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.

PbrbZIP15 promotes sugar accumulation in pear via activating the transcription of the glucose isomerase gene PbrXylA1.

The composition and abundance of soluble sugars in mature pear (Pyrus) fruit are important for its acceptance by consumers. However, our understanding of the genes responsible for soluble sugar accumulation remains limited. In this study, a S1-group member of bZIP gene family, PbrbZIP15, was characterized from pear genome through the combined analyses of metabolite and transcriptome data followed by experimental validation. PbrbZIP15, located in nucleus, was found to function in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli. After analyzing the expression profiles of sugar-metabolism-related genes and the distribution of cis-acting elements in their promoters, the glucose isomerase 1 gene (PbrXylA1), whose corresponding protein catalyzed the isomerization of glucose and fructose in vitro, was identified as a downstream target gene of PbrbZIP15. PbrbZIP15 could directly bind to the G-box element in PbrXylA1 promoter and activate its transcription, as evidenced by chromatin immunoprecipitation-quantitative PCR, yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase assay. PbrXylA1, featuring a leucine-rich signal peptide in its N-terminal, was localized to the endoplasmic reticulum. It was validated to play a significant role in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli, which was associated with the upregulated fructose/glucose ratio. Further studies revealed a positive correlation between the sucrose content and the expression levels of several sucrose-biosynthesis-related genes (PbrFRK3/8, PbrSPS1/3/4/8, and PbrSPP1) in PbrbZIP15-/PbrXylA1-transgenic fruit/calli. In conclusion, our results suggest that PbrbZIP15-induced soluble sugar accumulation during pear development is at least partly attributed to the activation of PbrXylA1 transcription.

Genome-wide identification of the mitogen-activated protein kinase kinase kinase (MAPKKK) in pear (Pyrus bretschneideri) and their functional analysis in response to black spot.

MAIN CONCLUSION: Identification of MAPKKK genes in pear and functional characterization of PbrMAPKKK82 in response to pear black spot. Mitogen-activated protein kinase kinase kinase (MAPKKK) is located upstream of the MAPK cascade pathway. This region senses extracellular stimuli via the signaling molecule or by themselves and is activated by phosphorylation. In this study, we identified 108 PbrMAPKKK genes from the pear genome. The genes were divided into three subfamilies and contained the conserved domain. Except for chromosome 7, there were 93 PbrMAPKKK genes randomly distributed on 16 out of the 17 chromosomes, while 15 PbrMAPKKK genes were detected on unknown chromosomes. They largely originated from whole-genome duplication (WGD) and dispersed events. In the expression analysis of PbrMAPKKK genes in seven pear tissue types by using a database, 20 PbrMAPKKK genes were selected to verify the expression associated with different resistance in two varieties by quantitative real-time PCR (qRT-PCR). The results showed that PbrMAPKKK12, PbrMAPKKK13, PbrMAPKKK53, PbrMAPKKK60, PbrMAPKKK65, PbrMAPKKK82, PbrMAPKKK83, and PbrMAPKKK96 were correlated with black spot resistance. PbrMAPKKK3, PbrMAPKKK9, PbrMAPKKK11, PbrMAPKKK34, PbrMAPKKK80, PbrMAPKKK81, PbrMAPKKK99, and PbrMAPKKK100 were correlated with black spot susceptibility, while the PbrMAPKKK gene positively responded to the life process of pear resistance to black spot. Furthermore, virus-induced gene silencing (VIGS) indicated that the PbrMAPKKK82 gene enhanced resistance to pear black spot disease.

Transcriptome and Resequencing Analyses Provide Insight into Differences in Organic Acid Accumulation in Two Pear Varieties.

Fruit acidity is one of the main determinants of fruit flavor and a target trait in fruit breeding. However, the genomic mechanisms governing acidity variation among different pear varieties remain poorly understood. In this study, two pear varieties with contrasting organic acid levels, 'Dangshansuli' (low-acidity) and 'Amute' (high-acidity), were selected, and a combination of transcriptome and population genomics analyses were applied to characterize their patterns of gene expression and genetic variation. Based on RNA-seq data analysis, differentially expressed genes (DEGs) involved in organic acid metabolism and accumulation were identified. Weighted correlation network analysis (WGCNA) revealed that nine candidate TCA (tricarboxylic acid)-related DEGs and three acid transporter-related DEGs were located in three key modules. The regulatory networks of the above candidate genes were also predicted. By integrating pear resequencing data, two domestication-related genes were found to be upregulated in 'Amute', and this trend was further validated for other pear varieties with high levels of organic acid, suggesting distinct selective sweeps during pear dissemination and domestication. Collectively, this study provides insight into organic acid differences related to expression divergence and domestication in two pear varieties, pinpointing several candidate genes for the genetic manipulation of acidity in pears.

Physiological and de novo transcriptome analysis of the fermentation mechanism of Cerasus sachalinensis roots in response to short-term waterlogging.

BACKGROUND: Cerasus sachalinensis is widely used in cool regions as a sweet cherry rootstock and is known for its sensitivity to soil waterlogging and waterlogging stress. However, the limited availability of Cerasus genomic resources has considerably restricted the exploration of its waterlogging response mechanism. To understand its reaction to short-term waterlogging, we analyzed the physiology and transcriptomes of C. sachalinensis roots in response to different waterlogging durations. RESULTS: In this study, 12,487 differentially expressed genes (DEGs) were identified from Cerasus sachalinensis roots under different waterlogging durations. Carbon metabolism and energy maintenance formed the first coping mechanism stage of C. sachalinensis in response to low oxygen conditions. Root energy processes, including root respiration and activities of the fermentation enzymes alcohol dehydrogenase, pyruvate decarboxylase, and lactate dehydrogenase, showed unique changes after 0 h, 3 h, 6 h, and 24 h of waterlogging exposure. Ribonucleic acid sequencing was used to analyze transcriptome changes in C. sachalinensis roots treated with 3 h, 6 h, and 24 h of waterlogging stress. After de novo assembly, 597,474 unigenes were recognized, of which 355,350 (59.47%) were annotated. To identify the most important pathways represented by DEGs, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to compare these genes. The first stage of root reaction to waterlogging stress was activation of carbohydrate metabolism to produce more glucose and maintain energy levels. At 3 h, the glycolytic and fermentation pathways were activated to maintain adenosine triphosphate production. At 24 h, pathways involved in the translation of proteins were activated to further assist the plant in tolerating waterlogging stress. These findings will facilitate a further understanding of the potential mechanisms of plant responses to waterlogging at physiological and transcriptome levels. CONCLUSIONS: Carbon metabolism and energy maintenance formed the first coping mechanism C. sachalinensis in response to low oxygen conditions, and they may be responsible for its short-term waterlogging response. Our study not only provides the assessment of genomic resources of Cerasus but also paves the way for probing the metabolic and molecular mechanisms underlying the short-term waterlogging response in C. sachalinensis.