Deleterious Effects of Heat Stress on the Tomato, Its Innate Responses, and Potential Preventive Strategies in the Realm of Emerging Technologies.

The tomato is a fruit vegetable rich in nutritional and medicinal value grown in greenhouses and fields worldwide. It is severely sensitive to heat stress, which frequently occurs with rising global warming. Predictions indicate a 0.2 °C increase in average surface temperatures per decade for the next three decades, which underlines the threat of austere heat stress in the future. Previous studies have reported that heat stress adversely affects tomato growth, limits nutrient availability, hammers photosynthesis, disrupts reproduction, denatures proteins, upsets signaling pathways, and damages cell membranes. The overproduction of reactive oxygen species in response to heat stress is toxic to tomato plants. The negative consequences of heat stress on the tomato have been the focus of much investigation, resulting in the emergence of several therapeutic interventions. However, a considerable distance remains to be covered to develop tomato varieties that are tolerant to current heat stress and durable in the perspective of increasing global warming. This current review provides a critical analysis of the heat stress consequences on the tomato in the context of global warming, its innate response to heat stress, and the elucidation of domains characterized by a scarcity of knowledge, along with potential avenues for enhancing sustainable tolerance against heat stress through the involvement of diverse advanced technologies. The particular mechanism underlying thermotolerance remains indeterminate and requires further elucidatory investigation. The precise roles and interplay of signaling pathways in response to heat stress remain unresolved. The etiology of tomato plants' physiological and molecular responses against heat stress remains unexplained. Utilizing modern functional genomics techniques, including transcriptomics, proteomics, and metabolomics, can assist in identifying potential candidate proteins, metabolites, genes, gene networks, and signaling pathways contributing to tomato stress tolerance. Improving tomato tolerance against heat stress urges a comprehensive and combined strategy including modern techniques, the latest apparatuses, speedy breeding, physiology, and molecular markers to regulate their physiological, molecular, and biochemical reactions.

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon.

Proanthocyanidins (PAs) are specialized metabolites that influence persimmon fruit quality. Normal astringent (A)-type and non-astringent (NA)-type mutants show significant variation in PA accumulation, but the influencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by DkDTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pattern of DkDTX5/MATE5 was correlated with PA accumulation in NA-type fruit. Upregulation and downregulation of DkDTX5/MATE5 promoted and inhibited PA accumulation, respectively, in the NA-type fruit. Interestingly, transporter assays of Xenopus laevis oocytes indicated that DkDTX5/MATE5 preferentially transported the PA precursors catechin, epicatechin, and epicatechin gallate, resulting in their increased ratios relative to the total PAs, which was the main source of variation in PA accumulation between the A-type and NA-type. The allele lacking Ser-84 in DkDTX5/MATE5 was identified as a dominantly expressed gene in the A-type and lost its transport function. Site-directed mutagenesis revealed that DkDTX5/MATE5 binds to PA precursors via Ser-84. These findings clarify the association between the transporter function of DkDTX5/MATE5 and PA variation, and can contribute to the breeding of new cultivars with improved fruit quality.

A Chromosome-Level Genome of 'Xiaobaixing' (Prunus armeniaca L.) Provides Clues to Its Domestication and Identification of Key bHLH Genes in Amygdalin Biosynthesis.

Apricot is a widely cultivated fruit tree of the drupe family, and its sweet/bitter kernel traits are important indicators of the quality and merchantability of apricots. The sweetness/bitterness traits were mainly determined by amygdalin content. However, the lack of high-quality genomes has limited insight into the traits. In this study, a high-quality genome of 'Xiaobaixing' was obtained by using single-molecule sequencing and chromosome-conformation capture techniques, with eight chromosomes of 0.21 Gb in length and 52.80% repetitive sequences. A total of 29,157 protein-coding genes were predicted with contigs N50 = 3.56 Mb and scaffold N50 = 26.73 Mb. Construction of phylogenetic trees of 15 species of Rosaceae fruit trees, with 'Xiaobaixing' differentiated by 5.3 Ma as the closest relative to 'Yinxiangbai'. GO functional annotation and KEGG enrichment analysis identified 227 specific gene families to 'Xiaobaixing', with 569 expansion-gene families and 1316 contraction-gene families, including the significant expansion of phenylalanine N-monooxygenase and ß-glucosidase genes associated with amygdalin synthesis, significant contraction of wild black cherry glucoside ß-glucosidase genes, amygdalin ß-glucosidase genes, and ß-glucosidase genes, and significant enrichment of positively selected genes in the cyanogenic amino acid metabolic pathway. The 88 bHLH genes were identified in the genome of 'Xiaobaixing', and ParbHLH66 (rna-Par24659.1) was found to be a key gene for the identification of sweet/bitter kernels of apricots. The amino acid sequence encoded by its gene is highly conserved in the species of Prunus mume, Prunus dulcis, Prunus persica, and Prunus avium and may be participating in the regulation of amygdalin biosynthesis, which provides a theoretical foundation for the molecular identification of sweet/bitter kernels of apricots.

Promoter Variation of the Key Apple Fruit Texture Related Gene MdPG1 and the Upstream Regulation Analysis.

MdPG1 encoding polygalacturonase in apple (Malus × domestica) is a key gene associated with fruit firmness and texture variations among apple cultivars. However, the causative variants of MdPG1 are still not known. In this study, we identified a SNPA/C variant within an ERF-binding element located in the promoter region of MdPG1. The promoter containing the ERF-binding element with SNPA, rather than the SNPC, could be strongly bound and activated by MdCBF2, a member of the AP2/ERF transcription factor family, as determined by yeast-one-hybrid and dual-luciferase reporter assays. We also demonstrated that the presence of a novel long non-coding RNA, lncRNAPG1, in the promoter of MdPG1 was a causative variant. lncRNAPG1 was specifically expressed in fruit tissues postharvest. lncRNAPG1 could reduce promoter activity when it was fused to the promoter of MdPG1 and a tobacco gene encoding Mg-chelatase H subunit (NtCHLH) in transgenic tobacco cells but could not reduce promoter activity when it was supplied in a separate gene construct, indicating a cis-regulatory effect. Our results provide new insights into genetic regulation of MdPG1 allele expression and are also useful for the development of elite apple cultivars.

Physiological and Full-Length Transcriptome Analyses Reveal the Dwarfing Regulation in Trifoliate Orange (Poncirus trifoliata L.).

Dwarfing rootstocks are capable of high-density planting and are therefore urgently needed in the modern citrus cultivation system. However, little is known about the physiological relevance and molecular basis underlying citrus height. This study aimed to comprehensively analyze phytohormone, carbohydrate, and associated transcriptome changes in the stem of two weak growth rootstocks ('TO' and 'FD') relative to the vigorous 'CC' rootstock. The phenotypic observation revealed that the plant height, plant weight, and internode length were reduced in dwarfing rootstocks. Moreover, the contents of indole-3-acetic acid (IAA), trans-zeatin (tZ), and abscisic acid (ABA), were higher in TO and FD rootstocks, whereas the gibberellin 3 (GA3) content was higher in the CC rootstocks. The carbohydrate contents, including sucrose, fructose, glucose, starch, and lignin significantly decreased in both the TO and FD rootstocks. The full-length transcriptome analysis revealed a potential mechanism regulating dwarfing phenotype that was mainly related to the phytohormone signaling transduction, sugar and starch degradation, lignin synthesis, and cellulose and hemicellulose degradation processes. In addition, many transcription factors (TFs), long non-coding RNAs (lncRNAs), and alternative splicing (AS) events were identified, which might act as important contributors to control the stem elongation and development in the weak growth rootstocks. These findings might deepen the understanding of the complex mechanisms of the stem development responsible for citrus dwarfing and provide a series of candidate genes for the application in breeding new rootstocks with intensive dwarfing.

Genome editing of SlMYB3R3, a cell cycle transcription factor gene of tomato, induces elongated fruit shape.

Fruit shape is an important trait that attracts consumers, and the regulation of genes related to cell division is crucial for shaping multicellular organs. In Arabidopsis, MYB3R transcription factors, which harbor three imperfect repeats in the N-terminus, control organ growth by regulating cell division. However, the function of MYB3Rs in tomato remains unknown. Here, we characterized tomato SlMYB3R3, which was preferentially expressed in flowers and placed in a subclade with two Arabidopsis cell cycle suppressors (MYB3R3/5). slmyb3r3 knockout mutants were generated using the CRISPR/Cas9 system. Morphological observation of the slmyb3r3 mutants showed that fruits that were elongated and occasionally peanut-like in shape were formed, which was caused by significantly increased cell numbers in the longitudinal direction. Transcriptome and yeast one-hybrid assay results suggested that SlMYB3R3 acted as a suppressor of cell-cycle-related genes by binding to the mitosis-specific activator (MSA) motifs in their promoters. Taken together, knock out of the suppressor SlMYB3R3 leads to elongated fruit, which results from the altered cell division pattern at the ovary stage, by regulating cell-cycle-related genes in an MSA-dependent manner. Our results suggest that SlMYB3R3 and its orthologs have the potential to change fruit shape as part of the molecular breeding of fruit crops.

H3K4me3 plays a key role in establishing permissive chromatin states during bud dormancy and bud break in apple.

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

DkmiR397 Regulates Proanthocyanidin Biosynthesis via Negative Modulating DkLAC2 in Chinese PCNA Persimmon.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, DkLAC2, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene DkLAC2 in 'Eshi 1' persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves. Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of DkLAC2. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2, whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type (WT) and overexpression of DkLAC2, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.

Hierarchical Encoder-Decoder With Soft Label-Decomposition for Mitochondria Segmentation in EM Images.

Semantic segmentation of mitochondria from electron microscopy (EM) images is an essential step to obtain reliable morphological statistics about mitochondria. However, automatically delineating plenty of mitochondria of varied shapes from complex backgrounds with sufficient accuracy is challenging. To address these challenges, we develop a hierarchical encoder-decoder network (HED-Net), which has a three-level nested U-shape architecture to capture rich contextual information. Given the irregular shape of mitochondria, we introduce a novel soft label-decomposition strategy to exploit shape knowledge in manual labels. Rather than simply using the ground truth label maps as the unique supervision in the model training, we introduce additional subcategory-aware supervision by softly decomposing each manual label map into two complementary label maps according to mitochondria's ovality. The three label maps are integrated with our HED-Net to supervise the model training. While the original label map guides the network to segment all the mitochondria of varied shapes, the auxiliary label maps guide the network to segment subcategories of mitochondria of circular shape and elliptic shape, respectively, which are much more manageable tasks. Extensive experiments on two public benchmarks show that our HED-Net performs favorably against state-of-the-art methods.

Comparative transcriptome analysis reveals regulatory network and regulators associated with proanthocyanidin accumulation in persimmon.

BACKGROUND: Proanthocyanidins (PAs) are important plant secondary metabolites that confer flavor, nutritional value, and resistance to pathogens. Persimmon is one of the PA richest crops. Mature fruits can be inedible because of the astringency caused by high PA levels and need to go through a de-astringency treatment before consumption. The molecular basis for PA accumulation is poorly known, particularly transcriptional regulators. We characterised three genotypes ('Luotiantianshi' (LT), 'Mopanshi' (MP), and 'Youhou' (YH)) with different PA accumulation patterns using an approach that combined PacBio full-length sequencing and Illumina-based RNA sequencing to build high-quality full-length transcriptomes. Additionally, we analysed transcriptome dynamics of the three genotypes (LT, MP, and YH) at four key fruit developmental stages. RESULTS: A total of 96,463 transcripts were obtained. We identified 80,075 protein-coding sequences (CDSs), 71,137 simple sequence repeats (SSRs), and 27,845 long noncoding RNAs (lncRNAs). Pearson correlation coefficient (PCC), principal component analysis (PCA), and differentially expressed transcripts (DETs) analyses indicated that the four different developmental stages within a genotype exhibited similar transcriptome activities. A total of 2,164 transcripts specific to each fruit developmental stage were detected. The transcripts specific to early stages were attributed to phenylpropanoid and flavonoid biosynthesis. Co-expression network analyses revealed MEbrown and MEblue modules were strongly associated to PA accumulation. From these two modules, 20 hub TFs are potential regulators for PA accumulation. Among them, Cluster_78388 (SBP protein), Cluster_63454 (bZIP protein), and Cluster_66595 (MYB protein) appear to involve in the PA biosynthesis in Chinese genotypes. CONCLUSIONS: This is the first high-quality reference transcriptome for commercial persimmon. Our work provides insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout fruit development.

The Analysis of Phenolic Compounds in Walnut Husk and Pellicle by UPLC-Q-Orbitrap HRMS and HPLC.

Husk and pellicle as the agri-food waste in the walnut-product industry are in soaring demand because of their rich polyphenol content. This study investigated the differential compounds related to walnut polyphenol between husk and pellicle during fruit development stage. By using ultra-high performance liquid chromatography-quadrupole-orbitrap (UHPLC-Q-Orbitrap), a total of 110 bioactive components, including hydrolysable tannins, flavonoids, phenolic acids and quinones, were tentatively identified, 33 of which were different between husk and pellicle. The trend of dynamic content of 16 polyphenols was clarified during walnut development stage by high-performance liquid chromatography (HPLC). This is the first time to comprehensive identification of phenolic compounds in walnut husk and pellicle, and our results indicated that the pellicle is a rich resource of polyphenols. The dynamic trend of some polyphenols was consistent with total phenols. The comprehensive characterization of walnut polyphenol and quantification of main phenolic compounds will be beneficial for understanding the potential application value of walnut and for exploiting its metabolism pathway.

DkMYB14 is a bifunctional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit.

Proanthocyanidins (PAs) are phenolic secondary metabolites that contribute to the protection of plant and human health. Persimmon (Diospyros kaki Thunb.) can accumulate abundant PAs in fruit, which cause a strong sensation of astringency. Proanthocyanidins can be classified into soluble and insoluble PAs; the former cause astringency but the latter do not. Soluble PAs can be converted into insoluble PAs upon interacting with acetaldehydes. We demonstrate here that DkMYB14, which regulates the accumulation of PA in persimmon fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting its insolubilization. We show that the amino acid Gly39 in the R2 domain and the ethylene response factor-associated amphiphilic repression-like motif in the C-terminal of DkMYB14 are essential for the regulation of both PA and acetaldehyde synthesis. The repressive function of DkMYB14 was lost after the mutation of either motif, and all activities of DkMYB14 were eliminated following the mutation of both motifs. Our results demonstrate that DkMYB14 functions as both a transcriptional activator and a repressor, directly repressing biosynthesis of PA and promoting its insolubilization, resulting in non-astringency in persimmon.

HIVE-Net: Centerline-aware hierarchical view-ensemble convolutional network for mitochondria segmentation in EM images.

BACKGROUND AND OBJECTIVE: With the advancement of electron microscopy (EM) imaging technology, neuroscientists can investigate the function of various intracellular organelles, e.g, mitochondria, at nano-scale. Semantic segmentation of electron microscopy (EM) is an essential step to efficiently obtain reliable morphological statistics. Despite the great success achieved using deep convolutional neural networks (CNNs), they still produce coarse segmentations with lots of discontinuities and false positives for mitochondria segmentation. METHODS: In this study, we introduce a centerline-aware multitask network by utilizing centerline as an intrinsic shape cue of mitochondria to regularize the segmentation. Since the application of 3D CNNs on large medical volumes is usually hindered by their substantial computational cost and storage overhead, we introduce a novel hierarchical view-ensemble convolution (HVEC), a simple alternative of 3D convolution to learn 3D spatial contexts using more efficient 2D convolutions. The HVEC enables both decomposing and sharing multi-view information, leading to increased learning capacity. RESULTS: Extensive validation results on two challenging benchmarks show that, the proposed method performs favorably against the state-of-the-art methods in accuracy and visual quality but with a greatly reduced model size. Moreover, the proposed model also shows significantly improved generalization ability, especially when training with quite limited amount of training data. Detailed sensitivity analysis and ablation study have also been conducted, which show the robustness of the proposed model and effectiveness of the proposed modules. CONCLUSIONS: The experiments highlighted that the proposed architecture enables both simplicity and efficiency leading to increased capacity of learning spatial contexts. Moreover, incorporating shape cues such as centerline information is a promising approach to improve the performance of mitochondria segmentation.

HDC-Net: Hierarchical Decoupled Convolution Network for Brain Tumor Segmentation.

Accurate segmentation of brain tumor from magnetic resonance images (MRIs) is crucial for clinical treatment decision and surgical planning. Due to the large diversity of the tumors and complex boundary interactions between sub-regions, it is of a great challenge. Besides accuracy, resource constraint is another important consideration. Recently, impressive improvement has been achieved for this task by using deep convolutional networks. However, most of state-of-the-art models rely on expensive 3D convolutions as well as model cascade/ensemble strategies, which result in high computational overheads and undesired system complexity. For clinical usage, the challenge is how to pursue the best accuracy within very limited computational budgets. In this study, we segment 3D volumetric image in one-pass with a hierarchical decoupled convolution network (HDC-Net), which is a light-weight but efficient pseudo-3D model. Specifically, we replace 3D convolutions with a novel hierarchical decoupled convolution (HDC) module, which can explore multi-scale multi-view spatial contexts with high efficiency. Extensive experiments on the BraTS 2018 and 2017 challenge datasets show that our method performs favorably against state of the art in accuracy yet with greatly reduced computational complexity.

MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon.

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5' RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon.

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.

Plant tannin immobilized Fe3O4@SiO2 microspheres: A novel and green magnetic bio-sorbent with superior adsorption capacities for gold and palladium.

In this paper, a new core-shell nanostructured magnetic bio-based composite was prepared by immobilizing persimmon tannin (PT) onto Fe3O4@SiO2 microspheres, and the as designed Fe3O4@SiO2@PT was utilized for adsorptive recovery of Au(III) and Pd(II). The preparation, morphology, composition and magnetic property of Fe3O4@SiO2@PT were characterized. Adsorption parameters of Fe3O4@SiO2@PT towards Au(III) and Pd(II) including initial pH, reaction time, initial concentration of metal ions, effect of acidity and interference of coexisting metal ions were investigated. It is sufficiently confirmed that silica was coated on Fe3O4 and persimmon tannin was immobilized on aminated Fe3O4@SiO2. The thickness of silica and loaded persimmon tannin are around 18 nm and 14 nm, respectively. With only 1.00 wt% of persimmon tannin, however, the maximum adsorption capacities of Fe3O4@SiO2@PT for Au(III) and Pd(II) were as high as 917.43 and 196.46 mg·g-1, respectively. In addition, after adsorption of Au(III) and Pd(II), the magnetization saturation values (Ms) of Fe3O4@SiO2@PT were high enough to guarantee efficient magnetic seperation. Metallic gold could be facilely recovered from wastewaters containing Au(III).

Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit.

Removal of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de-astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia-responsive ethylene response factors (ERFs) participate in anaerobic de-astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO2 (95%) was ameliorated by adding the ethylene inhibitor 1-MCP (1 µL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO2 -induced softening involves ethylene signalling. Among the hypoxia-responsive genes, expression of eight involved in fruit cell wall metabolism (Dkß-gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual-luciferase assay indicated that DkERF8/16/19 could trans-activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C-repeat/dehydration-responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia-responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1-MCP. Fruit of the Japanese cultivar 'Tonewase' provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO2 treatment without 1-MCP and changes in cell wall enzymes and ERFs did not occur.

An integrated analysis based on transcriptome and proteome reveals deastringency-related genes in CPCNA persimmon.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs) during development. PAs cause a dry or puckering sensation due to its astringency. Pollination constant and non-astringent (PCNA) persimmon fruits can lose astringency during fruit ripening. However, little is known about the mechanism of natural de-astringency of Chinese PCNA (CPCNA). To gain insight into the molecular events of CPCNA natural de-astringency, we used mRNA-seq and iTRAQ-based quantitative proteomic analysis to measure changes in genes and proteins expression at two key stages of natural astringency removal (i.e. 10 and 20 weeks after bloom) and water-treated (i.e. 40 °C·12 h) de-astringency fruits. Our analyses show that the three predominantly process in CPCNA de-astringency: (1) water treatment strongly up-regulates glycolysis/acetaldehyde metabolism, (2) expression of genes/proteins involved in PA biosynthetic pathway was remarkably reduced in natural and water-treated de-astringency, (3) sugar metabolism and ethylene related pathway were quite abundant in natural de-astringency. We also found ethylene-related TFs were quite abundant in natural de-astringency, followed by WRKY and NAC transcription factors. These results provide an initial understanding of the predominantly biological processes underlying the natural de-astringency and "coagulation effect" in CPCNA.

DkPK Genes Promote Natural Deastringency in C-PCNA Persimmon by Up-regulating DkPDC and DkADH Expression.

The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree. This process is controlled by a single locus and is dominant against other types of persimmons; therefore, this variant is an important candidate for commercial cultivation and the breeding of PCNA cultivars. In our previous study, six full-length coding sequences (CDS) for pyruvate kinase genes (DkPK1-6) were isolated, and DkPK1 is thought to be involved in the natural deastringency of C-PCNA persimmon fruit. Here, we characterize the eight other DkPK genes (DkPK7-14) from C-PCNA persimmon fruit based on transcriptome data. The transcript changes in DkPK7-14 genes and correlations with the proanthocyanidin (PA) content were investigated during different fruit development stages in C-PCNA, J-PCNA, and non-PCNA persimmon; DkPK7 and DkPK8 exhibited up-regulation patterns during the last developmental stage in C-PCNA persimmon that was negatively correlated with the decrease in soluble PAs. Phylogenetic analysis and subcellular localization analysis revealed that DkPK7 and DkPK8 are cytosolic proteins. Notably, DkPK7 and DkPK8 were ubiquitously expressed in various persimmon organs and abundantly up-regulated in seeds. Furthermore, transient over-expression of DkPK7 and DkPK8 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the pyruvate decarboxylase (DkPDC) and alcohol dehydrogenase genes (DkADH), which are closely related to acetaldehyde metabolism. The accumulated acetaldehyde that results from the up-regulation of the DkPDC and DkADH genes can combine with soluble PAs to form insoluble PAs, resulting in the removal of astringency from persimmon fruit. Thus, we suggest that both DkPK7 and DkPK8 are likely to be involved in natural deastringency via the up-regulation of DkPDC and DkADH expression during the last developmental stage in C-PCNA persimmon.