Genome-wide characterisation of HD-Zip transcription factors and functional analysis of PbHB24 during stone cell formation in Chinese white pear (Pyrus bretschneideri).

BACKGROUND: The homodomain-leucine zipper (HD-Zip) is a conserved transcription factor family unique to plants that regulate multiple developmental processes including lignificaion. Stone cell content is a key determinant negatively affecting pear fruit quality, which causes a grainy texture of fruit flesh, because of the lignified cell walls. RESULTS: In this study, a comprehensive bioinformatics analysis of HD-Zip genes in Chinese white pear (Pyrus bretschneideri) (PbHBs) was performed. Genome-wide identification of the PbHB gene family revealed 67 genes encoding PbHB proteins, which could be divided into four subgroups (I, II, III, and IV). For some members, similar intron/exon structural patterns support close evolutionary relationships within the same subgroup. The functions of each subgroup of the PbHB family were predicted through comparative analysis with the HB genes in Arabidopsis and other plants. Cis-element analysis indicated that PbHB genes might be involved in plant hormone signalling and external environmental responses, such as light, stress, and temperature. Furthermore, RNA-sequencing data and quantitative real-time PCR (RT-qPCR) verification revealed the regulatory roles of PbHB genes in pear stone cell formation. Further, co-expression network analysis revealed that the eight PbHB genes could be classified into different clusters of co-expression with lignin-related genes. Besides, the biological function of PbHB24 in promoting stone cell formation has been demonstrated by overexpression in fruitlets. CONCLUSIONS: This study provided the comprehensive analysis of PbHBs and highlighted the importance of PbHB24 during stone cell development in pear fruits.

Transcription factors Pbr3RAV2 and PbrTTG1 regulate pear resistance to Botryosphaeria dothidea via the autophagy pathway.

Pear ring rot, caused by Botryosphaeria dothidea, is the most serious disease of pear (Pyrus spp.) trees. However, the molecular mechanisms underlying pear resistance to B. dothidea remain elusive. Herein, we demonstrated that the pear AuTophagy-related Gene 1a (PbrATG1a) plays a key role in autophagic activity and resistance to B. dothidea. Stable overexpression of PbrATG1a enhanced resistance to B. dothidea in pear calli. Autophagy activity was greater in PbrATG1a overexpressing calli than in WT calli. We used yeast one-hybrid screening to identify a transcription factor, Related to ABI3 and VP1 (Pbr3RAV2), that binds the promoter of PbrATG1a and enhances pear resistance to B. dothidea by regulating autophagic activity. Specifically, overexpression of Pbr3RAV2 enhanced resistance to B. dothidea in pear calli, while transient silencing of Pbr3RAV2 resulted in compromised resistance to B. dothidea in Pyrus betulaefolia. In addition, we identified Transparent Testa Glabra 1 (PbrTTG1), which interacts with Pbr3RAV2. Pathogen infection enhanced the interaction between Pbr3RAV2 and PbrTTG1. The Pbr3RAV2-PbrTTG1 complex increased the binding capacity of Pbr3RAV2 and transcription of PbrATG1a. In addition to providing insights into the molecular mechanisms underlying pear disease resistance, these findings suggest potential genetic targets for enhancing disease resistance in pear.

Pyrus betulaefolia ERF3 interacts with HsfC1a to coordinately regulate aquaporin PIP1;4 and NCED4 for drought tolerance.

Environmental disasters like drought reduce agricultural output and plant growth. Redox management significantly affects plant stress responses. An earlier study found that PbPIP1;4 transports H2O2 and promotes H2O2 downstream cascade signaling to restore redox equilibrium. However, this regulatory mechanism requires additional investigation. In this search, the AP2 domain-containing transcription factor was isolated by screening Y1H from the wild pear (Pyrus betulaefolia) cDNA library, named PbERF3. The overexpression of PbERF3 in pear callus and Arabidopsis enhanced plant resistance to drought and re-established redox balance. The transcripts of the NCEDs gene were upregulated under drought stress. The drought stress-related abscisic acid (ABA) signaling pathway modulates PbERF3. PbERF3 silencing lowered drought tolerance. Furthermore, yeast 2-hybrid, luciferase, bimolecular fluorescence complementation, and co-immunoprecipitation assays verified that PbERF3 physically interacted with PbHsfC1a. The PbERF3-PbHsfC1a heterodimer coordinately bound to PbPIP1;4 and PbNCED4 promoter, therefore activating both the H2O2 and the ABA signaling pathway. This work revealed a novel PbERF3-PbHsfC1a-PbNCED4-PbPIP1;4 regulatory module, in which PbERF3 interacts with PbHsfC1a to trigger the expression of target genes. This module establishes an interaction between the H2O2 signaling component PbPIP1;4 and the ABA pathways component PbNCED4, enabling a response to drought.

Interaction among homeodomain transcription factors mediates ethylene biosynthesis during pear fruit ripening.

Fruit ripening is manipulated by the plant phytohormone ethylene in climacteric fruits. While the transcription factors (TFs) involved in ethylene biosynthesis and fruit ripening have been extensively studied in tomato, their identification in pear remains limited. In this study, we identified and characterized a HOMEODOMAIN TF, PbHB.G7.2, through transcriptome analysis. PbHB.G7.2 could directly bind to the promoter of the ethylene biosynthetic gene, 1-aminocyclopropane-1-carboxylic acid synthase (PbACS1b), thereby enhancing its activity and resulting in increased ethylene production during pear fruit ripening. Yeast-two-hybrid screening revealed that PbHB.G7.2 interacted with PbHB.G1 and PbHB.G2.1. Notably, these interactions disrupted the transcriptional activation of PbHB.G7.2. Interestingly, PbHB.G1 and PbHB.G2.1 also bind to the PbACS1b promoter, albeit different regions from those bound by PbHB.G7.2. Moreover, the regions of PbHB.G1 and PbHB.G2.1 involved in their interaction with PbHB.G7.2 differ from the regions responsible for binding to the PbACS1b promoter. Nonetheless, these interactions also disrupt the transcriptional activation of PbHB.G1 and PbHB.G2.1. These findings offer a new mechanism of ethylene biosynthesis during climacteric fruit ripening.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

Sleep Patterns and Traditional Cardiovascular Health Metrics: Joint Impact on Major Adverse Cardiovascular Events in a Prospective Cohort Study.

BACKGROUND: This study examines the association between traditional cardiovascular health (CVH) metrics and major adverse cardiovascular events (MACE) incidence in individuals with diverse sleep patterns. METHODS AND RESULTS: We analyzed data from 208 621 participants initially free of cardiovascular disease (CVD) in the UK Biobank study. Sleep patterns were assessed using scores for chronotype, duration, insomnia, snoring, and daytime dozing. Traditional CVH scores were derived from the Life's Simple 7 metrics. Cox proportional hazards multivariate regression assessed associations between distinct combinations of CVH and sleep scores and MACE, including nonfatal myocardial infarction, nonfatal stroke, and CVD mortality. Over a mean follow-up of 12.73 years, 9253 participants experienced incident MACE. Individuals with both a healthy sleep pattern and ideal CVH levels had the lowest MACE risk compared with those with a poor sleep pattern and poor CVH levels (hazard ratio, 0.306 [95% CI, 0.257-0.365]; P<0.001). Elevated CVH scores were associated with a reduced risk of MACE across different sleep patterns. Similar trends were observed for individual MACE components, heart failure, and all-cause mortality. These findings remained robust in sensitivity analyses and across various subgroups. CONCLUSIONS: In individuals without known CVD, maintaining a favorable sleep pattern and achieving optimal CVH levels, as measured by traditional metrics, were associated with the lowest MACE risk. Enhanced CVH significantly reduced CVD risk, even in individuals with a poor sleep pattern. These results emphasize the importance of considering multiple dimensions of sleep health alongside CVH to mitigate CVD risk. REGISTRATION: URL: https://www.ukbiobank.ac.uk; Unique identifier: 91090.

PbARF19-mediated auxin signaling regulates lignification in pear fruit stone cells.

The stone cells in pear fruits cause rough flesh and low juice, seriously affecting the taste. Lignin has been demonstrated as the main component of stone cells. Auxin, one of the most important plant hormone, regulates most physiological processes in plants including lignification. However, the concentration effect and regulators of auxin on pear fruits stone cell formation remains unclear. Here, endogenous indole-3-acetic acid (IAA) and stone cells were found to be co-localized in lignified cells by immunofluorescence localization analysis. The exogenous treatment of different concentrations of IAA demonstrated that the application of 200 µM IAA significantly reduced stone cell content, while concentrations greater than 500 µM significantly increased stone cell content. Besides, 31 auxin response factors (ARFs) were identified in pear genome. Putative ARFs were predicted as critical regulators involved in the lignification of pear flesh cells by phylogenetic relationship and expression analysis. Furthermore, the negative regulation of PbARF19 on stone cell formation in pear fruit was demonstrated by overexpression in pear fruitlets and Arabidopsis. These results illustrated that the PbARF19-mediated auxin signal plays a critical role in the lignification of pear stone cell by regulating lignin biosynthetic genes. This study provides theoretical and practical guidance for improving fruit quality in pear production.

Cardiovascular health metrics defined by Life's Essential 8 scores and subsequent macrovascular and microvascular complications in individuals with type 2 diabetes: A prospective cohort study.

AIM: To investigate the association between cardiovascular health metrics defined by Life's Essential 8 (LE8) scores and vascular complications among individuals with type 2 diabetes (T2D). MATERIALS AND METHODS: This prospective study included 11 033 participants with T2D, all devoid of macrovascular diseases (including cardiovascular and peripheral artery disease) and microvascular complications (e.g. diabetic retinopathy, neuropathy and nephropathy) at baseline from the UK Biobank. The LE8 score comprised eight metrics: smoking, body mass index, physical activity, non-high-density lipoprotein cholesterol, blood pressure, glycated haemoglobin, diet and sleep duration. Cox proportional hazards models were established to assess the associations of LE8 scores with incident macrovascular and microvascular complications. RESULTS: During a median follow-up of 12.1 years, we identified 1975 cases of incident macrovascular diseases and 1797 cases of incident microvascular complications. After adjusting for potential confounders, each 10-point increase in the LE8 score was associated with an 18% lower risk of macrovascular diseases and a 15% lower risk of microvascular complications. Comparing individuals in the highest and lowest quartiles of LE8 scores revealed hazard ratios of 0.55 (95% confidence interval 0.47-0.62) for incident macrovascular diseases, and 0.61 (95% confidence interval 0.53-0.70) for incident microvascular complications. This association remained robust across a series of sensitivity analyses and nearly all subgroups. CONCLUSION: Higher LE8 scores were associated with a lower risk of incident macrovascular and microvascular complications among individuals with T2D. These findings underscore the significance of adopting fundamental strategies to maintain optimal cardiovascular health and curtail the risk of developing diabetic vascular complications.

Predictive factors and the management of hyperglycemia in patients with acromegaly and Cushing's disease receiving pasireotide treatment: post hoc analyses from the SOM230B2219 study.

Introduction: Pasireotide, a somatostatin receptor ligand, is approved for treating acromegaly and Cushing's disease (CD). Hyperglycemia during treatment can occur because of the drug's mechanism of action, although treatment discontinuation is rarely required. The prospective, randomized, Phase IV SOM230B2219 (NCT02060383) trial was designed to assess optimal management of pasireotide-associated hyperglycemia. Here, we investigated predictive factors for requiring antihyperglycemic medication during pasireotide treatment. Methods: Participants with acromegaly or CD initiated long-acting pasireotide 40 mg/28 days intramuscularly (acromegaly) or pasireotide 600 µg subcutaneously twice daily during pre-randomization (≤16 weeks). Those who did not need antihyperglycemic medication, were managed with metformin, or received insulin from baseline entered an observational arm ending at 16 weeks. Those who required additional/alternative antihyperglycemic medication to metformin were randomized to incretin-based therapy or insulin for an additional 16 weeks. Logistic-regression analyses evaluated quantitative and qualitative factors for requiring antihyperglycemic medication during pre-randomization. Results: Of 190 participants with acromegaly and 59 with CD, 88 and 15, respectively, did not need antihyperglycemic medication; most were aged <40 years (acromegaly 62.5%, CD 86.7%), with baseline glycated hemoglobin (HbA1c) <6.5% (<48 mmol/mol; acromegaly 98.9%, CD 100%) and fasting plasma glucose (FPG) <100 mg/dL (<5.6 mmol/L; acromegaly 76.1%, CD 100%). By logistic regression, increasing baseline HbA1c (odds ratio [OR] 3.6; P=0.0162) and FPG (OR 1.0; P=0.0472) and history of diabetes/pre-diabetes (OR 3.0; P=0.0221) predicted receipt of antihyperglycemic medication in acromegaly participants; increasing baseline HbA1c (OR 12.6; P=0.0276) was also predictive in CD participants. Investigator-reported hyperglycemia-related adverse events were recorded in 47.9% and 54.2% of acromegaly and CD participants, respectively, mainly those with diabetes/pre-diabetes. Conclusion: Increasing age, HbA1c, and FPG and pre-diabetes/diabetes were associated with increased likelihood of requiring antihyperglycemic medication during pasireotide treatment. These risk factors may be used to identify those who need more vigilant monitoring to optimize outcomes during pasireotide treatment.

Multi-omics provide insights into the regulation of DNA methylation in pear fruit metabolism.

BACKGROUND: Extensive research has been conducted on fruit development in crops, but the metabolic regulatory networks underlying perennial fruit trees remain poorly understood. To address this knowledge gap, we conduct a comprehensive analysis of the metabolome, proteome, transcriptome, DNA methylome, and small RNAome profiles of pear fruit flesh at 11 developing stages, spanning from fruitlet to ripening. Here, we systematically investigate the metabolic landscape and regulatory network involved. RESULTS: We generate an association database consisting of 439 metabolites and 14,399 genes to elucidate the gene regulatory network of pear flesh metabolism. Interestingly, we detect increased DNA methylation in the promoters of most genes within the database during pear flesh development. Application of a DNA methylation inhibitor to the developing fruit represses chlorophyll degradation in the pericarp and promotes xanthophyll, ß-carotene, and abscisic acid (ABA) accumulation in the flesh. We find the gradual increase in ABA production during pear flesh development is correlated with the expression of several carotenoid pathway genes and multiple transcription factors. Of these transcription factors, the zinc finger protein PbZFP1 is identified as a positive mediator of ABA biosynthesis in pear flesh. Most ABA pathway genes and transcription factors are modified by DNA methylation in the promoters, although some are induced by the DNA methylation inhibitor. These results suggest that DNA methylation inhibits ABA accumulation, which may delay fruit ripening. CONCLUSION: Our findings provide insights into epigenetic regulation of metabolic regulatory networks during pear flesh development, particularly with regard to DNA methylation.

Structure-Piezoelectric Property Relationships of Thin Films Composed of Electrospun Aligned Poly(vinylidene fluoride) Nanofibers.

In the past two decades, many studies on piezoelectric nanofibers (NFs) prepared from poly(vinylidene fluoride) (PVDF) and its copolymers, including single NFs, randomly oriented nonwoven mats, and aligned NFs, have been reported. However, studies on the relationships between the PVDF NF diameter, the orientation of the ß-phase crystals inside NFs, and the piezoelectric properties of the NFs are still limited. In this study, the effect of the fiber diameter on the internal molecular packing/orientation and piezoelectric properties of aligned PVDF NF thin films was investigated. Herein, piezoelectric thin films composed of densely packed, uniaxially aligned, PVDF NFs with diameters ranging from 228 to 1315 nm were prepared by means of electrospinning with a rotating collector and successive hot-pressing and poling. The effect of the diameters of PVDF NFs on their internal structures, as well as the piezoelectric properties of the thin films, was investigated. All prepared NFs mainly contained ß-phase crystals with a similar total crystallinity. The orientation of the ß-phase crystals inside the NFs increased with an increase in the fiber diameter, resulting in an improved transverse piezoelectric coefficient (d31) for the thin films. The output voltage of the prepared thin films reached a maximum of 2.7 V at 104 Hz.

PbrWRKY62-PbrADC1 module involves in superficial scald development of Pyrus bretschneideri Rehd.fruit via regulating putrescine biosynthesis.

Putrescine plays a role in superficial scald development during the cold storage of pear fruit. However, the molecular mechanism behind this phenomenon has not been un-fully clarified until recently. In this study, a conjoint analysis of metabolites and gene expression profiles in the putrescine-metabolic pathway of P. bretschneideri Rehd. fruit followed by experimental validation revealed that PbrADC1, forming a homodimer in the chloroplast, was involved in putrescine biosynthesis and thus fruit chilling resistance. Additionally, the substrate-binding residue Cys546 in PbrADC1, whose activity was modified by H2O2, played a crucial role in arginine decarboxylation into agmatine. Through a combined analysis of the distribution of cis-acting elements in the PbrADC1 promoter as well as the expression profiles of related transcription factors (TFs), several TFs were identified as upstream regulators of PbrADC1 gene. Further investigation revealed that the nuclear PbrWRKY62 could directly bind to the W-box elements in the PbrADC1 promoter, activate its expression, enhance putrescine accumulation, and thus increase fruit chilling tolerance. In conclusion, our results suggest that the PbrWRKY62-PbrADC1 module is involved in the development of superficial scald in P. bretschneideri Rehd. fruit via regulating putrescine biosynthesis. Consequently, these findings could serve as valuable genetic resources for breeding scald-resistant pear fruit.

PuNDH9, a subunit of ETC Complex I regulates plant defense by interacting with PuPR1.

NAD+ and NADH play critical roles in energy metabolism, cell death, and gene expression. The NADH-ubiquinone oxidoreductase complex (Complex I) has been long known as a key enzyme in NAD+ and NADH metabolism. In the present study, we found and analyzed a new subunit of Complex I (NDH9), which was isolated from Pyrus ussuriensis combined with RT-PCR. Following infection with A. alternata, RT-qPCR analysis demonstrated an increase in the expression of PuNDH9. Genetic manipulation of PuNDH9 levels suggested that PuNDH9 plays key roles in NADH/NAD+ homeostasis, defense enzyme activities, ROS generation, cell death, gene expression, energy metabolism, and mitochondrial functions during the pear- A. alternata interaction. Furthermore, Y2H, GST-pull down, and a split-luciferase complementation imaging assays revealed that PuNDH9 interacts with PuPR1. We discover that PuNDH9 and PuPR1 synergistically activate defense enzyme activities, ROS accumulation, cell death, and plant defenses. Collectively, our findings reveal that PuNDH9 is likely important for plant defenses.

Identification and expression analysis of ATP-binding cassette (ABC) transporters revealed its role in regulating stress response in pear (Pyrus bretchneideri).

BACKGROUND: ATP-binding cassette (ABC) transporter proteins constitute a plant gene superfamily crucial for growth, development, and responses to environmental stresses. Despite their identification in various plants like maize, rice, and Arabidopsis, little is known about the information on ABC transporters in pear. To investigate the functions of ABC transporters in pear development and abiotic stress response, we conducted an extensive analysis of ABC gene family in the pear genome. RESULTS: In this study, 177 ABC transporter genes were successfully identified in the pear genome, classified into seven subfamilies: 8 ABCAs, 40 ABCBs, 24 ABCCs, 8 ABCDs, 9 ABCEs, 8 ABCFs, and 80 ABCGs. Ten motifs were common among all ABC transporter proteins, while distinct motif structures were observed for each subfamily. Distribution analysis revealed 85 PbrABC transporter genes across 17 chromosomes, driven primarily by WGD and dispersed duplication. Cis-regulatory element analysis of PbrABC promoters indicated associations with phytohormones and stress responses. Tissue-specific expression profiles demonstrated varied expression levels across tissues, suggesting diverse functions in development. Furthermore, several PbrABC genes responded to abiotic stresses, with 82 genes sensitive to salt stress, including 40 upregulated and 23 downregulated genes. Additionally, 91 genes were responsive to drought stress, with 22 upregulated and 36 downregulated genes. These findings highlight the pivotal role of PbrABC genes in abiotic stress responses. CONCLUSION: This study provides evolutionary insights into PbrABC transporter genes, establishing a foundation for future research on their functions in pear. The identified motifs, distribution patterns, and stress-responsive expressions contribute to understanding the regulatory mechanisms of ABC transporters in pear. The observed tissue-specific expression profiles suggest diverse roles in developmental processes. Notably, the significant responses to salt and drought stress emphasize the importance of PbrABC genes in mediating adaptive responses. Overall, our study advances the understanding of PbrABC transporter genes in pear, opening avenues for further investigations in plant molecular biology and stress physiology.

PbrCSP1, a pollen tube-specific cold shock domain protein, is essential for the growth and cold resistance of pear pollen tubes.

Cold shock domain proteins (CSPs), initially identified in Escherichia coli, have been demonstrated to play a positive role in cold resistance. Previous studies in wheat, rice, and Arabidopsis have indicated the functional conservation of CSPs in cold resistance between bacteria and higher plants. However, the biological functions of PbrCSPs in pear pollen tubes, which represent the fragile reproductive organs highly sensitive to low temperature, remain largely unknown. In this study, a total of 22 CSPs were identified in the seven Rosaceae species, with a focus on characterizing four PbrCSPs in pear (Pyrus bretschneideri Rehder). All four PbrCSPs were structurally conserved and responsive to the abiotic stresses, such as cold, high osmotic, and abscisic acid (ABA) treatments. PbrCSP1, which is specifically expressed in pear pollen tubes, was selected for further research. PbrCSP1 was localized in both the cytoplasm and nucleus. Suppressing the expression of PbrCSP1 significantly inhibited the pollen tube growth in vitro. Conversely, overexpression of PbrCSP1 promoted the growth of pear pollen tubes under the normal condition and, notably, under the cold environment at 4 °C. These findings highlight an essential role of PbrCSP1 in facilitating the normal growth and enhancing cold resistance in pear pollen tubes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01457-w.

PbWRKY18 promotes resistance against black spot disease by activation of the chalcone synthase gene PbCHS3 in pear.

Flavonoids are plant pigments that play a major role in plant defense and have significant health benefits to humans. Chalcone synthase (CHS) is an important enzyme in flavonoid biosynthesis and investigation transcription factors (TFs) regulating its expression and downstream targets is critical to understanding its mechanism. Here, a novel TF, PbWRKY18, was isolated from the pear Pyrus betulaefolia. Its expression was evaluated in various tissues by RT-PCR, particularly in response to Alternaria alternata, the pathogen responsible for black spot disease, and exogenous hormone administration. The PbWRKY18 protein was primarily found in the nucleus where it regulated transcriptional activity. Yeast one-hybrid and dual-luciferase reporter assays showed a strong association between PbWRKY18 and the PbCHS3 promoter, which drives PbCHS3 expression. It was also found that PbCHS3 was critical for the development of resistance against black spot disease. In addition, PbWRKY18 was found to significantly increase the expression of PbCHS3 and salicylic acid-related genes, as well as defense enzyme activity and tolerance to black spot disease. PbWRKY18 or PbCHS3 knockdown in pear attenuates resistance to Alternaria alternata. In summary, the study identified a novel WRKY18-CHS3 axis involved in resistance against black spot disease in pear.

PbRbohH/J mediates ROS generation to regulate the growth of pollen tube in pear.

Respiratory burst oxidase homolog (Rboh) family genes play crucial functions in development and growth. However, comprehensive and systematic investigation of Rboh family members in Rosaceae and their specific functions during pear pollen development are still limited. In the study, 63 Rboh genes were identified from eight Rosaceae genomes (Malus domestica, Pyrus bretschneideri, Pyrus communis, Prunus persica, Rubus occidentalis, Fragaria vesca, Prunus mume and Prunus avium) and divided into seven main subfamilies (I-VII) according to phylogenetic and structural features. Different modes of gene duplication led to the expansion of Rboh family, with purifying selection playing a vital role in the evolution of Rboh genes. In addition, RNA sequencing and qRT-PCR results indicated that PbRbohH and PbRbohJ were specifically high-expressed in pear pollen. Subsequently, subcellular localization revealed that PbRbohH/J distributed at the plasma membrane. Furthermore, by pharmacological analysis and antisense oligodeoxynucleotide assay, PbRbohH/J were demonstrated to mediate the formation of reactive oxygen species (ROS) to manage pollen tube growth. In conclusion, our results provide useful insights into the functions, expression patterns, evolutionary history of the Rboh genes in pear and other Rosaceae species.

Septin9 DNA methylation is associated with breast cancer recurrence or metastasis.

OBJECTIVE: We aimed to explore the prognostic value of Septin9 DNA methylation in breast cancer. METHODS: Breast cancer patients with and without recurrence or metastasis and matched non-breast cancer patients were screened retrospectively from 2014 to 2016. Bisulfite conversion and fluorescence quantitative methylation-specific polymerase chain reaction were used to detect the Septin9 methylation status and distribution levels in patient breast tissues. RESULTS: Septin9 DNA methylation was more frequent in breast cancer tissues than in non-breast cancer tissues, but was not significantly correlated with any relevant breast cancer patient clinicopathological characteristic. Septin9 methylation rates were higher in patients with recurrence or metastasis. Septin9 methylation, tumor size, lymph node status, and progesterone receptor (PR) expression could influence prognosis. Septin9 methylation was significantly associated with worse disease-free survival in breast cancer patients, with receiver operating characteristic curve analysis indicating that it had good prognostic ability, with an area under the curve (AUC) value of 0.719. The AUC values increased when Septin9 methylation was combined with tumor size, lymph node status, and PR to predict prognosis. CONCLUSIONS: Septin9 DNA methylation was an independent predictors of breast cancer prognostic risk. This could possibly help improve comprehensive prognosis prediction methods when combined with other risk factors.

The transcription factor PbbHLH164 is destabilized by PbRAD23C/D.1 and mediates ethylene biosynthesis during pear fruit ripening.

The phytohormone ethylene plays an important role in climacteric fruit ripening. However, the knowledge on molecular regulation of ethylene biosynthesis remains limited in pear fruit. Herein, a new basic helix-loop-helix transcription factor, PbbHLH164, was identified based on the transcriptome analysis of different developing and ripening fruits of two pear cultivars 'Sucui No. 1' and 'Cuiguan'. PbbHLH164 was more highly expressed in ripening fruit than in developing fruit and positively correlated with ethylene production in both cultivars. PbbHLH164 could directly bind to the promoter of 1-aminocyclopropane-1-carboxylate synthase, PbACS1b, to enhance the expression, leading to the increase of ethylene production and the acceleration of fruit ripening. Interestingly, PbbHLH164 physically interacted with an ubiquitin-like/ubiquitin-associated protein PbRAD23C/D.1, and the interaction of PbbHLH164 with PbRAD23C/D.1 attenuated the function of PbbHLH164 in enhancing the activity of the PbACS1b promoter. Notably, PbRAD23C/D.1 was involved in the degradation of PbbHLH164, and this degradation was inhibited by an ubiquitin proteasome inhibitor MG132. Different from PbbHLH164, PbRAD23C/D.1 was more highly expressed in developing fruit than in ripening fruit of both cultivars. These results suggest that the increase of ethylene production during pear fruit ripening results from the up-regulated expression of PbbHLH164 and the down-regulated expression of PbRAD23C/D.1. This information provided new insights into the molecular regulation of ethylene biosynthesis during fruit ripening.