The Fungal Diversity and Potential Pathogens Associated with Postharvest Fruit Rot of 'Huangguan' Pear (Pyrus bretschneideri) in Hebei Province, China.

Postharvest fruit rot caused by pathogens is a serious problem in the pear industry. This study investigated the fungal diversity and main pathogens and identified a new pathogen in the stored 'Huangguan' pear (Pyrus bretschneideri Rehd.), the dominant pear variety in northern China. We sampled 20 refrigeration houses from five main producing regions in Hebei Province and used Illumina sequencing technology to detect the fungal composition. Alternaria (56.3%) was the most abundant fungus, followed by Penicillium (9.2%) and Monilinia (6.2%). We also isolated and identified nine strains of Alternaria and four strains of Penicillium. Moreover, we observed a new postharvest fruit disease in 'Huangguan' pear caused by Stemphylium eturmiunum, which was confirmed by phylogenetic analysis by combining the sequences of three conserved genes, including internal transcribed spacer, gapdh, and calmodulin. This study marks the first documentation of S. eturmiunum causing fruit rot in 'Huangguan' pears, offering valuable insights for identifying and controlling this newly identified postharvest disease.

Time-Series Transcriptome Analysis Reveals the Molecular Mechanism of Ethylene Reducing Cold Sensitivity of Postharvest 'Huangguan' Pear.

'Huangguan' pear (Pyrus bretschneideri Rehd) fruit is susceptible to cold, characterized by developing peel browning spots (PBS) during cold storage. Additionally, ethylene pretreatment reduces chilling injury (CI) and inhibits PBS occurrence, but the mechanism of CI remains unclear. Here, we deciphered the dynamic transcriptional changes during the PBS occurrence with and without ethylene pretreatment via time-series transcriptome. We found that ethylene suppressed the cold-signaling gene expression, thereby decreasing the cold sensitivity of the 'Huangguan' fruit. Moreover, the "Yellow" module closely correlated with PBS occurrence was identified via weighted gene co-expression network analysis (WGCNA), and this module was related to plant defense via Gene Ontology (GO) enrichment analysis. Local motif enrichment analysis suggested that the "Yellow" module genes were regulated by ERF and WRKY transcription factors. Functional studies demonstrated that PbWRKY31 has a conserved WRKY domain, lacks transactivation activity, and localizes in the nucleus. PbWRKY31-overexpressed Arabidopsis were hypersensitive to cold, with higher expression levels of cold signaling and defense genes, suggesting that PbWRKY31 participates in regulating plant cold sensitivity. Collectively, our findings provide a comprehensive transcriptional overview of PBS occurrence and elucidate the molecular mechanism by which ethylene reduces the cold sensitivity of 'Huangguan' fruit as well as the potential role of PbWRKY31 in this process.

Comparative transcriptome analysis reveals the mechanism involving ethylene and cell wall modification related genes in Diospyros kaki fruit firmness during ripening.

Rapid loss of firmness is a major handicap for persimmon (Diospyros kaki Thunb.) transportation and retail. The present study employed a comparative transcriptomic approach to elucidate the mechanism involving ethylene and cell wall modification related genes in fruit firmness control of two cultivars during post harvest ripening. In contrast to the short shelf life cultivar (Mopan), the long shelf life cultivar (Yoho) kept high firmness during ripening. Extensive loss of firmness in Mopan drove an intense transcriptional activity. Globally, Mopan and Yoho shared very few common differentially expressed structural genes and regulators. Yoho strongly repressed the expression of ACC synthase and several classes of cell wall degradation genes at the onset of ripening and only induced them during late ripening period. Various ERF, WRKY, MYB, bHLH transcription factors were found highly active during fruit ripening. Overall, this study generates novel gene resources as important tools for extending persimmon shelf life.

RNA-Seq Analysis Identifies Transcription Factors Involved in Anthocyanin Biosynthesis of 'Red Zaosu' Pear Peel and Functional Study of PpPIF8.

Red-skinned pears are favored by people for their attractive appearance and abundance of anthocyanins. However, the molecular basis of anthocyanin biosynthesis in red pears remains elusive. Here, a comprehensive transcriptome analysis was conducted to explore the potential regulatory mechanism of anthocyanin biosynthesis in 'Red Zaosu' pear (Pyrus pyrifolia × Pyrus communis). Gene co-expression analysis and transcription factor mining identified 263 transcription factors, which accounted for 6.59% of the total number of transcription factors in the pear genome in two gene modules that are highly correlated with anthocyanin biosynthesis. Clustering, gene network modeling with STRING-DB, and local motif enrichment analysis (CentriMo) analysis suggested that PpPIF8 may play a role in anthocyanin biosynthesis. Furthermore, eight PIFs were identified in the pear genome, of which only PpPIF8 was rapidly induced by light. Functional studies showed that PpPIF8 localizes in the nucleus and is preferentially expressed in the tissue of higher levels of anthocyanin. The overexpression of PpPIF8 in pear peel and pear calli promotes anthocyanin biosynthesis and upregulates the expression of anthocyanin biosynthesis genes. Yeast-one hybrid and transgenic analyses indicated that PpPIF8 binds to the PpCHS promoter to induce PpCHS expression. The positive effect of PpPIF8 on anthocyanin biosynthesis is different from previously identified negative regulators of PyPIF5 and MdPIF7 in pear and apple. Taken together, our data not only provide a comprehensive view of transcription events during the coloration of pear peel, but also resolved the regulatory role of PpPIF8 in the anthocyanin biosynthesis pathway.

Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification.

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

Influence of Bagging on Fruit Quality, Incidence of Peel Browning Spots, and Lignin Content of 'Huangguan' Pears.

The 'Huangguan' pear is one of the high-quality pear cultivars produced in China. However, the bagged fruit of the 'Huangguan' pear often suffers from peel browning spots after rain during their mature period. In this study, in an effort to discover the impact of bagging treatments on the occurrence of peel browning spots and fruit quality, fruits were covered by single-layer, two-layer, or triple-layer paper bags six weeks after reaching full bloom. The results showed that the bagged fruits were characterized by smooth surfaces and reduced lenticels compared with the unbagged ones. The unbagged and the two-layer bagged fruits had yellow/green peels, while the single- and triple-layer bagged ones had yellow/white peels. Compared with the unbagged fruits, the bagged fruits had higher vitamin C (Vc) contents and values of peel color indexes L and a and lower soluble solid contents (SSCs), titratable acid (TA) contents, absorbance index differences (IAD), and b values. Additionally, the triple-layer bagged group was superior to other groups in terms of fruit quality, but it also had the maximum incidence of peel browning spots. Before and after the appearance of peel browning spots, the bagged fruits had smoother and thinner cuticles compared with the unbagged ones. Furthermore, the triple-layer bagged fruits had minimum lignin contents and maximum phenolic contents in their peels, with minimum activity of lignin synthesis-related enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO), as well as minimum expressions of relevant genes such as cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA reductase (CCR), 4-coumarate: coenzyme A ligase (4CL6), and cinnamate 4-hydroxylase (C4H1). It was deduced that POD activity and the relative expressions of CAD9, CCR3, CCR4, and CCR5 may play key roles in the occurrence of peel browning spots. In summary, lignin synthesis affected the incidence of peel browning spots in bagged 'Huangguan' pears. This study provides a theoretical basis for understanding the incidence of peel browning spots in 'Huangguan' pears.

Photoregulatory protein kinases fine-tune plant photomorphogenesis by directing a bifunctional phospho-code on HY5 in Arabidopsis.

Photomorphogenesis is a light-dependent plant growth and development program. As the core regulator of photomorphogenesis, ELONGATED HYPOCOTYL 5 (HY5) is affected by dynamic changes in its transcriptional activity and protein stability; however, little is known about the mediators of these processes. Here, we identified PHOTOREGULATORY PROTEIN KINASE 1 (PPK1), which interacts with and phosphorylates HY5 in Arabidopsis, as one such mediator. The phosphorylation of HY5 by PPK1 is essential to establish high-affinity binding with B-BOX PROTEIN 24 (BBX24) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which inhibit the transcriptional activity and promote the degradation of HY5, respectively. As such, PPKs regulate not only the binding of HY5 to its target genes under light conditions but also HY5 degradation when plants are transferred from light to dark. Our data identify a PPK-mediated phospho-code on HY5 that integrates the molecular mechanisms underlying the regulation of HY5 to precisely control plant photomorphogenesis.

The BAS chromatin remodeler determines brassinosteroid-induced transcriptional activation and plant growth in Arabidopsis.

Brassinosteroid (BR) signaling leads to the nuclear accumulation of the BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor, which plays dual roles in activating or repressing the expression of thousands of genes. BZR1 represses gene expression by recruiting histone deacetylases, but how it activates transcription of BR-induced genes remains unclear. Here, we show that BR reshapes the genome-wide chromatin accessibility landscape, increasing the accessibility of BR-induced genes and reducing the accessibility of BR-repressed genes in Arabidopsis. BZR1 physically interacts with the BRAHMA-associated SWI/SNF (BAS)-chromatin-remodeling complex on the genome and selectively recruits the BAS complex to BR-activated genes. Depletion of BAS abrogates the capacities of BZR1 to increase chromatin accessibility, activate gene expression, and promote cell elongation without affecting BZR1's ability to reduce chromatin accessibility and expression of BR-repressed genes. Together, these data identify that BZR1 recruits the BAS complex to open chromatin and to mediate BR-induced transcriptional activation of growth-promoting genes.

Ethylene Signal Is Involved in the Regulation of Anthocyanin Accumulation in Flesh of Postharvest Plums (Prunus salicina Lindl.).

Ethylene is positively correlated with the anthocyanin accumulation in postharvest plum fruit, but the regulation mechanism has not been fully clarified. In this work, the 'Friar' plum fruit under different storage temperatures (0, 10 and 25 °C) and treatments (100.0 µL L-1 ethylene and 1.0 µL L-1 1-MCP) were applied to study the relationship between anthocyanin accumulation and ethylene signal pathway. The fruits stored at 10 °C had higher ethylene production rate and more anthocyanin in flesh than those stored at 0 °C and 25 °C. Ten ethylene biosynthesis associated genes and forty-one ethylene signal transduction related genes were obtained from the previous transcriptome data. Among them, the expression levels of ethylene biosynthesis associated genes (PsACS1, PsACS4 and PsACO1), and ethylene signal transduction related genes (PsERS1s, PsETR2, PsERF1a, and PsERF12) were markedly higher in the fruits stored at 10 °C than those at 0 °C and 25 °C. Exogenous ethylene treatment enhanced while 1-MCP treatment inhibited the anthocyanin accumulation in the flesh under storage at 10 °C. In addition, exogenous ethylene treatment markedly increased the expression levels of PsACS1, PsACS4, PsACO1, PsETR2, PsERF1a, and PsERF12 in the flesh once it turning red, as well as the anthocyanin biosynthesis related genes (PsPAL, PsCHS, PsF3H, PsDRF, PsANS, PsUFGT and PsMYB10), whereas 1-MCP treatment manifested the contrary effects. Correlation analysis indicated that there was a significant positive correlation between genes expression related to ethylene signal pathway and anthocyanin biosynthesis, except for PsERF11. In conclusion, ethylene signal pathway is involved in the flesh reddening by up-regulating the anthocyanin biosynthesis related genes.

Dynamic Microbiome Changes Reveal the Effect of 1-Methylcyclopropene Treatment on Reducing Post-harvest Fruit Decay in "Doyenne du Comice" Pear.

Pathogen-induced decay is one of the most common causes of fruit loss, resulting in substantial economic loss and posing a health risk to humans. As an ethylene action inhibitor, 1-methylcyclopropene (1-MCP) can significantly reduce fruit decay, but its effect on fruit pathogens remains unclear. Herein, the change in microbial community structure was analyzed using the high-throughput sequencing technology, and characteristics related to fruit quality were determined after 1-MCP (1.0 M l L-1) treatment in "Doyenne du Comiceis" pear fruit during storage at ambient temperature. Overall, 1-MCP was highly effective in reducing disease incidence and induced multiple changes of the fungal and bacterial microbiota. At day 15, the microbial diversity of fungi or bacteria was reduced significantly in the control fruit (non-treated with 1-MCP), which had the most severe decay incidence. For fungi, in addition to Alternaria being the most abundant in both 1-MCP treatment (59.89%) and control (40.18%), the abundances of Botryosphaeria (16.75%), Penicillium (8.81%), and Fusarium (6.47%) increased significantly with the extension of storage time. They became the primary pathogens to cause fruit decay in control, but they were markedly decreased in 1-MCP treatment, resulting in reduced disease incidence. For bacteria, the abundance of Gluconobacter (50.89%) increased dramatically at day 15 in the control fruit, showing that it also played a crucial role in fruit decay. In addition, Botryosphaeria, Fusarium fungi, and Massilia, Kineococcus bacteria were identified as biomarkers to distinguish 1-MCP treatment and control using Random Forest analysis. The redundancy analysis (RDA) result showed that the amount of Botryosphaeria, Penicillium, and Fusarium were positively correlated with disease incidence and respiration rate of pear fruits while negatively correlated with fruit firmness. This investigation is the first comprehensive analysis of the microbiome response to 1-MCP treatment in post-harvest pear fruit, and reveals the relationship between fruit decay and microbial composition in pear fruit.

The Arabidopsis B-box protein BZS1/BBX20 interacts with HY5 and mediates strigolactone regulation of photomorphogenesis.

Plant growth is controlled by integration of hormonal and light-signaling pathways. BZS1 is a B-box zinc finger protein previously characterized as a negative regulator in the brassinosteroid (BR)-signaling pathway and a positive regulator in the light-signaling pathway. However, the mechanisms by which BZS1/BBX20 integrates light and hormonal pathways are not fully understood. Here, using a quantitative proteomic workflow, we identified several BZS1-associated proteins, including light-signaling components COP1 and HY5. Direct interactions of BZS1 with COP1 and HY5 were verified by yeast two-hybrid and co-immunoprecipitation assays. Overexpression of BZS1 causes a dwarf phenotype that is suppressed by the hy5 mutation, while overexpression of BZS1 fused with the SRDX transcription repressor domain (BZS1-SRDX) causes a long-hypocotyl phenotype similar to hy5, indicating that BZS1's function requires HY5. BZS1 positively regulates HY5 expression, whereas HY5 negatively regulates BZS1 protein level, forming a feedback loop that potentially contributes to signaling dynamics. In contrast to BR, strigolactone (SL) increases BZS1 level, whereas the SL responses of hypocotyl elongation, chlorophyll and HY5 accumulation are diminished in the BZS1-SRDX seedlings, indicating that BZS1 is involved in these SL responses. These results demonstrate that BZS1 interacts with HY5 and plays a central role in integrating light and multiple hormone signals for photomorphogenesis in Arabidopsis.

Concerted genomic targeting of H3K27 demethylase REF6 and chromatin-remodeling ATPase BRM in Arabidopsis.

SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM-REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.

BZS1, a B-box protein, promotes photomorphogenesis downstream of both brassinosteroid and light signaling pathways.

Photomorphogenesis is controlled by multiple signaling pathways, including the light and brassinosteroid (BR) pathways. BR signaling activates the BZR1 transcription factor, which is required for suppressing photomorphogenesis in the dark. We identified a suppressor of the BR hypersensitive mutant bzr1-1D and named it bzr1-1D suppressor1-Dominant (bzs1-D). The bzs1-D mutation was caused by overexpression of a B-box zinc finger protein BZS1, which is transcriptionally repressed by BZR1. Overexpression of BZS1 causes de-etiolation in the dark, short hypocotyls in the light, reduced sensitivity to BR treatment, and repression of many BR-activated genes. Knockdown of BZS1 by co-suppression partly suppressed the short hypocotyl phenotypes of BR-deficient or insensitive mutants. These results support that BZS1 is a negative regulator of BR response. BZS1 overexpressors are hypersensitive to different wavelengths of light and loss of function of BZS1 reduces plant sensitivity to light and partly suppresses the constitutive photomorphogenesis 1 (cop1) mutant in the dark, suggesting a positive role in light response. BZS1 protein accumulates at an increased level after light treatment of dark-grown BZS1-OX plants and in the cop1 mutants, and BZS1 interacts with COP1 in vitro, suggesting that light regulates BZS1 through COP1-mediated ubiquitination and proteasomal degradation. These results demonstrate that BZS1 mediates the crosstalk between BR and light pathways.