Apple Fruit Ripening Research Articles

Melatonin suppresses ethylene biosynthesis by inhibiting transcription factor MdREM10 during apple fruit ripening

Abstract Ethylene, a plant hormone, is essential for apple (Malus domestica) ripening. The precise molecular mechanism by which melatonin (MT) influences ethylene biosynthesis during apple fruit ripening remains unclear. This study found that exogenous MT treatment inhibited ethylene production and postponed apple fruit ripening. The endogenous MT content of apple fruits exhibited an inverse correlation with ethylene production during fruit ripening, suggesting that MT functions as a ripening suppressor in apple fruits. MT treatment suppressed the expression of key ethylene biosynthesis genes, MdACS1 and MdACO1, during apple fruit ripening. MT treatment decreased the expression levels of transcription factors MdREM10 and MdZF32. MdREM10 binds to the MdERF3 promoter, enhancing its expression and subsequently promoting MdACS1 transcription. Furthermore, MdREM10 directly bound to the MdZF32 promoter, promoting its transcription. MdZF32 directly bound to the MdACO1 promoter, inducing its expression. The findings suggested that MT suppresses ethylene biosynthesis and fruit ripening by inhibiting MdREM10, which indirectly promotes MdACS1 transcription via MdERF3 upregulation, and MdACO1 transcription via MdZF32 upregulation.

An InDel variant in the promoter of the NAC transcription factor MdNAC18.1 plays a major role in apple fruit ripening.

A complex regulatory network governs fruit ripening, but natural variations and functional differentiation of fruit ripening genes remain largely unknown. Utilizing a genome-wide association study (GWAS), we identified the NAC family transcription factor MdNAC18.1, whose expression is closely associated with fruit ripening in apple (Malus × domestica Borkh.). MdNAC18.1 activated the transcription of genes related to fruit softening (Polygalacturonase, PG) and ethylene biosynthesis (1-aminocyclopropane-1-carboxylic acid synthase, ACS), thereby promoting fruit ripening of apple and tomato (Solanum lycopersicum). There were two single-nucleotide polymorphisms (SNP-1,545 and SNP-2,002) and a 58-bp insertion-deletion (InDel-58) in the promoter region of MdNAC18.1. Among these, InDel-58 serves as the main effector in activating the expression of MdNAC18.1 and driving fruit ripening. InDel-58 determines the binding affinity of the class D MADS-box protein AGAMOUS-LIKE 11 (MdAGL11), a negative regulator of fruit ripening. The InDel-58 deletion in the early-ripening genotype reduces the inhibitory effect of MdAGL11 on MdNAC18.1. Moreover, MdNAC18.1 and its homologous genes originated from a common ancestor across 61 angiosperms, with functional diversification attributed to tandem replications that occurred in basal angiosperms. In summary, our study revealed how a set of natural variations influence fruit ripening and explored the functional diversification of MdNAC18.1 during evolution.

Impacts of 1-methylcyclopropene (1-MCP) and ethephon treatments on expression of aquaporin (AQP) genes during apple fruit ripening

The main aim of the present study was to determine the effects of ethephon (ET) and 1-methylcyclopropene (1-MCP) treatments on expression of aquaporin (AQP) genes during apple fruit ripening. The harvested apples were treated with 1-MCP (1 μL L−1) and ET (0.2 mmol L−1), respectively, and then ripened at room temperature (20 ± 1 °C). Our results suggested that 1-MCP application reduced weight loss, total soluble solids (TSS) content, respiration rate and ethylene production, maintained flesh firmness, and slowed down the increase in content of soluble sugar and the decrease in content of titratable acidity (TA) and dry matter concentration (DMC) in flesh and peel during ripening compared with the control, while ET treatment had the opposite effects. Quantitative real-time PCR (qPCR) analysis indicated that MdAQPs expression was differentially regulated by both treatments in flesh and peel during ripening. Expression of MdSIP1;2 and MdNIP5;3 in flesh and MdPIP1;3 and MdPIP2;7 in peel was generally up-regulated by both treatments, while MdTIP4;2 expression in peel was down-regulated by both treatments during ripening. Expression of MdNIP1;1, MdNIP2;1 and MdTIP1;1 in flesh and MdTIP2;2, MdTIP3;1 and MdNIP5;1 in peel was differentially down-regulated by ET treatment while differentially up-regulated by 1-MCP application during ripening. Transcript levels of MdTIP2;2, MdPIP1;2, MdPIP2;4, MdPIP2;3 and MdNIP5;2 in flesh and MdNIP2;1, MdSIP2;2, MdSIP1;2 and MdNIP1;1 in peel were differentially up-regulated by ET application while differentially reduced by 1-MCP application during ripening. After transient over-expression of MdERF2, MdAQPs exhibited varying expression profiles in both tissues during ripening. The results indicated that MdAQPs expression was modulated by ethylene signal in ripening apple fruit.

Epigenomic mechanism regulating the quality and ripeness of apple fruit with differing harvest maturity.

Harvest maturity significantly affects the quality of apple fruit in post-harvest storage process. Although the regulatory mechanisms underlying fruit ripening have been studied, the associated epigenetic modifications remain unclear. Thus, we compared the DNA methylation changes and the transcriptional responses of mature fruit (MF) and immature fruit (NF). There were significant correlations between DNA methylation and gene expression. Moreover, the sugar contents (sucrose, glucose, and fructose) were higher in MF than in NF, whereas the opposite pattern was detected for the starch content. The expression-level differences were due to DNA methylations and ultimately resulted in diverse fruit textures and ripeness. Furthermore, the higher ethylene, auxin, and abscisic acid levels in MF than in NF, which influenced the fruit texture and ripening, were associated with multiple differentially expressed genes in hormone synthesis, signaling, and response pathways (ACS, ACO, ZEP, NCED, and ABA2) that were regulated by DNA methylations. Multiple transcription factor genes involved in regulating fruit ripening and quality via changes in DNA methylation were identified, including MIKCC-type MADS-box genes and fruit ripening-related genes (NAP, SPL, WRKY, and NAC genes). These findings reflect the diversity in the epigenetic regulation of gene expression and may be relevant for elucidating the epigenetic regulatory mechanism underlying the ripening and quality of apple fruit with differing harvest maturity.

Transcriptome Analysis of White- and Red-Fleshed Apple Fruits Uncovered Novel Genes Related to the Regulation of Anthocyanin Biosynthesis.

The red flesh coloration of apples is a result of a biochemical pathway involved in the biosynthesis of anthocyanins and anthocyanidins. Based on apple genome analysis, a high number of regulatory genes, mainly transcription factors such as MYB, which are components of regulatory complex MYB-bHLH-WD40, and several structural genes (PAL, 4CL, CHS, CHI, F3H, DFR, ANS, UFGT) involved in anthocyanin biosynthesis, have been identified. In this study, we investigated novel genes related to the red-flesh apple phenotype. These genes could be deemed molecular markers for the early selection of new apple cultivars. Based on a comparative transcriptome analysis of apples with different fruit-flesh coloration, we successfully identified and characterized ten potential genes from the plant hormone transduction pathway of auxin (GH3); cytokinins (B-ARR); gibberellins (DELLA); abscisic acid (SnRK2 and ABF); brassinosteroids (BRI1, BZR1 and TCH4); jasmonic acid (MYC2); and salicylic acid (NPR1). An analysis of expression profiles was performed in immature and ripe fruits of red-fleshed cultivars. We have uncovered genes mediating the regulation of abscisic acid, salicylic acid, cytokinin, and jasmonic acid signaling and described their role in anthocyanin biosynthesis, accumulation, and degradation. The presented results underline the relationship between genes from the hormone signal transduction pathway and UFGT genes, which are directly responsible for anthocyanin color transformation as well as anthocyanin accumulation during apple-fruit ripening.

Expression Analysis of Metacaspase (MC) Gene Family in Response to Ethylene Signal During Apple Fruit Ripening

Expression Analysis of Metacaspase (MC) Gene Family in Response to Ethylene Signal During Apple Fruit Ripening

Optimization of apple fruit flavor by MdVHP1-2 via modulation of soluble sugar and organic acid accumulation

For fleshy fruits, the content and ratio of organic acids and soluble sugars are key factors for their flavor. Therefore, a better understanding of soluble sugar and organic acid accumulation in vacuoles is essential to the improvement of fruit quality. Vacuolar-type inorganic pyrophosphatase (V-PPase) has been found in various plants with crucial functions based on the hydrolysis of PPi. However, the effects of V-PPase on the soluble sugar and organic acid accumulation in apple fruit remain unclear. In this study, MdVHP1-2, a V-PPase protein in the vacuolar membrane, was identified. The results showed a positive correlation between the expression of MdVHP1-2 and the sugar/acid ratio during ripening of apple fruits. A series of transgenic analyses showed that overexpression of MdVHP1-2 significantly elevated the contents of soluble sugars and organic acids as well as the sugar/acid ratio in apple fruits and calli. Additionally, transient interference induced by MdVHP1-2 expression inhibited the accumulation of soluble sugars and organic acids in apple fruits. In summary, this study provides insight into the mechanisms by which MdVHP1-2 modulates fruit flavor through mediation of soluble sugar and organic acid accumulation, thereby facilitating improvement of the overall quality of apple and other fruits.

Role of an ATP binding cassette (ABC) transporter MdABCI17 in the anthocyanin accumulation of apple

As a key colorant and antioxidant in human diets, anthocyanin is biosynthesized in cytosols and transported to vacuoles and other sites for storage. Therefore, the tonoplast transporters of anthocyanin play a key role in the anthocyanin storage and accumulation of plants. In this study, an ATP-binding cassette (ABC) transporter MdABCI17, which is located in the tonoplast, was identified. There was a positive correlation between the MdABCI17 expression and the anthocyanin accumulation during apple fruit ripening. Transgenic analysis showed that up-regulation of MdABCI17 expression in apple fruit and calli significantly increased anthocyanin content. Additionally, the expression of multiple anthocyanin biosynthesize genes, such as MdANS, MdCHS, MdCHI, MdDFR, and MdUFGT, was remarkably upregulated in apple fruit and calli with upregulated expression of MdABCI17, further demonstrating the critical role of MdABCI17 in the anthocyanin accumulation of apple. In summary, the expression of MdABCI17 had positive effects on anthocyanin accumulation, which will be useful for improving the quality of apple and other plants.

Cultivar differences in the hormonal crosstalk regulating apple fruit development and ripening: Relationship with flavour components and postharvest susceptibility to Penicillium expansum

The hormonal interplay during the on-tree development and ripening of three apple cultivars with known differences in their postharvest ripening patterns was studied, at the biochemical and targeted gene expression level, along with characterizing the changes in main sugars, acids, phenylpropanoids and volatile organic compounds (VOCs). Our findings suggest that in ‘Royal Gala’ and ‘Opal®’ apples, a peak in indole 3-acetic acid (IAA) seems necessary to activate ethylene metabolism, being its intensity proportional to the ethylene production. The interplay between IAA and ethylene appears to be mediated by MdARF5, responsible for activating the expression of MdACS3 and triggering ethylene metabolism. On the other hand, the lack of ethylene production observed in ‘Granny Smith’ apples was likely related to the absence of an IAA peak and possibly caused by the over activation of IAA conjugation mechanisms leading to a greater accumulation of IAA inactive conjugates such as indole-3-acetyl-aspartate (IAAsp). Abscisic acid (ABA) accumulation was only observed in cultivars with the ability to accumulate sucrose and produce ethylene, suggesting a possible crosstalk among those hormones and sucrose in orchestrating apple on-tree ripening. While differences in hormone levels among cultivars led to noticeable differences in some specific VOCs, no evident associations were found between hormone changes and the accumulation or degradation of monosaccharides, organic acids or phenolic compounds during fruit development and ripening. Likewise, no clear relationship was found between the fruit susceptibility to blue mould and hormonal levels yet certain specific biochemical compounds (i.e., procyanidins and sucrose) could be acting as a source of resistance or susceptibility, respectively, to blue mould development. Overall, understanding the cultivar specific hormonal regulation of apple on-tree ripening provides valuable insights to optimize fruit quality at the time of harvest as well as to develop strategies for improved postharvest management.

Heterologous Overexpression of Apple MdKING1 Promotes Fruit Ripening in Tomato.

Fruit ripening is governed by a complex regulatory network, and ethylene plays an important role in this process. MdKING1 is a γ subunit of SNF1-related protein kinases (SnRKs), but the function was unclear. Here, we characterized the role of MdKING1 during fruit ripening, which can promote fruit ripening through the ethylene pathway. Our findings reveal that MdKING1 has higher expression in early-ripening cultivars than late-ripening during the early stage of apple fruit development, and its transcription level significantly increased during apple fruit ripening. Overexpression of MdKING1 (MdKING1 OE) in tomatoes could promote early ripening of fruits, with the increase in ethylene content and the loss of fruit firmness. Ethylene inhibitor treatment could delay the fruit ripening of both MdKING1 OE and WT fruits. However, MdKING1 OE fruits turned fruit ripe faster, with an increase in carotenoid content compared with WT. In addition, the expression of genes involved in ethylene biosynthesis (SlACO1, SlACS2, and SlACS4), carotenoid biosynthesis (SlPSY1 and SlGgpps2a), and fruit firmness regulation (SlPG2a, SlPL, and SlCEL2) was also increased in the fruits of MdKING1 OE plants. In conclusion, our results suggest that MdKING1 plays a key role in promoting tomato fruit ripening, thus providing a theoretical basis for apple fruit quality improvement by genetic engineering in the future.

Concentration-dependent impacts of exogenous methyl jasmonate (MeJA) on chlorophyll degradation of apple fruit during ripening

Although jasmonates (JAs) have been proved to regulate degreening of apple fruit, the relevant molecular mechanisms have not yet been clarified. Previous reports revealed that the effects of JAs on degreening of harvested horticultural crops might be dependent on concentration applied. Whether a similar result can be obtained from apple fruit is still unclear. This research explored the impacts of different concentrations of methyl jasmonate (MeJA) treatment on chlorophyll (Chl) degradation of postharvest apple fruit. Apples (Malus × domestica Borkh.) of commercial maturity were treated with 10 or 1500 µM MeJA and then stored at 20 ± 0.5 °C. The results indicated that exogenous 10 µM MeJA delayed degreening and ripening of apple fruit, whereas 1500 µM MeJA treatment had the opposite effects. Treatment with 10 µM MeJA alleviated Chl-oxidation by reducing Chl-peroxidase (POX) activity and reactive oxygen species (ROS) levels in chloroplast of apple peel during ripening, whereas 1500 µM MeJA treatment had the opposite effects. Activities of Magnesium-dechelatase (MDcase) and pheophytinase (PPH) in apple peel were differentially suppressed by 10 µM MeJA treatment while promoted by 1500 µM MeJA treatment during ripening. Application of 10 µM MeJA delayed the peak of endogenous jasmonic acid (JA) content, reduced jasmonate resistant 1 (JAR1) content and allene oxide synthase (AOS) activity, and differentially down-regulated expression of MdNYC3, MdNYE1a, MdPPH1, MdPAO6, MdPAO8, MdRCCR2 and MdMYC2a in apple peel during ripening, whereas opposite results were obtained from the 1500 µM MeJA-treated apple fruit. At the late ripening stage, MdHCAR expression in apple peel was promoted by 10 µM MeJA treatment while reduced by 1500 µM MeJA treatment. Transcript levels of MdNYC1 and MdNOL2 were generally reduced by both treatments at the late ripening stage. Transient overexpression of MdMYC2a accelerated Chl degradation of apple peel by promoting PPH and MDcase activities and enhancing expression of the above Chl catabolic genes (CCGs), except MdHCAR whose expression was down-regulated by it during ripening. The AOS activity, JA and JAR1 contents in apple peel were increased after transient overexpression of MdMYC2a during ripening. These data indicated that the PAO pathway and Chl-oxidation were regulated by JA signal in apple peel during ripening, and exogenous MeJA regulated Chl degradation of apple fruit in a concentration-dependent manner.

A complex network regulating malate contents during fruit ripening in climacteric fruits.

This article is a Commentary on Wang et al. (2023), 239: 1014–1034.

LED white light-activated transcription factor MdHY5 inhibits ethylene biosynthesis during apple fruit ripening

Ethylene is one of the most important plant hormones involved in the climacteric fruit ripening process. Apple is a climacteric fruit, and its ripening process is regulated by ethylene. The effect of light-emitting diode (LED) light on plant growth and development has been extensively investigated; however, it remains unclear how LED light affects apple fruit ripening. Herein, we observed that the ripening of apple fruit treated with eight kinds of LED light was significantly inhibited compared with that of the dark control, with inhibition by white light being the most significant. Furthermore, the optimal light intensity for the inhibition of ethylene production was 20 μmol·m−2s−1, with an optimal light duration of 12 h per day. LED white light treatment enhanced the expression of two ELONGATED HYPOCOTYL 5 (HY5) genes, MdHY5L and MdHY5S, which encode transcription factors involved in the light signaling pathway. These two HY5s inhibited the transcription of MdACS1, a crucial rate-limiting enzyme in the ethylene synthesis pathway, by directly binding to its promoter. Collectively, these findings imply that LED white light induces the expression of MdHY5L and MdHY5S and delays the expression of the ethylene synthesis-related gene MdACS1 to prevent fruit ripening. This study preliminarily elucidates the molecular mechanism underlying LED white light-induced inhibition of ethylene synthesis in apple fruit, providing an important theoretical basis for studying how to improve the storage ability and prolong the shelf life of apple fruit.

Exogenous Ca2+ promotes transcription factor phosphorylation to suppress ethylene biosynthesis in apple.

Ethylene biosynthesis in apple (Malus domestica) fruit can be suppressed by calcium ions (Ca2+) during storage; however, the underlying mechanisms are unclear. In this study, we identified the apple transcription factor MCM1-AGAMOUS-DEFICIENS-SRF5 (MdMADS5), which functions as a transcriptional activator of the ethylene biosynthesis-related gene 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1), a partner of the calcium sensor CALCIUM-DEPENDENT PROTEIN KINASES7 (MdCDPK7). Ca2+ promoted the MdCDPK7-mediated phosphorylation of MdMADS5, which resulted in the degradation of MdMADS5 via the 26S proteasome pathway. MdCDPK7 also phosphorylated 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE1 (MdACO1), the key enzyme in ethylene biosynthesis, leading to MdACO1 degradation and inhibition of ethylene biosynthesis. Our results reveal that Ca2+/MdCDPK7-MdMADS5 and Ca2+/MdCDPK7-MdACO1 are involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. These findings provide insights into fruit ripening, which may lead to the development of strategies for extending the shelf life of fruit.

1-Methylcyclopropene (1-MCP) treatment differentially mediated expression of vacuolar processing enzyme (VPE) genes and delayed programmed cell death (PCD) during ripening and senescence of apple fruit

• 1-MCP reduced VPE activity and delayed vacuolar-mediated PCD in apple fruit. • MdVPE s had varying expression patterns in response to 1-MCP during ripening. • MdVPE s were differentially expressed after transient over-expression of MdERF2. The main purpose of this research was to evaluate the impacts of 1-methylcyclopropene (1-MCP) treatment on vacuolar processing enzyme ( VPE ) genes and programmed cell death (PCD) during ripening and senescence of apple fruit ( Malus × domestica Borkh.). The ethylene antagonists 1-MCP (1 μL L −1 ) was applied to apples after harvest. Both treated and control fruit were stored at 20 ± 2°C. The data showed that 1-MCP treatment maintained flesh firmness, delayed climacteric peaks of respiration and ethylene, reduced VPE activity, relative electrical conductivity (REC), malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation during ripening. Transmission electron microscopy (TEM) and DNA ladder assay revealed that 1-MCP delayed vacuolar-mediated PCD in ripening apple fruit. Generally, expression of MdVPEγ1, MdVPEγ2, MdVPEδ4 and MdVPEδ7 was down-regulated by 1-MCP treatment, while expression of MdVPEα4 and MdVPEβ2 was enhanced by it during ripening compared with controls. 1-MCP treatment differentially reduced expression of MdVPEα1, MdVPEα2 and MdVPEα5 at the early ripening stage but promoted their expression at the late ripening stage, while it had the opposite effects on MdVPEβ1, MdVPEβ3 and MdVPEγ4 expression. After transient overexpression of MdERF2, MdVPEs displayed varying expression patterns, among which MdVPEα2 transcripts increased greater than the other genes. Our results indicated that MdVPEs expression and vacuolar-mediated PCD were mediated by ethylene signal during ripening and senescence of apple fruit.

Impact of the B-Plus white pearl (Belyi Zhemchug) preparation on the spring frost tolerance, yield and quality of apple crops

In 2021–2022, experiments were conducted at the Laboratory of Fruit Crop Resistance Physiology, All-Russia Institute for Fruit Crops Breeding (VNIISPK), to investigate the action of the “White Pearl Antifreeze” (Belyi Zhemchug Antifriz) phytomodulator and the “White Pearl Drip Ca + Mg” (Belyi Zhemchug Drip Ca+Mg) phytocorrector produced by the AgroPlus Group of Companies, LLC. Experimental and control plots were characterized by dark-gray forest soils with a humus content of 3-4 % and a humus horizon thickness of 30–35 cm. Apple trees cv. Sinap Orlovsky (VNIISPK) grafted on semi-dwarf rootstock 54-118 were planted in 2013 according to a 6 x 3 m scheme. Natural grassing was used in row spacings; herbicides were applied around the seedlings. The experimental design included: 1) control (water treatment) and 2) foliar treatment with a 1 % solution of “White Pearl Antifreeze” + a 1 % solution of “White Pearl Drip Ca+Mg”. The experiment was conducted in three replications with five observed trees in each. Our aim was to assess the effect of the mentioned preparations on the spring frost tolerance, yield, and quality of apple fruit under the weather conditions of 2021. Foliar treatments with the studied preparations reduced the frost damage to Sinap Orlovsky apple buds by 6.3 % at -3.5 °С and by 10.4 % at -4 °С. The conducted summer foliar treatments with the organomineral mixture under study intensified the growth and ripening of apple fruit. The improved protein-carbohydrate metabolism, water regime, photosynthetic activity, and donor-acceptor leaf–fruit relations increased the fruit weight by 10 g and the yield by 1.8 times. The conclusion is made that the B-PLUS “White Pearl Antifreeze” preparation exhibits a complex effect in terms of increasing plant tolerance to low-temperature spring damage, thus improving the yield and quality of apple crops.

Comparative transcriptome analysis of the climacteric of apple fruit uncovers the involvement of transcription factors affecting ethylene biosynthesis

Apple (Malus domestica) fruit generally undergoes a climacteric. During its ripening process, there is a peak in ethylene release and its firmness simultaneously decreases. Although more in-depth research into the mechanism of climacteric-type fruit ripening is being carried out, some aspects remain unclear. In this study, we compared the transcriptomes of 0-Pre and 15-Post (pre- and post-climacteric fruit), and 15-Post and 15-MCP [fruit treated with 1-MCP (1-methylcyclopropene)]. Various transcription factors, such as MADS-box, ERF, NAC, Dof and SHF were identified among the DEGs (differential gene expressions). Furthermore, these transcription factors were selected for further validation analysis by qRT-PCR. Moreover, yeast one hybrid (Y1H), β-glucuronidase (GUS) transactivation assay and dual-luciferase reporter assay showed that MdAGL30, MdAGL104, MdERF008, MdNAC71, MdDof1.2, MdHSFB2a and MdHSFB3 bound to MdACS1 promoter and directly regulated its transcription, thereby regulating ethylene biosynthesis in apple fruit. Our results provide useful information and new insights for research on apple fruit ripening.

Phosphorylation of MdCYTOKININ RESPONSE FACTOR4 suppresses ethylene biosynthesis during apple fruit ripening.

The plant hormone ethylene plays a central role in the ripening of climacteric fruits, such as apple (Malus domestica). Ethylene biosynthesis in apple fruit can be suppressed by calcium ions (Ca2+); however, the underlying mechanism is largely unknown. In this study, we identified an apple APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor, MdCYTOKININ RESPONSE FACTOR4 (MdCRF4), which functions as a transcriptional activator of ethylene biosynthesis- and signaling-related genes, including Md1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1) and MdETHYLENE-RESPONSIVE FACTOR3 (MdERF3), as a partner of the calcium sensor, calmodulin. Ca2+ promoted the Ca2+/CaM2-mediated phosphorylation of MdCRF4, resulting in MdCRF4 recognition by the E3 ubiquitin ligase MdXB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (MdXBAT31), and consequently its ubiquitination and degradation via the 26S proteasome pathway. This in turn resulted in lower expression of MdACS1 and MdERF3 and reduced ethylene biosynthesis. Transiently overexpressing various MdCRF4 proteins with specific mutated phosphorylation sites revealed that the phosphorylation state of MdCRF4 affects the ripening of apple fruit. The results reveal that a Ca2+/CaM-MdCRF4-MdXBAT31 module is involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. This provides insights into fruit ripening that may result in strategies for extending fruit shelf life.

Integrated multi-omic analysis of fruit maturity identifies biomarkers with drastic abundance shifts spanning the harvest period in ‘Royal Gala’ apple

Accurate assessment of apple fruit maturity at harvest is required since fruit harvested too early or too late are susceptible to physiological disorders or excessive softening during subsequent storage. Biological markers of early fruit maturity allow forecasting of optimal harvest time, contributing significant industry value through more accurate management of harvest logistics. This study investigated the changes in cortex of apple (Malus x domestica ‘Royal Gala’) fruit at four harvests: very early (H1), early (H2), commercial (H3) and late (H4), using a combination of transcriptomics, metabolomics, hormone abundances and enzyme activity profiles. Harvest times were discriminated based on several sets of variates, showing that metabolism was very active within this short time period. Good discrimination between H1 and H2 and between H2 and H3 was observed in the declining abundance of a range of photosystem transcripts and the increasing abundance of early ripening markers. Degradation of the photosynthetic apparatus was correlated with ethylene production. Multi-omics analysis using mixOmics identified groups of variates whose abundance declined or increased during the harvest period, and strong correlations between components of different pathways were evident. We identify a suite of biomarkers, including Chl a/b binding protein of LHCII, Xyloglucan glycosyltransferase 5, PG1, ACO1, internal ethylene concentration and starch pattern index, for orchardists to accurately predict harvest time several weeks in advance, thus providing time to mobilise the necessary logistical resources.

Genome-Wide Identification and Expression Profiling Reveal the Potential Functions of the SWEET Gene Family during the Sink Organ Development Period in Apple (Malus × domestica Borkh.)

Sugars Will Eventually be Exported Transporters (SWEETs) play important roles during plant growth and development. Bioinformatics revealed 27 SWEET genes in the apple (Malus × domestica Borkh.) genome and classified them into four clades. These genes are unevenly located on 12 chromosomes, and 8 tandem-repeat genes and 18 fragment-repeat genes are present in the MdSWEET family. PlantCARE-based prediction of cis-acting elements of the MdSWEET promoter suggested that most MdSWEETs may be expressed specifically in the phloem and respond to hormones and stresses. qRT-PCR results showed that expression patterns of MdSWEETs displayed pronounced differences in different tissues/organs and different stages of apple fruit development. MdSWEET5, MdSWEET7, and MdSWEET21 were strongly expressed in mature leaves, MdSWEET2, MdSWEET12, MdSWEET13, MdSWEET14, MdSWEET17, and MdSWEET22 were highly expressed in peduncles, MdSWEET4 was highly expressed in young leaves, MdSWEET3, MdSWEET11, MdSWEET15, MdSWEET16, MdSWEET19, MdSWEET24, and MdSWEET25 were highly expressed in different parts of flowers, and MdSWEET1, MdSWEET6, MdSWEET8, MdSWEET9, MdSWEET10, MdSWEET18, MdSWEET20, MdSWEET23, and MdSWEET26 were strongly expressed in fruits. MdSWEET8 showed higher expression in the early stage of fruit development, MdSWEET1, MdSWEET10, and MdSWEET27 were highly expressed in the middle stage of fruit development, and MdSWEET6, MdSWEET9, MdSWEET18, MdSWEET20, MdSWEET23, and MdSWEET26 were sharply upregulated in the late developmental period. Our study could facilitate SWEET functional analysis in different tissue/organs and in sugar accumulation throughout the development and ripening of apple fruits. These findings provide potential opportunities to increase sugar accumulation in fruit, thereby improving fruit quality and yield.