAR were considerably higher in yellow fruits than in dark-red fruits, especially at the early stage. Eight regulatory genes (CpMYB4, CpMYB10, CpMYB20, CpMYB306, bHLH1, CpNAC10, CpERF106, and CpbZIP4) were also identified as determinants of fruit color in Chinese cherry. Liquid chromatography-tandem mass spectrometry identified 33 and 3 differential expressed metabolites related to anthocyanins and procyanidins between mature dark-red and yellow fruits. Cyanidin-3-O-rutinoside was the predominant anthocyanin compound in both fruits, while it was 6.23-fold higher in dark-red than in yellow fruits. More accumulated flavanol and procyanidin contents resulted in less anthocyanin content in flavonoid pathway in yellow fruits due to the higher expression level of CpLAR. These findings can help understand the coloring mechanism of dark-red and yellow fruits in Chinese cherry, and provide genetic basis for breeding new cultivars.

The regulation of detached ripening is significant for prolonging fruit shelf life. Although light quality and sucrose affecting strawberry fruit ripening have been widely reported, little information is available about how they co-regulate the strawberry detached ripening process. In this study, different light qualities (red light-RL, blue light-BL, and white light-WL) and 100 mM sucrose were applied to regulate the ripening of initial red fruits detached from the plant. The results showed RL-treated samples (RL + H2O, RL + 100 mM sucrose) had brighter and purer skin color with a higher L*, b*, and C* value, and promoted the ascorbic acid. Almost all light treatments significantly decreased TSS/TA (total soluble solid/titratable acid) and soluble sugar/TA ratio, which is exacerbated by the addition of sucrose. Blue or red light in combination with sucrose notably increased total phenolic content and decreased malondialdehyde (MDA) accumulation. In addition, blue or red light combined with sucrose increased abscisic acid (ABA) content and promoted ABA signaling by inducing ABA-INSENSITIVE 4 (ABI4) expression and inhibiting SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 2.6 (SnRK2.6) expression. The strawberries exposed to blue and red light significantly improved auxin (IAA) content compared to the control (0 d), whereas the addition of sucrose inhibited IAA accumulation. Moreover, sucrose treatment suppressed the AUXIN/INDOLE-3-ACETIC ACID 11 (AUX/IAA11) and AUXIN RESPONSE FACTOR 6 (ARF6) expression under different light qualities. Overall, these results indicated that RL/BL + 100 mM sucrose might promote the detached ripening of strawberries by regulating abscisic acid and auxin signaling.

Anthocyanins are responsible for the red color of strawberry, they are a subclass of flavonoids synthesized in cytosol and transferred to vacuole to form the visible color. Previous studies in model and ornamental plants indicated members of the glutathione S-transferase (GST) gene family were involved in vacuolar accumulation of anthocyanins. In the present study, a total of 130 FaGST genes were identified in the genome of cultivated strawberry (Fragaria × ananassa), which were unevenly distributed across the 28 chromosomes from the four subgenomes. Evolutionary analysis revealed the expansion of FaGST family was under stable selection and mainly drove by WGD/segmental duplication event. Classification and phylogenetic analysis indicated that all the FaGST genes were clarified into seven subclasses, among which FaGST1, FaGST37, and FaGST97 belonging to Phi class were closely related to FvRAP, an anthocyanin-related GST of wildwood strawberry, and this clade was clustered with other known anthocyanin-related GSTs. RNAseq-based expression analysis at different developmental stages of strawberry revealed that the expression of FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 was gradually increased during the fruit ripening, consistent with the anthocyanins accumulation. These expression patterns of those five FaGST genes were also significantly correlated with those of other anthocyanin biosynthetic genes such as FaCHI, FaCHS, and FaANS, as well as anthocyanin regulatory gene FaMYB10. These results indicated FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 may function in vacuolar anthocyanin accumulation in cultivated strawberry.

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.

Glucose-6-phosphate dehydrogenase (G6PDH) plays an important role in plant stress responses. Here, five FaG6PDH sequences were obtained in strawberry, designated as FaG6PDH-CY, FaG6PDH-P1, FaG6PDH-P1.1, FaG6PDH-P2 and FaG6PDH-P0, which were divided into cytosolic (CY) and plastidic (P) isoforms based on the bioinformatic analysis. The respective FaG6PDH genes had distinct expression patterns in all tissues and at different stages of fruit development. Notably, FaG6PDH-CY was the most highly expressed gene among five FaG6PDH members, indicating it encoded the major G6PDH isoform throughout the plant. FaG6PDH positively regulated cold tolerance in strawberry. Inhibition of its activity gave rise to greater cold-induced injury in plant. The FaG6PDH-CY transcript had a significant increase under cold stress, similar to the G6PDH enzyme activity, suggesting a principal participant in response to cold stress. Further study showed that the low-temperature responsiveness (LTR) element in FaG6PDH-CY promoter can promote the gene expression when plant encountered cold stimuli. Besides, FaG6PDH-CY was involved in regulating cold-induced activation of antioxidant enzyme genes (FaSOD, FaCAT, FaAPX and FaGR) and RBOH-dependent ROS generation. The elevated FaG6PDH-CY enhanced ROS-scavenging capability of antioxidant enzymes to suppress ROS excessive accumulation and relieved the oxidative damage, eventually improving the strawberry resistance to cold stress.

C2H2-type zinc finger proteins (C2H2-ZFPs) play a key role in various plant biological processes and responses to environmental stresses. In Arabidopsisthaliana, C2H2-ZFP members with two zinc finger domains have been well-characterized in response to abiotic stresses. To date, the functions of these genes in strawberries are still uncharacterized. Here, 126 C2H2-ZFPs in cultivated strawberry were firstly identified using the recently sequenced Fragaria × ananassa genome. Among these C2H2-ZFPs, 46 members containing two zinc finger domains in cultivated strawberry were further identified as the C1-2i subclass. These genes were unevenly distributed on 21 chromosomes and classified into five groups according to the phylogenetic relationship, with similar physicochemical properties and motif compositions in the same group. Analyses of conserved domains and gene structures indicated the evolutionary conservation of the C1-2i subclass. A Ka/Ks analysis indicated that the C1-2i members were subjected to purifying selection during evolution. Furthermore, FaZAT10, a typical C2H2-ZFP, was isolated. FaZAT10 was expressed the highest in roots, and it was induced by drought, salt, low-temperature, ABA, and MeJA treatments. It was localized in the nucleus and showed no transactivation activity in yeast cells. Overall, these results provide useful information for enriching the analysis of the ZFPs gene family in strawberry, and they provide support for revealing the mechanism of FaZAT10 in the regulatory network of abiotic stress.

Proanthocyanidins (PAs), also known as condensed tannins, are widespread throughout the plant kingdom, presenting diverse biological and biochemical activities. Being one of the most abundant groups of natural polyphenolic antioxidant, PAs are applied to improve plant tolerance to (a)biotic stresses and delay the senescence of fruit by scavenging the reactive oxygen species (ROS) and enhancing antioxidant responses. The effects of PAs on coloring and softening of strawberries (Fragaria × ananassa Duch.), a worldwide demanded edible fruit and typical material for studying non-climacteric fruit ripening, were firstly assessed in this work. The results showed that exogenous PAs delayed the decrease in fruit firmness and anthocyanins accumulation but improved the fruit skin brightness. Strawberries treated with PAs had similar total soluble solids, total phenolics, and total flavonoids, but lower titratable acidity content. Moreover, the contents of endogenous PAs, abscisic acid and sucrose, were somehow increased by PA treatment, while no obvious change was found in fructose and glucose content. In addition, the anthocyanin- and firmness-related genes were significantly repressed, while the PA biosynthetic gene (anthocyanin reductase, ANR) was highly up-regulated by PA treatment at the key point for fruit softening and coloring. In summary, the results presented in this study suggest that PAs slow down strawberry coloration and softening by inhibiting the expression of related genes, which could be helpful for a better understanding of the biological role of PAs and provide a new strategy to regulate strawberry ripening.

Fruit softening is a crucial factor that controls shelf life and commercial value. Pectate lyase (PL) has a major role in strawberry fruit softening. However, the PL gene family in strawberry has not been comprehensively analyzed. In this study, 65 FaPL genes were identified in the octoploid strawberry genome. Subcellular localization prediction indicated that FaPLs are mostly localized to the extracellular and cytoplasmic spaces. Duplication event analysis suggested that FaPL gene family expansion is mainly driven by whole genome or segmental duplication. The FaPL family members were classified into six groups according to the phylogenetic analysis. Among them, FaPL1, 3, 5, 20, 25, 42, and 57 had gradually increased expressions during strawberry fruit development and ripening and higher expression levels in the fruits with less firmness than that in firmer fruit. This result suggested that these members are involved in strawberry softening. Furthermore, overexpression of FaPL1 significantly reduced the fruit firmness, ascorbic acid (AsA), and malondialdehyde (MDA) content but obviously increased the anthocyanins, soluble proteins, and titratable acidity (TA), while it had no apparent effects on flavonoids, phenolics, and soluble sugar content. These findings provide basic information on the FaPL gene family for further functional research and indicate that FaPL1 plays a vital role in strawberry fruit softening.