Soybean symbiotic-nodule zonation and cell differentiation are defined by NIN2 signaling and GH3-dependent auxin homeostasis.

Symbiotic nodules comprise two classes, indeterminate and determinate, defined by the presence/absence of apical meristem and developmental zonation. Why meristem and zonation are absent from determinate nodules remains unclear. Here, we define cell types in developing soybean nodules, highlighting the undifferentiated infection zones and differentiated nitrogen-fixation zones. Auxin governs infection zone maintenance. GRETCHEN HAGEN 3 (GH3) enzymes deactivate auxin by conjugation and promote cell differentiation. gh3 mutants increased undifferentiated cells and enlarged infection zones. The central symbiosis-transcription factor NIN2a activates GH3.1 to reduce auxin levels and facilitates cell differentiation. High auxin promotes NIN2a protein accumulation and enhances signaling, further deactivating auxin and depleting infection zones. Our findings shed light on the NIN2a-GH3-auxin module that drives soybean nodule cell differentiation. This study challenges our understanding of determinate nodule development and proposes that the regulation of nodule zonation offers valuable insights into broader mechanisms of cell differentiation across plant species.

Thyroid and neurobehavioral effects of DiNP on GH3 cells and larval zebrafish (Danio rerio).

Diisononyl phthalate (DiNP) has been used to replace bis(2-ethylhexyl) phthalate (DEHP) and is frequently found in the environment and humans. DiNP is reported for its anti-androgenic activity; however, little is known about its effects on thyroid function and neurodevelopment. In the present study, the thyroid disruption and neurobehavioral alteration potential of DiNP and its major metabolites were assessed in a rat pituitary carcinoma cell line (GH3) and embryo-larval zebrafish (Danio rerio). In GH3 cells, exposure to DiNP and its metabolites not only increased proliferation but also induced transcriptional changes in several target genes, which were different from those observed with DEHP exposure. In larval fish, a 5-day exposure to DiNP caused significant increases in thyroid hormone levels, following a similar pattern to that reported for DEHP exposure. Following exposure to DiNP, the activity of the larval fish decreased, and neurodevelopment-related genes, such as c-fos, elavl3, and mbp, were down-regulated. These changes are generally similar to those observed for DEHP. Up-regulation of gap43 and down-regulation of elavl3 gene, which are important for both thyroid hormone production and neurodevelopment, respectively, support the potential for both thyroid and behavioral disruption of DiNP. Overall, these results emphasize the need to consider the adverse thyroid and neurodevelopmental effects in developing regulations for DEHP-replacing phthalates.

Use of xylosidase 3C from Segatella baroniae to discriminate xylan non-reducing terminus substitution characteristics.

OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) ß-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of ß-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

A novel GH3-ß-glucosidase from soda lake metagenomic libraries with desirable properties for biomass degradation.

Beta-glucosidases catalyze the hydrolysis of the glycosidic bonds of cellobiose, producing glucose, which is a rate-limiting step in cellulose biomass degradation. In industrial processes, ß-glucosidases that are tolerant to glucose and stable under harsh industrial reaction conditions are required for efficient cellulose hydrolysis. In this study, we report the molecular cloning, Escherichia coli expression, and functional characterization of a ß-glucosidase from the gene, CelGH3_f17, identified from metagenomics libraries of an Ethiopian soda lake. The CelGH3_f17 gene sequence contains a glycoside hydrolase family 3 catalytic domain (GH3). The heterologous expressed and purified enzyme exhibited optimal activity at 50 °C and pH 8.5. In addition, supplementation of 1 M salt and 300 mM glucose enhanced the ß-glucosidase activity. Most of the metal ions and organic solvents tested did not affect the ß-glucosidase activity. However, Cu2+ and Mn2+ ions, Mercaptoethanol and Triton X-100 reduce the activity of the enzyme. The studied ß-glucosidase enzyme has multiple industrially desirable properties including thermostability, and alkaline, salt, and glucose tolerance.

A peroxisomal cinnamate:CoA ligase-dependent phytohormone metabolic cascade in submerged rice germination.

The mechanism underlying the ability of rice to germinate underwater is a largely enigmatic but key research question highly relevant to rice cultivation. Moreover, although rice is known to accumulate salicylic acid (SA), SA biosynthesis is poorly defined, and its role in underwater germination is unknown. It is also unclear whether peroxisomes, organelles essential to oilseed germination and rice SA accumulation, play a role in rice germination. Here, we show that submerged imbibition of rice seeds induces SA accumulation to promote germination in submergence. Two submergence-induced peroxisomal Oryza sativa cinnamate:CoA ligases (OsCNLs) are required for this SA accumulation. SA exerts this germination-promoting function by inducing indole-acetic acid (IAA) catabolism through the IAA-amino acid conjugating enzyme GH3. The metabolic cascade we identified may potentially be adopted in agriculture to improve the underwater germination of submergence-intolerant rice varieties. SA pretreatment is also a promising strategy to improve submerged rice germination in the field.

Genome-wide identification and expression characterization of the GH3 gene family of tea plant (Camellia sinensis).

To comprehensively understand the characteristics of the GH3 gene family in tea plants (Camellia sinensis), we identified 17 CsGH3 genes and analyzed their physicochemical properties, phylogenetic relationships, gene structures, promoters, and expression patterns in different tissues. The study showed that the 17 CsGH3 genes are distributed on 9 chromosomes, and based on evolutionary analysis, the CsGH3 members were divided into three subgroups. Gene duplication analysis revealed that segmental duplications have a significant impact on the amplification of CsGH3 genes. In addition, we identified and classified cis-elements in the CsGH3 gene promoters and detected elements related to plant hormone responses and non-biotic stress responses. Through expression pattern analysis, we observed tissue-specific expression of CsGH3.3 and CsGH3.10 in flower buds and roots. Moreover, based on predictive analysis of upstream regulatory transcription factors of CsGH3, we identified the potential transcriptional regulatory role of gibberellin response factor CsDELLA in CsGH3.14 and CsGH3.15. In this study, we found that CsGH3 genes are involved in a wide range of activities, such as growth and development, stress response, and transcription. This is the first report on CsGH3 genes and their potential roles in tea plants. In conclusion, these results provide a theoretical basis for elucidating the role of GH3 genes in the development of perennial woody plants and offer new insights into the synergistic effects of multiple hormones on plant growth and development in tea plants.

Characterization and stabilization of GluLm and its application to deglycosylate dietary flavonoids and lignans.

This study describes the characterization of the recombinant GH3 aryl-ß-glucosidase "GluLm" from Limosilactobacillus mucosae INIA P508, followed by its immobilization on an agarose support with the aim of developing an efficient application to increase the availability and concentration of flavonoid and lignan aglycones in a vegetal beverage. In previous studies, heterologous GluLm-producing strains demonstrated a great capacity to deglycosylate flavonoids. Nevertheless, the physicochemical properties and substrate spectrum of the enzyme remained unknown up to now. A high production of purified GluLm was achieved (14 mg L-1). GluLm exhibited optimal activity at broad ranges of pH (5.0-8.0) and temperature (25-60°C), as well as high affinity (Km of 0.10 mmol L-1) and specific constant (86554.0 mmol L-1 s-1) against p-nitrophenyl-ß-D-glucopyranoside. Similar to other GH3 ß-glucosidases described in lactic acid bacteria, GluLm exhibited ß-xylosidase, ß-galactosidase, and ß-fucosidase activities. However, this study has revealed for the first time that a GH3 ß-glucosidase is capable to hydrolyze different families of glycosylated phenolics such as flavonoids and secoiridoids. Although it exhibited low thermal stability, immobilization of GluLm improved its thermostability and allowed the development of a beverage based on soybeans and flaxseed extract with high concentration of bioactive isoflavone (daidzein, genistein), lignan (secoisolariciresinol, pinoresinol, and matairesinol), and other flavonoid aglycones. KEY POINTS: • Limosilactobacillus mucosae INIA P508 GluLm was purified and biochemically characterized • Immobilized GluLm efficiently deglycosylated flavonoids and lignans from a vegetal beverage • A viable application to produce vegetal beverages with a high content of aglycones is described.

Auxin homeostasis is maintained by sly-miR167-SlARF8A/B-SlGH3.4 feedback module in the development of locular and placental tissues of tomato fruits.

The locular gel, produced by the placenta, is important for fruit flavor and seed development in tomato. However, the mechanism underlying locule and placenta development is not fully understood yet. Here, we show that two SlARF transcription factors, SlARF8B and SlARF8A (SlARF8A/B), promote the development of locular and placenta tissues. The expression of both SlARF8A and SlARF8B is repressed by sly-microRNA167 (sly-miR167), allowing for the activation of auxin downstream genes. In slarf8a, slarf8b, and slarf8a/b mutants, the auxin (IAA) levels are decreased, whereas the levels of inactive IAA conjugates including IAA-Ala, IAA-Asp, and IAA-Glu are increased. We further find that SlARF8B directly inhibits the expression of SlGH3.4, an acyl acid amino synthetase that conjugates the amino acids to IAA. Disruption of such auxin balance by the increased expression of SlGH3.4 or SlGH3.2 results in defective locular and placental tissues. Taken together, our findings reveal an important regulatory module constituted by sly-miR167-SlARF8A/B-SlGH3.4 during the development of locular and placenta tissues of tomato fruits.

Genome-wide identification, characterization, and expression analysis of the sweet potato (Ipomoea batatas [L.] Lam.) ARF, Aux/IAA, GH3, and SAUR gene families.

BACKGROUND: Auxins are known to have roles in the tuberization process in sweet potato (Ipomoea batatas [L.] Lam.) and these effects are mediated by various auxin signalling gene families. In this study, an analysis of the sweet potato genome was performed to identify the ARF, Aux/IAA, GH3, and SAUR auxin signalling gene family members in this crop. RESULTS: A total of 29 ARF, 39 Aux/IAA, 13 GH3, and 200 SAUR sequences were obtained, and their biochemical properties and gene expression profiles were analysed. The sequences were relatively conserved based on exon-intron structure, motif analysis, and phylogenetic tree construction. In silico expression analyses of the genes in fibrous and storage roots indicated that many sequences were not differentially expressed in tuberizing and non-tuberizing roots. However, some ARF, Aux/IAA, and SAUR genes were up-regulated in tuberizing storage roots compared to non-tuberizing fibrous roots while many GH3 genes were down-regulated. Additionally, these genes were expressed in a variety of plant parts, with some genes being highly expressed in shoots, leaves, and stems while others had higher expression in the roots. Some of these genes are up-regulated during the plant's response to various hormone treatments and abiotic stresses. Quantitative RT-PCR confirmation of gene expression was also conducted, and the results were concordant with the in silico analyses. A protein-protein interaction network was predicted for the differentially expressed genes, suggesting that these genes likely form part of a complex regulatory network that controls tuberization. These results confirm those of existing studies that show that auxin signalling genes have numerous roles in sweet potato growth and development. CONCLUSION: This study provides useful information on the auxin signalling gene families in Ipomoea batatas and suggests putative candidates for further studies on the role of auxin signalling in tuberization and plant development.

The Roles of GRETCHEN HAGEN3 (GH3)-Dependent Auxin Conjugation in the Regulation of Plant Development and Stress Adaptation.

The precise control of free auxin (indole-3-acetic acid, IAA) gradient, which is orchestrated by biosynthesis, conjugation, degradation, hydrolyzation, and transport, is critical for all aspects of plant growth and development. Of these, the GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetase family, pivotal in conjugating IAA with amino acids, has garnered significant interest. Recent advances in understanding GH3-dependent IAA conjugation have positioned GH3 functional elucidation as a hot topic of research. This review aims to consolidate and discuss recent findings on (i) the enzymatic mechanisms driving GH3 activity, (ii) the influence of chemical inhibitor on GH3 function, and (iii) the transcriptional regulation of GH3 and its impact on plant development and stress response. Additionally, we explore the distinct biological functions attributed to IAA-amino acid conjugates.

Comprehensive Analysis of GH3 Gene Family in Potato and Functional Characterization of StGH3.3 under Drought Stress.

As an important hormone response gene, Gretchen Hagen 3 (GH3) maintains hormonal homeostasis by conjugating excess auxin with amino acids during plant stress-related signaling pathways. GH3 genes have been characterized in many plant species, but they are rarely reported in potato. Here, 19 StGH3 genes were isolated and characterized. Phylogenetic analysis indicated that StGH3s were divided into two categories (group I and group III). Analyses of gene structure and motif composition showed that the members of a specific StGH3 subfamily are relatively conserved. Collinearity analysis of StGH3 genes in potato and other plants laid a foundation for further exploring the evolutionary characteristics of the StGH3 genes. Promoter analysis showed that most StGH3 promoters contained hormone and abiotic stress response elements. Multiple transcriptome studies indicated that some StGH3 genes were responsive to ABA, water deficits, and salt treatments. Moreover, qRT-PCR analysis indicated that StGH3 genes could be induced by phytohormones (ABA, SA, and MeJA) and abiotic stresses (water deficit, high salt, and low temperature), although with different patterns. Furthermore, transgenic tobacco with transient overexpression of the StGH3.3 gene showed positive regulation in response to water deficits by increasing proline accumulation and reducing the leaf water loss rate. These results suggested that StGH3 genes may be involved in the response to abiotic stress through hormonal signal pathways. Overall, this study provides useful insights into the evolution and function of StGH3s and lays a foundation for further study on the molecular mechanisms of StGH3s in the regulation of potato drought resistance.

GH3-mediated auxin inactivation attenuates multiple stages of lateral root development.

Lateral root (LR) positioning and development rely on the dynamic interplay between auxin production, transport but also inactivation. Nonetheless, how the latter affects LR organogenesis remains largely uninvestigated. Here, we systematically analyze the impact of the major auxin inactivation pathway defined by GRETCHEN HAGEN3-type (GH3) auxin conjugating enzymes and DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) in all stages of LR development using reporters, genetics and inhibitors in Arabidopsis thaliana. Our data demonstrate that the gh3.1/2/3/4/5/6 hextuple (gh3hex) mutants display a higher LR density due to increased LR initiation and faster LR developmental progression, acting epistatically over dao1-1. Grafting and local inhibitor applications reveal that root and shoot GH3 activities control LR formation. The faster LR development in gh3hex is associated with GH3 expression domains in and around developing LRs. The increase in LR initiation is associated with accelerated auxin response oscillations coinciding with increases in apical meristem size and LR cap cell death rates. Our research reveals how GH3-mediated auxin inactivation attenuates LR development. Local GH3 expression in LR primordia attenuates development and emergence, whereas GH3 effects on pre-initiation stages are indirect, by modulating meristem activities that in turn coordinate root growth with LR spacing.

The many faces of lysine acylation in proteins: Phytohormones as unexplored substrates.

Protein post-translational modification (PTM) is a ubiquitous process that occurs in most proteins. Lysine residues containing an ε-amino group are recognized as hotspots for the addition of different chemical groups. Lysine acetylation, extensively studied in histones, serves as an epigenetic hallmark capable of promoting changes in chromatin structure and availability. Acyl groups derived from molecules involved in carbohydrate and lipid metabolisms, such as lactate, succinate and hydroxybutyrate, were identified as lysine modifications of histones and other proteins. Lysine-acyltransferases do not exhibit significant substrate specificity concerning acyl donors. Furthermore, plant hormones harboring acyl groups often form conjugates with free amino acids to regulate their activity and function during plant physiological processes and responses, a process mediated by GH3 enzymes. Besides forming low-molecular weight conjugates, auxins have been shown to covalently modify proteins in bean seeds. Aside from auxins, other phytohormones with acyl groups are unexplored potential substrates for post-translational acylation of proteins. Using MS data searches, we revealed various proteins with lysine residues linked to auxin, abscisic acid, gibberellic acid, jasmonic acid, and salicylic acid. These findings raise compelling questions about the ability of plant hormones harboring carboxyl groups to serve as new candidates for protein acylation and acting in protein PTM and modulation.

Structural insights into a bacterial ß-glucosidase capable of degrading sesaminol triglucoside to produce sesaminol: toward the understanding of the aglycone recognition mechanism by the C-terminal lid domain.

The sesaminol triglucoside (STG)-hydrolyzing ß-glucosidase from Paenibacillus sp. (PSTG1), which belongs to glycoside hydrolase family 3 (GH3), is a promising catalyst for the industrial production of sesaminol. We determined the X-ray crystal structure of PSTG1 with bound glycerol molecule in the putative active site. PSTG1 monomer contained typical three domains of GH3 with the active site in domain 1 (TIM barrel). In addition, PSTG1 contained an additional domain (domain 4) at the C-terminus that interacts with the active site of the other protomer as a lid in the dimer unit. Interestingly, the interface of domain 4 and the active site forms a hydrophobic cavity probably for recognizing the hydrophobic aglycone moiety of substrate. The short flexible loop region of TIM barrel was found to be approaching the interface of domain 4 and the active site. We found that n-heptyl-ß-D-thioglucopyranoside detergent acts as an inhibitor for PSTG1. Thus, we propose that the recognition of hydrophobic aglycone moiety is important for PSTG1-catalyzed reactions. Domain 4 might be a potential target for elucidating the aglycone recognition mechanism of PSTG1 as well as for engineering PSTG1 to create a further excellent enzyme to degrade STG more efficiently to produce sesaminol.

Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism.

Processive and distributive catalysis defines the conversion continuum, thus underpinning the transformation of oligo- and polymeric substrates by enzymes. Distributive catalysis follows an association-transformation-dissociation pattern during the formation of enzyme-reactant complexes, whereas during processive catalysis, enzymes partner with substrates and complete multiple catalytic events before dissociation from an enzyme-substrate complex. Here, we focus on processive catalysis in glycoside hydrolases (GHs), which ensures efficient conversions of substrates with high precision, and has the advantage over distributive catalysis in efficiency. The work presented here examines a recent discovery of substrate-product-assisted processive catalysis in the GH3 family enzymes with enclosed pocket-shaped active sites. We detail how GH3 ß-d-glucan glucohydrolases exploit a transiently formed lateral pocket for product displacement and reactants sliding (or translocation motion) through the catalytic site without dissociation, including movements during nanoscale binding/unbinding and sliding. The phylogenetic tree of putative 550 Archaean, bacterial, fungal, Viridiplantae, and Metazoan GH3 entries resolved seven lineages that corresponded to major substrate specificity groups. This analysis indicates that two tryptophan residues in plant ß-d-glucan glucohydrolases that delineate the catalytic pocket, and infer broad specificity, high catalytic efficiency, and substrate-product-assisted processivity, have evolved through a complex evolutionary process, including horizontal transfer and neo-functionalisation. We conclude that the definition of thermodynamic and mechano-structural properties of processive enzymes is fundamentally important for theoretical and practical applications in bioengineering applicable in various biotechnologies.

The novel polyfluoroalkyl benzenesulfonate OBS exposure induces cell cycle arrest and senescence of rat pituitary cell GH3 via the p53/p21/RB pathway.

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS), an economical alternative to perfluorooctane sulfonate (PFOS) in multiple industrial fields, is widely detected in the environment. The toxicity of OBS has received increasing attention. Pituitary cells are components of the endocrine system and act as vital regulators of homeostatic endocrine balance. However, the effects of OBS on pituitary cells remain unknown. The present study explores the effects of OBS (0.5, 5, and 50 µM) on GH3 rat pituitary cells after treatment for 24, 48, and 72 h. We found that OBS significantly inhibited cell proliferation in GH3 cells with remarkable senescent phenotypes, including enhanced SA-ß-gal activity and expression of senescence-associated secretory phenotype (SASP)-related genes, cell cycle arrest, and upregulation of the senescence-related proteins γ-H2A.X and Bcl-2. OBS caused significant cell cycle arrest of GH3 cells at the G1-phase and concomitantly downregulated the expression of some key proteins for the G1/S transition, including cyclin D1 and cyclin E1. Consistently, the phosphorylation of retinoblastoma (RB), which plays a central role in regulating the cell cycle, was prominently reduced after OBS exposure. Furthermore, OBS notably activated the p53-p21 signalling pathway in GH3 cells, as evidenced by increased p53 and p21 expressions, enhanced p53 phosphorylation, and augmented p53 nuclear import. To our knowledge, this study is the first to reveal that OBS triggers senescence in pituitary cells via the p53-p21-RB signalling pathway. Our study demonstrates a novel toxic effect of OBS in vitro, and provides new perspectives for understanding the potential toxicity of OBS.

Evolutionary Analysis of the Melon (Cucumis melo L.) GH3 Gene Family and Identification of GH3 Genes Related to Fruit Growth and Development.

The indole-3-acetic acid (IAA) auxin is an important endogenous hormone that plays a key role in the regulation of plant growth and development. In recent years, with the progression of auxin-related research, the function of the Gretchen Hagen 3 (GH3) gene has become a prominent research topic. However, studies focusing on the characteristics and functions of melon GH3 family genes are still lacking. This study presents a systematic identification of melon GH3 gene family members based on genomic data. The evolution of melon GH3 family genes was systematically analyzed by means of bioinformatics, and the expression patterns of the GH3 family genes in different melon tissues during different fruit developmental stages and with various levels of 1-naphthaleneacetic acid (NAA) induction were analyzed with transcriptomics and RT-qPCR. The melon genome contains 10 GH3 genes distributed across seven chromosomes, and most of these genes are expressed in the plasma membrane. According to evolutionary analysis and the number of GH3 family genes, these genes can be divided into three subgroups, and they have been conserved throughout the evolution of melon. The melon GH3 gene has a wide range of expression patterns across distinct tissue types, with expression generally being higher in flowers and fruit. Through promoter analysis, we found that most cis-acting elements contained light- and IAA-responsive elements. Based on the RNA-seq and RT-qPCR analyses, it can be speculated that CmGH3-5, CmGH3-6 and CmGH3-7 may be involved in the process of melon fruit development. In conclusion, our findings suggest that the GH3 gene family plays an important role in the development of melon fruit. This study provides an important theoretical basis for further research on the function of the GH3 gene family and the molecular mechanism underlying the development of melon fruit.

SlGH3.15, a member of the GH3 gene family, regulates lateral root development and gravitropism response by modulating auxin homeostasis in tomato.

Multiple Gretchen Hagen 3 (GH3) genes have been implicated in a range of processes in plant growth and development through their roles in maintaining hormonal homeostasis. However, there has only been limited study on the functions of GH3 genes in tomato (Solanum lycopersicum). In this work, we investigated the important function of SlGH3.15, a member of the GH3 gene family in tomato. Overexpression of SlGH3.15 led to severe dwarfism in both the above- and below-ground sections of the plant, accompanied by a substantial decrease in free IAA content and reduction in the expression of SlGH3.9, a paralog of SlGH3.15. Exogenous supply of IAA negatively affected the elongation of the primary root and partially restored the gravitropism defects in SlGH3.15-overexpression lines. While no phenotypic change was observed in the SlGH3.15 RNAi lines, double knockout lines of SlGH3.15 and SlGH3.9 were less sensitive to treatments with the auxin polar transport inhibitor. Overall, these findings revealed important roles of SlGH3.15 in IAA homeostasis and as a negative regulator of free IAA accumulation and lateral root formation in tomato.

CsAP2-09 confers resistance against citrus bacterial canker by regulating CsGH3.1L-mediated phytohormone biosynthesis.

Citrus bacterial canker (CBC) is a serious bacterial disease affecting citrus plantations and the citrus industry all over the world. We have previously shown that an apetala 2/ethylene response factor in Citrus sinensis, CsAP2-09, positively regulated resistance to CBC, although the regulatory mechanisms remained undetermined. Here, we demonstrated that CsAP2-09 positively and sustainably controlled resistance to CBC in three-year transgenic plants. CsAP2-09 was found to be a transcriptional activator, and qRT-PCR and dual luciferase assays showed that it controlled the expression CsGH3.1L. CsAP2-09 bound directly to the promotor of CsGH3.1L, shown by yeast one-hybrid assay, with the binding site confirmed by electrophoretic mobility shift assay. Biochemical assays showed that CsAP2-09 negatively regulated the biosynthesis of indole acetic acid (IAA) and positively regulated that of salicylic acid (SA) and ethylene, verified with transient overexpression of CsGH3.1L. The combination of these results with those of previous reports indicated that SA, ethylene, and IAA can directly regulate CBC resistance. Overall, we revealed a pathway whereby CsAP2-09 conferred CBC resistance by direct binding to the CsGH3.1L promoter, activating its expression and modulating IAA, SA, and ethylene biosynthesis. Our study indicates the potential value of manipulating CsAP2-09 and CsGH3.1L in the breeding of CBC-resistant citrus.

Evaluating the expression pattern of the opioid receptor in pituitary neuroendocrine tumors (PitNET) and the role of morphine and naloxone in the regulation of pituitary cell line growth and apoptosis.

PURPOSE: The expression pattern of the opioid receptor (MOR) in pituitary neuroendocrine tumors (PitNET) and the possible effect of morphine and naloxone on GH3 cell growth and apoptosis were evaluated. METHODS: The 114 pituitary tissues including non-functioning, GH-producing and ACTH-producing PitNET and healthy cadaver pituitary tissues were included. The expression level of the MOR gene and protein was assessed using real-time PCR and Western blot. The association with patient demographic characteristics was assessed. Morphine and naloxone were applied to assess their possible pharmacological role in GH3 pituitary adenoma cell death. The cytotoxic effect, the apoptosis rate, the cell cycle distribution, the content of reactive oxygen species and the caspase 3 activity were measured. RESULTS: MOR gene levels increased significantly in pituitary neuroendocrine tumors (PitNET) compared to the healthy pituitary samples. The increased level of MOR gene expression was prominent in invasive functional and non-functional pituitary tumors. A consistent expression pattern was demonstrated for MOR protein levels in PitNET samples. A dose- and time-dependent reduction in the rate of GH3 pituitary cells was observed after morphine treatment with an IC50 of 483 µM after 24 h of incubation. Morphine induced early apoptosis, accumulation of cells in sub-G1 phase, increase in cellular ROS levels and caspase-3 activity. The observed effects of morphine were reversed after MOR blockade using 10 and 25 µM naloxone. CONCLUSION: The possible contributing role of the MOR in pituitary tumor cell growth and the putative pharmaceutical effect of morphine in pituitary neuroendocrine tumor cell death (PitNET) is illustrated.