Apple skin color is an important quality trait affecting marketability. The anthocyanin accumulation in 'Hongro' apple skins was analyzed by evaluating different defoliation timing treatments of fruit cluster leaves applied two, three, and four weeks before harvest (WBH). Non-defoliated plants served as controls. Defoliation did not affect the fruit weight, size, flesh firmness, soluble solid content, titratable acidity, or the starch pattern index at harvest. However, the fruit sunburn rate was higher for defoliation at 3 and 4 WBH compared to defoliation at 2 WBH and non-defoliation treatments. The red-colored area and a* value for both the sun-exposed and sun-shaded sides of apple skin were highest in the defoliation treatments at 3 and 4 WBH, intermediate at 2 WBH, and lowest in the non-defoliation treatment case. On the sun-exposed side, the L* value was lower after the defoliation treatments compared to the non-defoliation treatment, but the b* value was not affected by any of the defoliation treatments. On the sun-shaded side, the L* and b* values were lower under defoliation at 4 WBH compared to the other treatments. The anthocyanin content on the sun-exposed side was highest after defoliation at 4 WBH, followed by 3 and 2 WBH, and was lowest in the non-defoliation treatment. Similarly, the anthocyanin content on the sun-shaded side was highest in defoliated plants at 4 and 3 WBH, intermediate at 2 WBH, and lowest in the nondefoliated plants. Additionally, the expression levels of major anthocyanin biosynthetic genes (MdPAL, MdCHS, MdCHI, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB10) were upregulated in all defoliation treatments on both the sun-exposed and sun-shaded sides, particularly during early defoliation at 4 and 3 WBH. Therefore, this study showed that the defoliation timing influences the development of red skin coloration in 'Hongro' apples and that defoliation at 4 and 3 WBH achieves the optimum coloration for this cultivar.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+—NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

Increasing fruit crop production sustainability under climate change, particularly increasing temperatures, is a major challenge in modern agriculture. High temperatures affect apple fruit quality and decrease its color. Herein, we constructed an experimental field under temperature simulation to evaluate climate change mitigation strategies for apples. ‘Hongro’ apples were subjected to three treatments: (1) cultivation inside a vinyl house for heat treatment (heat induction), (2) cultivation under water irrigation (heat reduction), and (3) cultivation under normal atmospheric temperature (control). At harvest, the fruits of the heat treatment group exhibited poor coloration, with a lower gene expression and pigment accumulation than those of the water irrigation and control groups. Furthermore, the fruit quality of the heat treatment group decreased, with a lower soluble solid content (SSC) and titratable acidity (TA), and smaller fruits. Additionally, a higher fruit disorder (cracking and spots) ratio was observed in the heat treatment group than in the water irrigation and control groups. However, the fruits of the water irrigation group exhibited higher quality indexes (flesh firmness, SSC, and TA) and less cracking than those of the heat treatment and control groups. Heat reduction, including water irrigation, may be used for orchard management to prevent climate change-induced increasing temperatures.

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.

Methods by which to realize the postharvest preservation of the new apple cultivars 'Colorpple' and 'Manhong' are unknown. Hence, the objective of this study was to evaluate the effects of 1-methylcyclopropene (1 -MCP) on the postharvest physiological characteristics and storage quality of 'Colorpple' and 'Manhong' apples. Harvested apples were treated with 1 -MCP (1 mu LL -1 ) and stored for 40 days under warm temperature (20 +/- 1 degrees C) and for eight months under cold (0 +/- 1 degrees C) temperature conditions. Untreated fruits were used as control groups. Ethylene production and respiration rates were lower in all treated fruits stored at a warm temperature. However, 1 -MCP had minimal effects on flesh firmness, weight loss, or the soluble solid content (SSC), affecting only the titratable acidity (TA) at 40 days. Higher L * (20 and 30 days) and lower a * (30 and 40 days) values were observed only in the fruit skins of untreated 'Colorpple' apples. Additionally, skin greasiness increased in the untreated 'Colorpple' (30 and 40 days) and 'Manhong' (20 days) apples. Slight levels of fruit decay were observed in 'Colorpple' (30 days) and 'Manhong' (30 and 40 days). At a cold temperature, 1 -MCP induced lower ethylene production and respiration rates and higher flesh firmness and TA in both cultivars, while weight losses as well as skin color and SSC changes were not detected. A lower SSC/TA ratio was observed in treated fruits after 6-8 months of storage. Moderate to severe skin greasiness was observed in both untreated apple types. Overall, this study suggests that a 1 -MCP treatment improves the postharvest physiological characteristics and fruit quality of 'Colorpple' and 'Manhong' apples; 1 -MCP is more effective for long-term storage of these cultivars at cold temperatures but is less effective at ambient temperatures.

Fruit skin color and physical quality are important for customer acceptability and market value. Therefore, this study aimed to evaluate the effect of pneumatic defoliation on the fruit quality, coloration, and anthocyanin content of ‘Fuji’ apples. Apple trees were subjected to no defoliation (control) and defoliation at low (0.6 bar) and high (0.9 bar) air pressure 20 days before harvest at 1 km/h of tractor speed. High-defoliation treatment increased the leaf damage rate but did not significantly affect the defoliation rate compared to low-defoliation treatment. Additionally, photosynthetically active radiation and solar irradiance inside the tree canopies were highest in the high-defoliation group, followed by the low-defoliation and control groups. With the exception of higher firmness in the high-defoliation treatment, pneumatic defoliation treatments had little effect on fruit size and weight, titratable acidity, soluble solids content, the starch pattern index, and the sunburn incidence of fruit. Compared with that of the control group, both defoliation treatments significantly increased the a* and C values and decreased the ho values of the fruit color. Moreover, both defoliation treatments significantly increased anthocyanin content and upregulated the anthocyanin biosynthesis genes (MdPAL, MdCHS, MdCHI, MdF3H, MdANS, MdANS, MdUFGT) and the transcription factor (MdMYB10). A Pearson′s correlation analysis also showed that anthocyanin production was strongly correlated with each of the anthocyanin biosynthesis genes, especially in the pneumatic defoliation treatments. Conclusively, the results show that pneumatic defoliation at low pressure bars could be an effective strategy for improving the red coloration of ‘Fuji’ apples.

Coloration, a major factor contributing to apple quality, attracts consumer attraction, thereby increasing the sale of fruits in the market. Among the fruit pigments, anthocyanins and carotenoids are the most important pigments that impart coloration to apples. Pigment accumulation, which is regulated by transcriptional factors in the anthocyanin and carotenoid biosynthesis pathways, varies according to genetics, transcription, and developmental stage. To understand the regulation of color development in apples, we evaluated the expression of the genes in the anthocyanin and carotenoid biosynthetic pathways and analyzed the accumulation of pigment, including anthocyanin and carotenoid in the skin and flesh of the red-fleshed apple ‘Enbu’ cultivar at different fruit development stages (30, 60, 90, 120, and 150 days after full bloom, and ripe). Color development increased according to the developmental stage, with considerable variation in both the transcript levels and pigment concentrations observed in tissues, skin, and flesh. Moreover, we identified key transcription factors among the anthocyanin and carotenoid biosynthesis genes that regulated pigment accumulation. Pearson’s correlation analysis showed a strong correlation between the coloration patterns and the expression levels of anthocyanin biosynthesis-related genes (MdPAL, MdCHI, and MdF3H) and carotenoid biosynthesis-related genes (MdGGPPS, MdPSY, MdZDS, MdCRTISO, MdCRHβ, and MdZEP). This study provides insight into the molecular mechanisms underlying pigment biosynthesis for breeding high-quality red-fleshed apple varieties to cater to consumer attention and preference.

In grafted apple, rootstock-derived signals influence scion cold tolerance by initiating physiological changes to survive over the winter. To understand the underlying molecular interactions between scion and rootstock responsive to cold, we developed transcriptomics and metabolomics data in the stems of two scion/rootstock combinations, 'Gala'/'G202' (cold resistant rootstock) and 'Gala'/'M9' (cold susceptible rootstock). Outer layers of scion and rootstock stem, including vascular tissues, were collected from the field-grown grafted apple during the winter. The clustering of differentially expressed genes (DEGs) and gene ontology enrichment indicated distinct expression dynamics in the two graft combinations, which supports the dependency of scion cold tolerance on the rootstock genotypes. We identified 544 potentially mobile mRNAs of DEGs showing highly-correlated seasonal dynamics between scion and rootstock. The mobility of a subset of 544 mRNAs was validated by translocated genome-wide variants and the measurements of selected RNA mobility in tobacco and Arabidopsis. We detected orthologous genes of potentially mobile mRNAs in Arabidopsis thaliana, which belong to cold regulatory networks with RNA mobility. Together, our study provides a comprehensive insight into gene interactions and signal exchange between scion and rootstock responsive to cold. This will serve for future research to enhance cold tolerance of grafted tree crops.

Flowering time (in rice, termed the heading date), plant height, and grain number are crucial agronomic traits for rice productivity. The heading date is controlled via environmental factors (day length and temperature) and genetic factors (floral genes). TERMINAL FLOWER 1 (TFL1) encodes a protein that controls meristem identity and participates in regulating flowering. In this study, a transgenic approach was used to promote the heading date in rice. We isolated and cloned apple MdTFL1 for early flowering in rice. Transgenic rice plants with antisense MdTFL1 showed an early heading date compared with wild-type plants. A gene expression analysis suggested that introducing MdTFL1 upregulated multiple endogenous floral meristem identity genes, including the (early) heading date gene family FLOWERING LOCUS T and MADS-box transcription factors, thereby shortening vegetable development. Antisense MdTFL1 also produced a wide range of phenotypic changes, including a change in overall plant organelles that affected an array of traits, especially grain productivity. The transgenic rice exhibited a semi-draft phenotype, increased leaf inclination angle, restricted flag leaf length, reduced spikelet fertility, and fewer grains per panicle. MdTFL1 plays a central role in regulating flowering and in various physiological aspects. These findings emphasize the role of TFL1 in regulating flowering in shortened breeding and expanding its function to produce plants with semi-draft phenotypes.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

Apple skin color is essential for market value. Therefore, this study evaluated the efficiency of pneumatic defoliation (PD) on the enhancement of red skin color and anthocyanin pigments in 'Picnic' apples. Three weeks before harvesting, apple trees were treated with PD with low- (0.7 bar) and high- (0.9 bar) air pressure at a tractor driving speed of 2 km/h. Undefoliated trees served as controls. Higher leaf defoliation and leaf damage rates were observed in the high-PD treatment than those in the low-PD treatment. Photosynthetically active radiation inside the canopies was the highest in trees subjected to high-pressure PD than in those that underwent low-pressure PD and the controls. At harvest, the fruit color (a*)value, red-colored area, and anthocyanin content in the fruit skin were the highest in the high-PD treatment, intermediate in the low-PD treatment, and the lowest in the control treatments. Additionally, a higher expression of anthocyanin biosynthetic genes was observed in both defoliation treatments, especially under high PD. However, except for higher flesh firmness under low PD, the L* and b* values and fruit quality indices (fruit weight, starch pattern index, titratable acidity, soluble solids content, and sunburn occurrence) were not significantly affected by either PD treatment. In conclusion, PD can be used to enhance skin coloration and anthocyanin pigments in apples; further PD with high air pressure achieved optimum red skin coloration in 'Picnic' apples.

Apples (Malus × domestica Borkh.) require up to several years for flowering and bearing fruits. The transition from vegetative to reproductive phase is controlled by floral regulators such as TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT). TFL1 mediates the maintenance of vegetative phase, unlike the antagonistic function of FT to promote the transition into reproductive phase. In this study, we isolated apple TFL1-like gene (MdTFL1) to elucidate various phenotypic traits triggered by the antisense expression of MdTFL1 in tobacco apart from its floral induction function. Early flowering was observed in the tobacco line with MdTFL1 knockout, indicating the reduced time for transition to vegetative phases. Quantitative reverse-transcription PCR showed upregulation of genes involved in the regulation of floral induction, including NtAP1, NtSOC1, NFL1, and NtFTs, and downregulation of carotenoid cleavage dioxygenases (CCDs) and CEN-like genes in transgenic lines. Interestingly, transgenic tobacco expressing antisense MdTFL1 exhibited distinct morphological changes in lateral shoot outgrowth, internode length, and the development of leaves, flowers, and fruits. The results suggested that using the antisense expression of MdTFL1 gene is one of the approaches to shorten the vegetable phase and proposed improvement of plant architecture in horticultural crops.

The coloration of the apple fruit (Malus × domestica Borkh.) depends on pigment content. Light stimulus activates a broad range of photosynthesis-related genes, including carotenoids. The effect of light on two red commercial apple cultivars, ‘Summer Prince’ and ‘Arisoo’ at the juvenile stage were examined. Apple fruits were either bagged to reduce light irradiation or were exposed to direct, enhanced sunlight (reflected). The pigment content and the expression of carotenoid metabolism genes in the peel and flesh of apple fruits were significantly different between the shaded and the reflected parts. These parameters were also different in the two cultivars, highlighting the contribution of the genetic background. Further, a combination of light and transient overexpression of carotenogenic genes increased fruit coloration and pigment content in the variety ‘RubyS’. Western blot analysis showed the expression of small heat shock proteins (smHSP) in lysates extracted from the reflected part of the fruits but not in the bagged fruits, indicating the activation of smHSP in response to heat generated by the reflected light. Therefore, the synergy between the genes and the environment dictates the color of apple fruits.

Fruit abscission is a complex physiological process that is regulated by internal and environmental factors. During early development, apple fruit are exposed to extreme temperature fluctuations that are associated with premature fruit drop; however, their effect on fruit abscission is largely unknown. We hypothesized that fruit abscission is triggered by cold stress and investigated the molecular basis of premature fruit drop using RNA-Seq and metabolomics data from apple fruit undergoing abscission following cold stress in the field. Genes responsive to abscisic acid signaling and cell wall degradation were upregulated during abscission, consistent with the increased abscisic acid concentrations detected by liquid chromatography-mass spectrometry. We performed ex vivo cold shock experiments with excised tree subunits consisting of a branch, pedicel, and fruit. Abscission induction occurred in the cold-stressed subunits with concurrent upregulation of abscisic acid biosynthesis (MdNCED1) and metabolism (MdCYP707A) genes, and ethylene biosynthesis (MdACS1) and receptor (MdETR2) genes in the pedicel. Another key finding was the activation of cytoplasmic streaming in abscission-zone cells detected by electron microscopy. Our results provide a novel insight into the molecular basis of fruit abscission physiology in response to cold stress in apple.

ABSTRACT Anthocyanin accumulation is responsible for the red color of the skin of apple fruits (Malus × domestica Borkh.). Environmental stimuli, such as light, temperature, soil factors, tree factors, and the application of chemicals can alter the synthesis of anthocyanins. Herein, we examined the expression of genes related to anthocyanin biosynthesis in the fruit of apple cultivars, ‘Summer Prince’ and ‘Arisoo’ at the early stage of fruit development 60 DAFB (days after full bloom). The expression of genes, which included structural and regulatory genes involved in anthocyanin biosynthesis, and light-responsive genes was determined in fruits developed under the effect of sunlight exposure for seven days. Apple fruits were divided in to: ‘reflected sunlight’, ‘bagged’, and ‘control’ groups. The expression levels of the anthocyanin synthesis-related genes were significantly different among the groups and between the shaded and reflected sides. We also determined the difference in coloration patterns in the different groups using the colorimetric coordinates method. The shaded side of apple fruits quickly turned more redder after exposure to reflected sunlight and the coloration was almost similar to that on the exposed side after the treatment. Strong correlation coefficients between anthocyanin-related gene expression levels and coloration patterns were obtained. This study shows that sunlight plays an important role in stimulating the coloration of apple fruit by regulating the expression of genes involved in anthocyanin accumulation during the early stage of fruit development. The present study assumes importance because redder fruits, generated through a non-transgenic approach would be more acceptable to consumers.

Nitrogen (N) is an essential macronutrient that regulates diverse physiological processes for plant survival and development. In apple orchards, inappropriate N conditions can cause imbalanced growth and subsequent physiological disorders in trees. In order to investigate the molecular basis underlying the physiological signals for N stress responses, we examined the metabolic signals responsive to contrasting N stress conditions (deficient/excessive) in apple leaves using transcriptome approaches. The clustering of differentially expressed genes (DEGs) showed the expression dynamics of genes associated with each N stress group. Functional analyses of gene ontology and pathway enrichments revealed the potential candidates of metabolic signals responsible for N-deficient/excessive stress responses. The functional interactions of DEGs in each cluster were further explored by protein–protein interaction network analysis. Our results provided a comprehensive insight into molecular signals responsive to N stress conditions, and will be useful in future research to enhance the nutrition tolerance of tree crops.

Fruit abscission is a complex physiological process that is regulated by internal and environmental factors. During early development, apple fruit are exposed to extreme temperature fluctuations that are associated with premature fruit drop; however, its effect on fruit abscission is largely unknown. We hypothesized that fruit abscission is triggered by cold stress and investigated the molecular basis of premature fruit drop using RNA-seq and metabolomics data from apple fruit undergoing abscission following cold stress in the field. Genes responsive to abscisic acid signalling and cell wall degradation were up-regulated during abscission, consistent with the increased abscisic acid concentrations detected by LCMS. We performed ex-vivo cold shock experiments with excised tree subunits consisting of a branch, pedicel, and fruit. Abscission induction occurred in the cold-stressed subunits with concurrent up-regulation of abscisic acid biosynthesis (MdNCED1) and metabolism (MdCYP707A) genes, and ethylene biosynthesis (MdACS1) and receptor (MdETR2) genes in pedicel. Another key finding was the activation of cytoplasmic streaming of abscission-zone cells detected by electron microscopy. Our results provide a novel insight into the molecular basis of fruit abscission physiology in response to cold stress in apple.

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with this recombinant vector by the Agrobacterium mediated method and the integration of the target gene into the plant genome was confirmed by genomic PCR. The expression of rhVEGF165 in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF165 protein in the culture medium was 19 mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF165 was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF165 significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF165 could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter.