Transcription factor PbbZIP4 is targeted for proteasome-mediated degradation by the ubiquitin ligase PbATL18 to influence pear's resistance to Colletotrichum fructicola by regulating the expression of PbNPR3.

Pear anthracnose caused by Colletotrichum fructicola is one of the main fungal diseases in all pear-producing areas. The degradation of ubiquitinated proteins by the 26S proteasome is a regulatory mechanism of eukaryotes. E3 ubiquitin ligase is substrate specific and is one of the most diversified and abundant enzymes in the regulation mechanism of plant ubiquitination. Although numerous studies in other plants have shown that the degradation of ubiquitinated proteins by the 26S proteasome is closely related to plant immunity, there are limited studies on them in pear trees. Here, we found that an E3 ubiquitin ligase, PbATL18, interacts with and ubiquitinates the transcription factor PbbZIP4, and this process is enhanced by C. fructicola infection. PbATL18 overexpression in pear callus enhanced resistance to C. fructicola infection, whereas PbbZIP4 overexpression increased sensitivity to C. fructicola infection. Silencing PbATL18 and PbbZIP4 in Pyrus betulaefolia seedlings resulted in opposite effects, with PbbZIP4 silencing enhancing resistance to C. fructicola infection and PbATL18 silencing increasing sensitivity to C. fructicola infection. Using yeast one-hybrid screens, an electrophoretic mobility shift assay, and dual-luciferase assays, we demonstrated that the transcription factor PbbZIP4 upregulated the expression of PbNPR3 by directly binding to its promoter. PbNPR3 is one of the key genes in the salicylic acid (SA) signal transduction pathway that can inhibit SA signal transduction. Here, we proposed a PbATL18-PbbZIP4-PbNPR3-SA model for plant response to C. fructicola infection. PbbZIP4 was ubiquitinated by PbATL18 and degraded by the 26S proteasome, which decreased the expression of PbNPR3 and promoted SA signal transduction, thereby enhancing plant C. fructicola resistance. Our study provides new insights into the molecular mechanism of pear response to C. fructicola infection, which can serve as a theoretical basis for breeding superior disease-resistant pear varieties.

PbHsfC1a-coordinates ABA biosynthesis and H2O2 signalling pathways to improve drought tolerance in Pyrus betulaefolia.

Abiotic stresses have had a substantial impact on fruit crop output and quality. Plants have evolved an efficient immune system to combat abiotic stress, which employs reactive oxygen species (ROS) to activate the downstream defence response signals. Although an aquaporin protein encoded by PbPIP1;4 is identified from transcriptome analysis of Pyrus betulaefolia plants under drought treatments, little attention has been paid to the role of PIP and ROS in responding to abiotic stresses in pear plants. In this study, we discovered that overexpression of PbPIP1;4 in pear callus improved tolerance to oxidative and osmotic stresses by reconstructing redox homeostasis and ABA signal pathways. PbPIP1;4 overexpression enhanced the transport of H2O2 into pear and yeast cells. Overexpression of PbPIP1;4 in Arabidopsis plants mitigates the stress effects caused by adding ABA, including stomatal closure and reduction of seed germination and seedling growth. Overexpression of PbPIP1;4 in Arabidopsis plants decreases drought-induced leaf withering. The PbPIP1;4 promoter could be bound and activated by TF PbHsfC1a. Overexpression of PbHsfC1a in Arabidopsis plants rescued the leaf from wilting under drought stress. PbHsfC1a could bind to and activate AtNCED4 and PbNCED4 promoters, but the activation could be inhibited by adding ABA. Besides, PbNCED expression was up-regulated under H2O2 treatment but down-regulated under ABA treatment. In conclusion, this study revealed that PbHsfC1a is a positive regulator of abiotic stress, by targeting PbPIP1;4 and PbNCED4 promoters and activating their expression to mediate redox homeostasis and ABA biosynthesis. It provides valuable information for breeding drought-resistant pear cultivars through gene modification.

Genome-wide identification and expression analysis of the bZIP transcription factors, and functional analysis in response to drought and cold stresses in pear (Pyrus breschneideri).

BACKGROUND: Transcription factors (TFs) are involved in many important biological processes, including cell stretching, histological differentiation, metabolic activity, seed storage, gene regulation, and response to abiotic and biotic stresses. Little is known about the functions, evolutionary history, and expression patterns of basic region-leucine zipper TF family genes in pear, despite the release of the genome of Chinese white pears ("Dangshansuli"). RESULTS: Overall, 92 bZIP genes were identified in the pear genome (Pyrus breschneideri). Of these, 83 were randomly distributed on all 17 chromosomes except chromosome 4, and the other 9 genes were located on loose scaffolding. The genes were divided into 14 subgroups. Whole-genome duplications, dispersed duplication, and purifying selection for whole-genome duplications are the main reasons for the expansion of the PbrbZIP gene family. The analysis of functional annotation enrichment indicated that most of the functions of PbrbZIP genes were enriched in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways involved in the abiotic stress response. Next, expression analysis and virus-induced gene silencing results indicated that PbrbZIP genes might play critical roles in response to drought and cold stresses, especially for the genes from subgroups A, C, G, I, and S. CONCLUSIONS: Ninety-two PbrbZIP genes were identified from the pear genome and classified into 14 subgroups. PbrbZIP genes were mainly expanded from whole-genome duplications and dispersed duplications and retained by purifying selection. PbrbZIP genes were induced by cold and drought stresses and played important roles in drought and cold tolerance. These results provided useful information for further increasing the tolerance of pears to stresses and a foundation to study the cold and drought tolerance mechanism of PbrbZIP genes.

Genome-wide identification of PbrbHLH family genes, and expression analysis in response to drought and cold stresses in pear (Pyrus bretschneideri).

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factors play important roles in many processes in plant growth, metabolism and responses to abiotic stresses. Although, the sequence of Chinese white pear genome (cv. 'Dangshansuli') has already been reported, there is still a lack of clarity regarding the bHLH family genes and their evolutionary history. RESULTS: In this work, a genome-wide identification of the bHLH genes in Chinese white pear was performed, and we characterized the functional roles of these PbrbHLH genes in response to abiotic stresses. Based on the phylogenetic analysis and structural characteristics, 197 identified bHLH genes could be well classified into 21 groups. Expansion of PbrbHLH gene family was mainly driven by WGD and dispersed duplication with the purifying selection from the recent WGD. The functional annotation enrichment showed that the majority of PbrbHLHs were enriched in the GO terms and KEGG pathways involved in responds to stress conditions as TFs. Transcriptomic profiles and qRT-PCR revealed that PbrbHLH7, PbrbHLH8, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 were significantly up-regulated under cold and drought treatments. In addition, PbrbHLH195-silenced pear seedlings display significant reduced cold tolerance, exhibiting reduced chlorophyll content, as well as increased electrolyte leakage and concentrations of malondialdehyde and H2O2. CONCLUSION: For the first time, a comprehensive analysis identified the bHLH genes in Chinese white pear and demonstrated that PbrbHLH195 is involved in the production of ROS in response to cold stress, suggesting that members of the PbrbHLH family play an essential role in the stress tolerance of pear.

A WRKY transcription factor PbrWRKY53 from Pyrus betulaefolia is involved in drought tolerance and AsA accumulation.

WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report the identification and functional characterization of PbrWRKY53 isolated from Pyrus betulaefolia. PbrWRKY53 was greatly up-regulated by drought and abscisic acid, but slightly induced by salt and cold. Subcellar localization analyses showed that PbrWRKY53 was located in the nucleus. Ectopic expression of PbrWRKY53 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to drought stress. The transgenic plants exhibited better water status, less reactive oxygen species generation and higher levels of antioxidant enzyme activities and metabolites than the wild type. In addition, overexpression of PbrWRKY53 in transgenic tobacco resulted in enhanced expression level of PbrNCED1, and led to the increase in larger amount of vitamin C accumulation in comparison to WT. Knock-down of PbrWRKY53 in P. ussuriensis down-regulated PbrNCED1 abundance, accompanied by compromised drought tolerance. Yeast one-hybrid assay, EMSA and transient expression analysis demonstrated that PbrWRKY53 could bind to the W-box element in the promoter region of PbrNCED1. Taken together, these results demonstrated that PbrWRKY53 plays a positive role in drought tolerance, which might be, at least in part, promoting production of vitamin C via regulating PbrNCED1 expression.

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.

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.

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.

PbrChiA: a key chitinase of pear in response to Botryosphaeria dothidea infection by interacting with PbrLYK1b2 and down-regulating ROS accumulation.

Pear ring rot, caused by the pathogenic fungi Botryosphaeria dothidea, seriously affects pear production. While the infection-induced reactive oxygen species (ROS) burst of infected plants limits the proliferation of B. dothidea during the early infection stage, high ROS levels can also contribute to their growth during the later necrotrophic infection stage. Therefore, it is important to understand how plants balance ROS levels and resistance to pathogenic B. dothidea during the later stage. In this study, we identified PbrChiA, a glycosyl hydrolases 18 (GH18) chitinase-encoding gene with high infection-induced expression, through a comparative transcriptome analysis. Artificial substitution, stable overexpression, and virus induced gene silencing (VIGS) experiments demonstrated that PbrChiA can positively regulate pear resistance as a secreted chitinase to break down B. dothidea mycelium in vitro and that overexpression of PbrChiA suppressed infection-induced ROS accumulation. Further analysis revealed that PbrChiA can bind to the ectodomain of PbrLYK1b2, and this interaction suppressed PbrLYK1b2-mediated chitin-induced ROS accumulation. Collectively, we propose that the combination of higher antifungal activity from abundant PbrChiA and lower ROS levels during later necrotrophic infection stage confer resistance of pear against B. dothidea.

PbrWRKY70 increases pear (Pyrus bretschneideri Rehd) black spot disease tolerance by negatively regulating ethylene synthesis via PbrERF1B-2.

Various pear plant cultivars exhibit diverse abilities to resist pear black spot disease (BSD), while the precise molecular mechanisms of resistance against pear BSD remain unclear. This study proposed a profound expression of a WRKY gene, namely PbrWRKY70, derived from Pyrus bretschneideri Rehd, within a BSD-resistant pear cultivar. Comparative analysis against the wild-type revealed that the overexpression of PbrWRKY70 engendered augmented BSD resistance of transgenic Arabidopsis thaliana and pear calli. Notably, the transgenic plants exhibited higher activities of superoxide dismutase and peroxidase, along with an elevated capacity to counteract superoxide anions via increased anti-O2-. Additionally, these plants displayed diminished lesion diameter, as well as reduced levels of hydrogen peroxide, malondialdehyde and 1-aminocyclopropane-1-carboxylic acid (ACC) contents. We subsequently demonstrated that PbrWRKY70 selectively bound to the promoter region of ethylene-responsive transcription factor 1B-2 (PbrERF1B-2), a potential negative regulator of ACC, thereby downregulating the expression of ACC synthase gene (PbrACS3). Consequently, we confirmed that PbrWRKY70 could enhance pear resistance against BSD by reducing ethylene production via modulation of the PbrERF1B-2-PbrACS3 pathway. This study established the pivotal relationship among PbrWRKY70, ethylene synthesis and pear BSD resistance, fostering the development of novel BSD-resistant cultivars. Furthermore, this breakthrough holds the potential to enhance pear fruit yield and optimize storage and processing during the later stages of fruit maturation.

Maize bHLH55 functions positively in salt tolerance through modulation of AsA biosynthesis by directly regulating GDP-mannose pathway genes.

Ascorbic acid (AsA) is an antioxidant and enzyme co-factor that is vital to plant development and abiotic stress tolerance. However, the regulation mechanisms of AsA biosynthesis in plants remain poorly understood. Here, we report a basic helix-loop-helix 55 (ZmbHLH55) transcription factor that regulates AsA biosynthesis in maize. Analysis of publicly available transcriptomic data revealed that ZmbHLH55 is co-expressed with several genes of the GDP-mannose pathway. Experimental data showed that ZmbHLH55 forms homodimers localized to the cell nuclei, and it exhibits DNA binding and transactivation activity in yeast. Under salt stress conditions, knock down mutant (zmbhlh55) in maize accumulated lower levels of AsA compared with wild type, accompanied by lower antioxidant enzymes activity, shorter root length, and higher malondialdehyde (MDA) level. Gene expression data from the WT and zmbhlh55 mutant, showed that ZmbHLH55 positively regulates the expression of ZmPGI2, ZmGME1, and ZmGLDH, but negatively regulates ZmGMP1 and ZmGGP. Furthermore, ZmbHLH55-overexpressing Arabidopsis, under salt conditions, showed higher AsA levels, increased rates of germination, and elevated antioxidant enzyme activities. In conclusion, these results have identified previously unknown regulation mechanisms for AsA biosynthesis, indicating that ZmbHLH55 may be a potential candidate to enhance plant salt stress tolerance in the future.

Transcriptomic and evolutionary analyses of white pear (Pyrus bretschneideri) ß-amylase genes reveals their importance for cold and drought stress responses.

ß-amylase (BAM) genes play essential roles in plant abiotic stress responses. Although the genome of Chinese white pear (Pyrus bretschneideri) has recently been made available, knowledge regarding the BAM family in pear, including gene function, evolutionary history and patterns of gene expression remains limited. In this study, we identified 17 PbBAMs in the pear genome. Of these, 12 PbBAM members were mapped onto 9 chromosomes and 5 PbBAM genes were located on scaffold contigs. Based on gene structure, protein motif analysis, and the topology of the phylogenetic tree of the PbBAM family, we classified member genes into 4 groups. All PbBAM genes were found to contain typical glycosyl hydrolysis 14 domain motifs. Interfamilial comparisons revealed that the phylogenetic relationships of BAM genes in other Rosaceae species were similar those found in pear. We also found that whole-genome duplication (WGD)/segmental duplication events played critical roles in the expansion of the BAM family. Next, we used transcriptomic data to study gene expression during the response of drought and low temperate responses, and found that genes in Group B were related to drought and cold stress. We identified four PbBAM genes associated with abiotic stress in Pear. Finally, by analyzing co-expression networks and co-regulatory genes, we found that PbBAM1a and PbBAM1b were associated with the pear abiotic stress response.

Unbiased subgenome evolution following a recent whole-genome duplication in pear (Pyrus bretschneideri Rehd.).

Genome fractionation (also known as diploidization) frequently occurs following paleopolyploidization events. Biased fractionation between subgenomes has been found in some paleo-allopolyploids, while this phenomenon is absent in paleo-autopolyploids. Pear (Pyrus bretschneideri Rehd.) experienced a recent whole-genome duplication (WGD, ~30 million years ago); however, the evolutionary fate of the two subgenomes derived from this WGD event is not clear. In this study, we identified the two paleo-subgenomes in pear using peach (Prunus persica) as an outgroup and investigated differences in the gene loss rate, evolutionary rate, gene expression level, and DNA methylation level between these two subgenomes. Fractionation bias was not found between the two pear subgenomes, which evolved at similar evolutionary rates. The DNA methylation level of the two subgenomes showed little bias, and we found no expression dominance between the subgenomes. However, we found that singleton genes and homeologous genes within each subgenome showed divergent evolutionary patterns of selective constraints, expression and epigenetic modification. These results provide insights into subgenome evolution following paleopolyploidization in pear.

Overexpression of PbrNHX2 gene, a Na+/H+ antiporter gene isolated from Pyrus betulaefolia, confers enhanced tolerance to salt stress via modulating ROS levels.

Intracellular Na+/H+ antiporters (NHXs) play important roles in plant tolerance to salt stress. However, plant NHXs functioning in salt tolerance and the underlying physiological mechanisms remain poorly understood. In this report, we report the identification and functional characterization of PbrNHX2 isolated from Pyrus betulaefolia. PbrNHX2 expression levels were induced by salt, and dehydration, but was unaffected by cold. PbrNHX2 was localized in the tonoplast. Overexpression of PbrNHX2 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to salt tolerance, whereas down-regulation of PbrNHX2 in Pyrus betulaefolia by virus-induced gene silencing (VIGS) resulted in elevated salt sensitivity. The transgenic lines contained lower levels of Na+, higher levels of K+, and higher K/Na ratio, whereas they were changed in an opposite way when PbrNHX2 was silenced. In addition, the transgenic plants accumulated lower levels of reactive oxygen species compared with wild type, accompanied by higher activities of three antioxidant enzymes. Taken together, the data demonstrate that PbrNHX2 plays a positive role in salt tolerance and that it holds a great potential for engineering salt tolerance in crops.