Activities, Quantitative Changes and Subcellular Localization of α-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α_Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α_amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple ( Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α_amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular_localization studies via immunogold electron_ microscopy technique showed that α_amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid_distributed pattern of α_amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α_amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α_amylase is involved in starch hydrolysis in plastids of the fruit cells.

In Vivo Tissue-dependent Abscisic Acid Specific-binding Proteins with High Affinity in Cytosol of Developing Apple Fruits

The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sites localized in cytosol were identified and characterized in the flesh of developing apple ( Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the subcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in H-3-ABA binding medium, the flesh tissue discs were directly in vivo incubated in H-3-ABA binding medium, a high ABA binding activity to the cytosolic fraction isolated from these tissue discs was detected. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA binding needs a living state of tissue. The in vivo tissue-dependent binding sites were shown to possess protein nature with both active serine residua and thiol-group of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting gave evidence of two different classes of ABA binding proteins, one with a higher affinity ( Kd = 2.9 nmol/L) and the other with lower affinity ( Kd = 71.4 nmol/L). Phaseic acid, 2-trans-4-trans-ABA or cis-trans-(-)-ABA had substantially no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.

Using an improved lightweight YOLOv8 model for real-time detection of multi-stage apple fruit in complex orchard environments

For the purpose of monitoring apple fruits effectively throughout the entire growth period in smart orchards.A lightweight model named YOLOv8n-ShuffleNetv2-Ghost-SE was proposed.The ShuffleNetv2 basic modules and down-sampling modules were alternately connected,replacing the Backbone of YOLOv8n model.The Ghost modules replaced the Conv modules and the C2fGhost modules replaced the C2f modules in the Neck part of the YOLOv8n.ShuffleNetv2 reduced the memory access cost through channel splitting operations.The Ghost module combined linear and non-linear convolutions to reduce the network computation cost.The Wise-IoU(WIoU)replaced the CIoU for calculating the bounding box regression loss,which dynamically adjusted the anchor box quality threshold and gradient gain allocation strategy,optimizing the size and position of predicted bounding boxes.The Squeeze-and-Excitation(SE)was embedded in the Backbone and Neck part of YOLOv8n to enhance the representation ability of feature maps.The algorithm ensured high precision while having small model size and fast detection speed,which facilitated model migration and deployment.Using 9652 images validated the effectiveness of the model.The YOLOv8n-ShuffleNetv2-Ghost-SE model achieved Precision of 94.1%,Recall of 82.6%,mean Average Precision of 91.4%,model size of 2.6 MB,parameters of 1.18 M,FLOPs of 3.9 G,and detection speed of 39.37 fps.The detection speeds on the Jetson Xavier NX development board were 3.17 fps.Comparisons with advanced models including Faster R-CNN,SSD,YOLOv5s,YOLOv7‑tiny,YOLOv8s,YOLOv8n,MobileNetv3_small-Faster,MobileNetv3_small-Ghost,ShuflleNetv2-Faster,ShuflleNetv2-Ghost,ShuflleNetv2-Ghost-CBAM,ShuflleNetv2-Ghost-ECA,and ShuflleNetv2-Ghost-CA demonstrated that the method achieved smaller model and faster detection speed.The research can provide reference for the development of smart devices in apple orchards.

Replanting Affects the Tree Growth and Fruit Quality of Gala Apple

Apple replant disease（ARD） causes the inhibition of root system development, stunts tree growth and so on. To further investigate the effects of ARD on apple fruits, a 25-year-old apple orchard was remediated to establish a replant orchard between November 2008 and March 2009. A rotational cropping orchard was established on an adjacent wheat field. The cultivar and rootstock-scion combination used in the newly established orchards was Royal Gala/M26/Malus hupehensis Rehd. Ripe fruits were collected in mid-August 2011 and mid-August 2012, meanwhile, the following indices were measured： yield per plant; fruit weight; the fruit shape index; the contents of anthocyanin, carotenoid and chlorophyll; the soluble sugar content in the flesh; titratable acid; the sugar-acid ratio; firmness; and aroma components; apple plant ground diameter, plant height increment and the total length of the current-year shoots. The results showed that compared to rotational cropping, continuous cropping yielded statistically significant reductions in fruit weight and yield per plant of 39.8 and 76.5%, respectively. However, there were no changes in the fruit shape index. The anthocyanin and carotenoid contents decreased by 81.7 and 37.7%, respectively, while the chlorophyll content increased by 251.0%. All of these differences in content were statistically significant. The soluble sugar levels and sugar-acid ratio decreased by 25.4 and 60.9%, respectively, but the titratable acid levels and fruit firmness increased by 90.9 and 42.8%, respectively. Ten of the most important esters contributing to the apple aroma were analyzed, and the following changes were observed： hexyl acetate, butyl acetate, hexyl butyrate, acetate-2-methyl butyl, 2-methyl-hexyl butyrate, amyl acetate, butyl butyrate, 2-methyl-butyl butyrate, hexyl propionate and hexyl hexanoate decreased by 25.5, 78.4, 89.1, 55.5, 79.5, 77.2, 86.8, 69.9, 61.2, and 68.1%, respectively. The contents of three other aroma components,（E）-2-hexenal, hexanal and 1-hexanol, significantly increased. Eight characteristic aroma components were found in the rotational cropping fruits： hexyl acetate, butyl acetate, acetate-2-methyl butyl, 2-methyl-hexyl butyrate, amyl acetate, 2-methyl- butyl butyrate, hexyl acetate and hexyl propionate. There were four characteristic ester components（hexyl acetate, butyl acetate, acetate-2-methyl butyl, 2-methyl-hexyl butyrate） and two characteristic aldehyde aroma components（（E）-2-hexenal and hexanal） in the continuous cropping fruits. Compared with the rotational cropping fruits, four characteristic ester components were declined and two characteristic aldehyde aroma components were increased. Compared with the control, replanted apple plant ground diameter, plant height increment and the total length of the current-year shoots were reduced by 27.6, 40.6 and 72.2%, respectively.

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.

Identification, through transcriptome analysis, of transcription factors that regulate anthocyanin biosynthesis in different parts of red-fleshed apple 'May' fruit

Anthocyanins, a class of flavonoid compounds that provide pigmentation to plant organs, are beneficial components of the human diet and there is an interest in understanding the regulation of their formation in crop plants. The apple cultivar ‘May’ represents an excellent model for the study of anthocyanin metabolism due to the spatial color pattern of its flesh, which transitions sequentially from red to white to red again,from the outside to the inside of the apple fruit. To screen the transcription factors involved in the anthocyanin pathway, transcriptomes from differently colored sections of ‘May’ fruits were analyzed by RNA sequencing. Kyoto Encyclopedia of Genes and Genomes(KEGG) database was used to analyse the gene expression data. The results suggested differentially expressed genes(DEGs) that are related to phytohormones are involved in regulating anthocyanin biosynthesis. K-means clustering analysis revealed 167 common DEGs between different sections of fruit with the same expression pattern as candidates for regulating anthocyanin synthesis. Further analysis showed that nine of the 167 DEGs were annotated as transcription factors and quantitative real time PCR(qRT-PCR) confirmed that their expression was obviously higher in red regions of the fruit, consistent with their roles as hub genes that regulate anthocyanin synthesis. This study provides valuable results for future studies of anthocyanin synthesis in apple fruit.

Combined effect of Bacillus siamensis and chlorogenic acid on maintenance of quality and control of disease in stored wax apple fruit

Objectives:The purpose of this study was to evaluate the effect of combined postharvest use of Bacillus siamensis strain and chlorogenic acid on quality maintenance and disease control in wax apple fruit.Materials and Methods:Wax apple fruit were treated with Bacillus siamensis strain(N1),chlorogenic acid(CHA)and N1+CHA and preserved at 25℃ for 12 d.The appearance and quality parameters were evaluated,along with the disease index,content of total soluble solids(TSS),total acid(TA),vitamin C(Vc),total phenolic,and flavonoids during cold storage.Meanwhile,the activities ofβ-1,3-glucanase(GLU),phenylalanine ammonialyase(PAL),polyphenol oxidase(PPO),and peroxidase(POD)were determined.Furthermore,the transcriptome and the expression level of key defense enzyme genes were analyzed by RNA-seq and real-time quantitative reverse transcription polymerase chain reaction.Results:N1+CHA treatment significantly lowered DI and delayed fruit quality deterioration by slowing TSS and TA loss and enhancing anti-oxidant capacity,including Vc,total phenolic,and flavonoids content.Meanwhile,the activities of GLU,PAL,PPO,and POD were dramatically increased by N1+CHA treatment.Additionally,N1+CHA treatment modulated several metabolic pathways,including those involved in planthormone signal transduction and plant–pathogen interaction.The expression levels of key defense enzyme genes were significantly upregulated in stored wax apple fruit by the N1+CHA treatment,which were well coincided with the transcriptome data.Conclusions:The combined use of N1+CHA significantly prevents disease and maintains fruit quality of wax apple during storage.These findings indicate that it could serve as a promising biological technique for preserving wax apple fruit.

Relationship Between Stimulated Ethylene Production and Alternative Respiration Pathway in "Royal Gala" Apple Fruit

Endogenous ethylene production and alternative oxidase (AOX) protein expression in 'Royal Gala' apple fruits were investigated after treatments with cold ( 0℃ for 1 week) and heat ( 38℃ for 1 h). A monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum was used to identify the AOX protein in apple fruits. The molecular mass of AOX in 'Royal Gala' apple fruits is approximately 38 kDa, similar to those reported in tobacco and tomato. The cold treatment depressed the release of endogenous ethylene production before the climacteric ethylene production and obviously induced the expression of AOX protein expression. The heat treatment had the opposite effects on the ethylene production and AOX protein expression. In addition, the climax of endogenous ethylene production preceded the maximum AOX expression after the cold temperature treatment. It is therefore proposed that in climacteric fruits the production of induced ethylene is not coordinated with the level of AOX protein.

Effects of 1-MCP on Ethylene Synthesis and Metabolismof Apple During Storage

Red Fuji apple(Malus domestica Borkh var. Red Fuji)fruits were used to study the effect of 1-MCP on ethylene biosynthesis metabolism during storage. The results showed that 1-MCP maintained the firmness and inhibited the respiration rate, LOX activity and ethylene production rate of fruits. Further study indicated that 1-MCP inhibited ACS(ACC synthase)activity from the 15th day, increased ACC accumulation, and delayed the appearance of ACO(ACC oxidase)activity peak. The increase of protein kinase activity was also inhibited by 1-MCP during fruit ethylene climacteric time.

Key minerals influencing apple quality in Chinese orchard identified by nutritional diagnosis of leaf and soil analysis

We investigated the correlation between leaf/soil minerals and fruit quality in apple trees grown in orchards,with the ultimate goal of improving the latter.Leaf mineral nutrients;soil nutrients in the 0-20,20-40,and 40-60 cm layers;and fruit quality traits in 32 apple orchards in China were monitored for 2 years.Significant factors associated with fruit quality were identified via correlation analysis.An analysis of leaf data revealed that leaf nitrogen（N） and leaf magnesium（Mg） levels were extremely high in 75 and 89%of the orchards,respectively.In the Bohai Gulf region,94%of the orchards showed significantly higher values than the standard.The soil pH values of the orchards in eastern China like eastern Shandong or Liaoning were lower than 7.0,while the pH values in the Loess Plateau of northwestern China like Shaanxi were much higher than 7.Soil alkali-hydrolyzable N levels in 47%of the orchards were lower than the optimal level of 70 mg kg^-1.Generally,the soil alkali-hydrolyzable N levels of orchards in the Bohai Gulf region were significantly higher than those in the Loess Plateau region.The available P levels in the orchards of the Bohai Gulf region were up to three times higher than those of the Loess Plateau region.However,although the available potassium（K） in most orchards was sufficient（51.39-309.94 mg kg^-1）,leaf K content in 73%of the orchards was low,possibly due to fruit bagging or fruit overload.Approximately 63%of the orchards in Shandong and 29%of the orchards in Shannxi showed leaf Fe deficiencies.In the Loess Plateau,most orchards showed high leaf Ca levels,a strong correlation was observed between leaf and soil phosphorus/potassium（P/K）content and fruit organic acid content.The amounts of fruit soluble sugar or fructose were positively correlated with soil calcium/potassium（Ca/K） levels and leaf calcium/boron（Ca/B） levels in most orchards.The excessive leaf N levels caused by the extensive application of N fertilizers had a negative effect on fruit quality in most apple orchards in China.P,K,Ca,and B were key minerals associated with fruit quality.

Fruits of Labor

Soon apples will not only be for eating,but also for trading much like commodities such as gold,crude oil and stock indices.On December 22,2017,China started listing apple futures,the world’s first fresh fruit contract,on the Zhengzhou Commodity Exchange in Central China’s Henan Province.