Gretchen Hagen 3 Research Articles

The transcription factor TaFDL2-1A functions in auxin metabolism mediated by abscisic acid to regulate shoot growth in wheat.

Genetic strategies can be effective in improving wheat (Triticum aestivum L.) drought stress tolerance, but accumulating evidence suggests that overexpressing drought-resistance genes, especially genes related to the abscisic acid (ABA) signaling pathway, can retard plant growth. We previously characterized the positive roles of the wheat bZIP transcription factor TaFD-Like2-1A (TaFDL2-1A) in drought stress tolerance and ABA biosynthesis and response, whereas a dwarfing shoot exhibited under normal conditions. This study determined the underlying mechanisms that allow TaFDL2-1A to affect shoot growth. Overexpressing TaFDL2-1A decreased cell length, cell width, leaf size, shoot length, and biomass in wheat. The results of RNA-seq showed that multiple differently expressed transcripts are enriched in the auxin signaling pathway. Further analysis indicated higher expression levels of Gretchen Hagen3 (GH3) genes and lower indole-3-acetic acid (IAA) concentrations in the TaFDL2-1A overexpression lines. Exogenous IAA treatment restored the phenotypes of the TaFDL2-1A overexpression lines to wild-type levels. Transcriptional regulation analysis suggested that TaFDL2-1A enhances the expression of auxin metabolism genes, such as TaGH3.2-3A, TaGH3.2-3B, TaGH3.8-2A, and TaGH3.8-2D, by directly binding to ACGT core cis-elements. Furthermore, tafdl2 knock-out plants had lower expression levels of these GH3 genes and higher IAA levels than Fielder wheat. These GH3 gene expression and IAA levels were induced and reduced in Fielder wheat and tafdl2 knock-out plants treated with exogenous ABA. Our findings elucidate mechanisms underlying the functional redundancy of TaFDL2-1A in the crosstalk between ABA and IAA to affect shoot growth and provide insights into the balance between drought resistance and yield in wheat.

Genomic Analysis and Expression Dynamics of GH3 Genes Under Abiotic Stresses in Brassica rapa

ABSTRACTIn silico characterization of the Gretchen Hagen3 (GH3) gene family is required for understanding phytohormone regulation and stress responses in Brassica rapa to develop the improved stress‐tolerant cultivars. The main objective of this research was to identify the key BrGH3 genes under different abiotic stresses. Using bioinformatics tools, a total of 27 BrGH3 genes were identified in B. rapa for which phylogenetic relationship, syntenic pairing, conserved motifs, miRNA regulation, and cis‐regulatory elements of BrGH3s were also studied. BrGH3 gene expression has been examined in numerous tissues and under various abiotic and phytohormone stressors. Phylogenetic studies separated BrGH3 genes into three major classes and nine subclasses based on gene functional evolution. Collinearity analysis demonstrated that all BrGH3 genes had syntenic linkage with AtGH3, BnaGH3, and BolGH3, indicating that genome duplication mechanisms were crucial to BrGH3 gene evolution. The expression dynamics of BrGH3 under diverse phytohormonal stresses methyl jasmonate (MeJA), auxin (IAA), abscisic acid (ABA), gibberellic acid (GA), and salicylic acid (SA) at different time intervals suggest that BrGH3‐5.1 has a consistently greater expression than other BrGH3 genes. Under salt, drought, and low temperature stress, BrGH3‐1 and BrGH3‐4 genes expressed comparatively higher. The findings suggest that five genes (BrGH3‐5.1, BrGH3‐5.2, BrGH3‐15, BrGH3‐17.2, and BrGH3‐19) have a key role in inducing broad stress resilience. These results can be beneficial in developing stress‐tolerant varieties of Chinese cabbage.

Jasmonates Regulate Auxin-Mediated Root Growth Inhibition in Response to Rhizospheric pH in Arabidopsis thaliana.

Rhizospheric pH, an important environmental cue, severely impacts plant growth and fitness, therefore, has emerged as a major determinant of crop productivity. Despite numerous attempts, the key questions related to plants response against rhizospheric pH remains largely elusive. The present study provides a mechanistic framework for rhizospheric pH-mediated root growth inhibition (RGI). Utilizing various genetic resources combined with pharmacological agents and high-resolution confocal microscopy, the study provides direct evidence for the involvement of jasmonates and auxin in rhizospheric pH-mediated RGI. We show that auxin maxima at root tip is tightly regulated by the rhizospheric pH. In contrast, jasmonates (JAs) abundance inversely correlates with rhizospheric pH. Furthermore, JA-mediated regulation of auxin maxima through GRETCHEN HAGEN 3 (GH3) family genes explains the pattern of RGI observed over the range of rhizospheric pH. Our findings revealed auxin as the key regulator of RGI during severe pH conditions, while JAs antagonistically regulate auxin response against rhizospheric pH.

Genome-wide gene network uncover temporal and spatial changes of genes in auxin homeostasis during fruit development in strawberry (F. × ananassa)

BackgroundThe plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (Fragaria vesca), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle.ResultsGenes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Heat Shock Transcription Factor and Heat Shock Proteins (HSF/HSP), APETALA2/Ethylene Responsive Factor (AP2/ERF) and MYB transcription factor groups.ConclusionsThese results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.

Anchorene, a carotenoid-derived growth regulator, modulates auxin homeostasis by suppressing GH3-mediated auxin conjugation.

Anchorene, identified as an endogenous bioactive carotenoid-derived dialdehyde and diapocarotenoid, affects root development by modulating auxin homeostasis. However, the precise interaction between anchorene and auxin, as well as the mechanisms by which anchorene modulates auxin levels, remain largely elusive. In this study, we conducted a comparative analysis of anchorene's bioactivities alongside auxin and observed that anchorene induces multifaceted auxin-like effects. Through genetic and pharmacological examinations, we revealed that anchorene's auxin-like activities depend on the indole-3-pyruvate-dependent auxin biosynthesis pathway, as well as the auxin inactivation pathway mediated by Group II Gretchen Hagen 3 (GH3) proteins that mainly facilitate the conjugation of indole-3-acetic acid (IAA) to amino acids, leading to the formation of inactivated storage forms. Our measurements indicated that anchorene treatment elevates IAA levels while reducing the quantities of inactivated IAA-amino acid conjugates and oxIAA. RNA sequencing further revealed that anchorene triggers the expression of numerous auxin-responsive genes in a manner reliant on Group II GH3s. Additionally, our in vitro enzymatic assays and biolayer interferometry (BLI) assay demonstrated anchorene's robust suppression of GH3.17-mediated IAA conjugation with glutamate. Collectively, our findings highlight the significant role of carotenoid-derived metabolite anchorene in modulating auxin homeostasis, primarily through the repression of GH3-mediated IAA conjugation and inactivation pathways, offering novel insights into the regulatory mechanisms of plant bioactive apocarotenoids.

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.

Endogenous Hormone Levels and Transcriptomic Analysis Reveal the Mechanisms of Bulbil Initiation in Pinellia ternata.

Pinellia ternata is a medicinal plant that has important pharmacological value, and the bulbils serve as the primary reproductive organ; however, the mechanisms underlying bulbil initiation remain unclear. Here, we characterized bulbil development via histological, transcriptomic, and targeted metabolomic analyses to unearth the intricate relationship between hormones, genes, and bulbil development. The results show that the bulbils initiate growth from the leaf axillary meristem (AM). In this stage, jasmonic acid (JA), abscisic acid (ABA), isopentenyl adenosine (IPA), and salicylic acid (SA) were highly enriched, while indole-3-acetic acid (IAA), zeatin, methyl jasmonate (MeJA), and 5-dexoxystrigol (5-DS) were notably decreased. Through OPLS-DA analysis, SA has emerged as the most crucial factor in initiating and positively regulating bulbil formation. Furthermore, a strong association between IPA and SA was observed during bulbil initiation. The transcriptional changes in IPT (Isopentenyltransferase), CRE1 (Cytokinin Response 1), A-ARR (Type-A Arabidopsis Response Regulator), B-ARR (Type-B Arabidopsis Response Regulator), AUX1 (Auxin Resistant 1), ARF (Auxin Response Factor), AUX/IAA (Auxin/Indole-3-acetic acid), GH3 (Gretchen Hagen 3), SAUR (Small Auxin Up RNA), GA2ox (Gibberellin 2-oxidase), GA20ox (Gibberellin 20-oxidase), AOS (Allene oxide synthase), AOC (Allene oxide cyclase), OPR (Oxophytodienoate Reductase), JMT (JA carboxy l Methyltransferase), COI1 (Coronatine Insensitive 1), JAZ (Jasmonate ZIM-domain), MYC2 (Myelocytomatosis 2), D27 (DWARF27), SMAX (Suppressor of MAX2), PAL (Phenylalanine Ammonia-Lyase), ICS (Isochorismate Synthase), NPR1 (Non-expressor of Pathogenesis-related Genes1), TGA (TGACG Sequence-specific Binding), PR-1 (Pathogenesis-related), MCSU (Molybdenium Cofactor Sulfurase), PP2C (Protein Phosphatase 2C), and SnRK (Sucrose Non-fermenting-related Protein Kinase 2) were highly correlated with hormone concentrations, indicating that bulbil initiation is coordinately controlled by multiple phytohormones. Notably, eight TFs (transcription factors) that regulate AM initiation have been identified as pivotal regulators of bulbil formation. Among these, WUS (WUSCHEL), CLV (CLAVATA), ATH1 (Arabidopsis Thaliana Homeobox Gene 1), and RAX (Regulator of Axillary meristems) have been observed to exhibit elevated expression levels. Conversely, LEAFY demonstrated contrasting expression patterns. The intricate expression profiles of these TFs are closely associated with the upregulated expression of KNOX(KNOTTED-like homeobox), suggesting a intricate regulatory network underlying the complex process of bulbil initiation. This study offers a profound understanding of the bulbil initiation process and could potentially aid in refining molecular breeding techniques specific to P. ternata.

Fidelity in plant hormone modifications catalyzed by Arabidopsis GH3 acyl acid amido synthetases

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.

Elucidating the role of exogenous melatonin in mitigating alkaline stress in soybeans across different growth stages: a transcriptomic and metabolomic approach

BackgroundSoybean (Glycine max), a vital grain and oilseed crop, serves as a primary source of plant protein and oil. Soil salinization poses a significant threat to soybean planting, highlighting the urgency to improve soybean resilience and adaptability to saline stress. Melatonin, recently identified as a key plant growth regulator, plays crucial roles in plant growth, development, and responses to environmental stress. However, the potential of melatonin to mitigate alkali stress in soybeans and the underlying mechanisms remain unclear.ResultsThis study investigated the effects of exogenous melatonin on the soybean cultivar Zhonghuang 13 under alkaline stress. We employed physiological, biochemical, transcriptomic, and metabolomic analyses throughout both vegetative and pod-filling growth stages. Our findings demonstrate that melatonin significantly counteracts the detrimental effects of alkaline stress on soybean plants, promoting plant growth, photosynthesis, and antioxidant capacity. Transcriptomic analysis during both growth stages under alkaline stress, with and without melatonin treatment, identified 2,834 and 549 differentially expressed genes, respectively. These genes may play a vital role in regulating plant adaptation to abiotic stress. Notably, analysis of phytohormone biosynthesis pathways revealed altered expression of key genes, particularly in the ARF (auxin response factor), AUX/IAA (auxin/indole-3-acetic acid), and GH3 (Gretchen Hagen 3) families, during the early stress response. Metabolomic analysis during the pod-filling stage identified highly expressed metabolites responding to melatonin application, such as uteolin-7-O-(2''-O-rhamnosyl)rutinoside and Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside, which helped alleviate the damage caused by alkali stress. Furthermore, we identified 183 differentially expressed transcription factors, potentially playing a critical role in regulating plant adaptation to abiotic stress. Among these, the gene SoyZH13_04G073701 is particularly noteworthy as it regulates the key differentially expressed metabolite, the terpene metabolite Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside. WGCNA analysis identified this gene (SoyZH13_04G073701) as a hub gene, positively regulating the crucial differentially expressed metabolite of terpenoids, Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside. Our findings provide novel insights into how exogenous melatonin alleviates alkali stress in soybeans at different reproductive stages.ConclusionsIntegrating transcriptomic and metabolomic approaches, our study elucidates the mechanisms by which exogenous melatonin ameliorates the inhibitory effects of alkaline stress on soybean growth and development. This occurs through modulation of biosynthesis pathways for key compounds, including terpenes, flavonoids, and phenolics. Our findings provide initial mechanistic insights into how melatonin mitigates alkaline stress in soybeans, offering a foundation for molecular breeding strategies to enhance salt-alkali tolerance in this crop.

A novel mutation in IAA16 is associated with dicamba resistance in Chenopodium album.

Resistance to dicamba in Chenopodium album was first documented over a decade ago, however, the molecular basis of dicamba resistance in this species has not been elucidated. In this research, the resistance mechanism in a dicamba-resistant C. album phenotype was investigated using a transcriptomics (RNA-sequence) approach. The dose-response assay showed that the resistant (R) phenotype was nearly 25-fold more resistant to dicamba than a susceptible (S) phenotype of C. album. Also, dicamba treatment significantly induced transcription of the known auxin-responsive genes, Gretchen Hagen 3 (GH3), small auxin-up RNAs (SAURs), and 1-aminocyclopropane-1-carboxylate synthase (ACS) genes in the susceptible phenotype. Comparing the transcripts of auxin TIR/AFB receptors and auxin/indole-3-acetic acid (AUX/IAA) proteins identified from C. album transcriptomic analysis revealed that the R phenotype contained a novel mutation at the first codon of the GWPPV degron motif of IAA16, resulting in an amino acid substitution of glycine (G) with aspartic acid (D). Sequencing the IAA16 gene in other R and S individuals further confirmed that all the R individuals contained the mutation. In this research, we describe the dicamba resistance mechanism in the only case of dicamba-resistant C. album reported to date. Prior work has shown that the dicamba resistance allele confers significant growth defects to the R phenotype investigated here, suggesting that dicamba-resistant C. album carrying this novel mutation in the IAA16 gene may not persist at high frequencies upon removal of dicamba application. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Silencing of early auxin responsive genes MdGH3-2/12 reduces the resistance to Fusarium solani in apple

Apple replant disease (ARD) has led to severe yield and quality reduction in the apple industry. Fusarium solani (F. solani) has been identified as one of the main microbial pathogens responsible for ARD. Auxin (indole-3-acetic acid, IAA), an endogenous hormone in plants, is involved in almost all plant growth and development processes and plays a role in plant immunity against pathogens. Gretchen Hagen3 (GH3) is one of the early/primary auxin response genes. The aim of this study was to evaluate the function of MdGH3-2 and MdGH3-12 in the defense response of F. solani by treating MdGH3-2/12 RNAi plants with F. solani. The results show that under F. solani infection, RNAi of MdGH3-2/12 inhibited plant biomass accumulation and exacerbated root damage. After inoculation with F. solani, MdGH3-2/12 RNAi inhibited the biosynthesis of acid-amido synthetase. This led to the inhibition of free IAA combining with amino acids, resulting in excessive free IAA accumulation. This excessive free IAA altered plant tissue structure, accelerated fungal hyphal invasion, reduced the activity of antioxidant enzymes (SOD, POD and CAT), increased the reactive oxygen species (ROS) level, and reduced total chlorophyll content and photosynthetic ability, while regulating the expression of PR-related genes including PR1, PR4, PR5 and PR8. It also changed the contents of plant hormones and amino acids, and ultimately reduced the resistance to F. solani. In conclusion, these results demonstrate that MdGH3-2 and MdGH3-12 play an important role in apple tolerance to F. solani and ARD.

Identification of novel inhibitors of plant GH3 IAA-amido synthetases through molecular docking studies.

Auxins play a critical role in several plant developmental processes and their endogenous levels are regulated at multiple levels. The enzymes of the GRETCHEN HAGEN 3 (GH3) protein family catalyze the conjugation of amino acids to indoleacetic acid (IAA), the major endogenous auxin. The GH3 proteins are encoded by multiple redundant genes in plant genomes, making it difficult to perform functional genetic studies to understand their role in auxin homeostasis. To address these challenges, we used a chemical approach that exploits the reaction mechanism of GH3 proteins to identify small molecule inhibitors of their activity from a defined chemical library. The study evaluated receptor-ligand complexes based on their binding energy and classified them accordingly. Docking algorithms were used to correct any deviations, resulting in a list of the most important inhibitory compounds for selected GH3 enzymes based on a normalized sum of energy. The study presents atomic details of protein-ligand interactions and quantifies the effect of several of the identified small molecule inhibitors on auxin-mediated root growth processes in Arabidopsis thaliana. The direct effect of these compounds on endogenous auxin levels was measured using appropriate auxin sensors and endogenous hormone measurements. Our study has identified novel compounds of the flavonoid biosynthetic pathway that are effective inhibitors of GH3 enzyme-mediated IAA conjugation. These compounds play a versatile role in hormone-regulated plant development and have potential applications in both basic research and agriculture.

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.

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.

Transcriptional Effects of Rootstock on Scion after Drought: A Case Study of Using MdGH3 RNAi as the Rootstock

Drought stress is an important environmental factor limiting apple yield and fruit quality. Previously, we identified GRETCHEN HAGEN3.6 (GH3.6) as a negative regulator of drought stress in apple trees. Using transgenic MdGH3 RNAi (knocking down MdGH3.6 and its five homologs) plants as rootstock can increase drought tolerance, water use efficiency, flowering, and fruit quality of the Fuji scion after drought stress. However, the molecular mechanism behind this phenomenon is still unknown. Here, we performed transcriptome sequencing of the grafted plants (Fuji/GL-3 where Fuji was used as the scion and non-transgenic GL-3 was used as the rootstock, and Fuji/MdGH3 RNAi where MdGH3 RNAi was used as the rootstock) under control and drought conditions. Under control conditions, 667 up-regulated genes and 176 down-regulated genes were identified in the scion of Fuji/MdGH3 RNAi, as compared to the scion of Fuji/GL-3. Moreover, 941 up-regulated genes and 2226 down-regulated genes were identified in the rootstock of MdGH3 RNAi plants relative to GL-3. GO terms of these differentially expressed genes (DEGs) in scion and rootstock showed associations with plant growth, fruit development, and stress responses. After drought stress, 220 up-regulated and 452 down-regulated genes were identified in MdGH3 RNAi rootstock, as compared to GL-3. Significantly enriched GO terms included response to abiotic stimulus, cell division, microtubule-based process, metabolic and biosynthetic process of flavonoid, pigment, and lignin. The comparison between the scion of Fuji/MdGH3 RNAi and Fuji/GL-3 yielded a smaller number of DEGs; however, all of them were significantly enriched in stress-related GO terms. Furthermore, 365 and 300 mRNAs could potentially move from MdGH3 RNAi rootstock to scion under control and drought conditions, respectively, including FIDDLEHEAD (FDH), RESPONSIVE TO DESICCATION 26 (RD26), ARS-binding factor 2 (ABF2), WRKY75, and ferritin (FER). Overall, our work demonstrates the effects of rootstock on scion at the transcriptional level after drought stress and provides theoretical support for further understanding and utilization of MdGH3 RNAi plants.

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation

Auxin inactivation is critical for plant growth and development. To develop plant growth regulators functioning in auxin inactivation pathway, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that partially mimicked the effects of auxin. Genetic, pharmacological, and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

Genome-Wide Identification of Auxin-Responsive GH3 Gene Family in Saccharum and the Expression of ScGH3-1 in Stress Response.

Gretchen Hagen3 (GH3), one of the three major auxin-responsive gene families, is involved in hormone homeostasis in vivo by amino acid splicing with the free forms of salicylic acid (SA), jasmonic acid (JA) or indole-3-acetic acid (IAA). Until now, the functions of sugarcane GH3 (SsGH3) family genes in response to biotic stresses have been largely unknown. In this study, we performed a systematic identification of the SsGH3 gene family at the genome level and identified 41 members on 19 chromosomes in the wild sugarcane species, Saccharum spontaneum. Many of these genes were segmentally duplicated and polyploidization was the main contributor to the increased number of SsGH3 members. SsGH3 proteins can be divided into three major categories (SsGH3-I, SsGH3-II, and SsGH3-III) and most SsGH3 genes have relatively conserved exon-intron arrangements and motif compositions. Diverse cis-elements in the promoters of SsGH3 genes were predicted to be essential players in regulating SsGH3 expression patterns. Multiple transcriptome datasets demonstrated that many SsGH3 genes were responsive to biotic and abiotic stresses and possibly had important functions in the stress response. RNA sequencing and RT-qPCR analysis revealed that SsGH3 genes were differentially expressed in sugarcane tissues and under Sporisorium scitamineum stress. In addition, the SsGH3 homolog ScGH3-1 gene (GenBank accession number: OP429459) was cloned from the sugarcane cultivar (Saccharum hybrid) ROC22 and verified to encode a nuclear- and membrane-localization protein. ScGH3-1 was constitutively expressed in all tissues of sugarcane and the highest amount was observed in the stem pith. Interestingly, it was down-regulated after smut pathogen infection but up-regulated after MeJA and SA treatments. Furthermore, transiently overexpressed Nicotiana benthamiana, transduced with the ScGH3-1 gene, showed negative regulation in response to the infection of Ralstonia solanacearum and Fusarium solani var. coeruleum. Finally, a potential model for ScGH3-1-mediated regulation of resistance to pathogen infection in transgenic N. benthamiana plants was proposed. This study lays the foundation for a comprehensive understanding of the sequence characteristics, structural properties, evolutionary relationships, and expression of the GH3 gene family and thus provides a potential genetic resource for sugarcane disease-resistance breeding.

Molecular characterization of the GH3 family in alfalfa under abiotic stress

The phytohormone auxin plays a pivotal role in regulating plant growth, development, and abiotic stress responses by promptly controlling the expression of auxin response genes. The Gretchen Hagen3 (GH3) genes are a major early auxin response gene family that contribute to auxin homeostasis by conjugating excess auxins to amino acids. To our knowledge, a genome-wide investigation of the GH3 genes in alfalfa has never been reported. Here, we present a comprehensive bioinformatics analysis of the MsGH3 gene family, including chromosomal locations, phylogenetic relationships, gene structures, conserved motifs and Gene Ontology annotation. Interestingly, the analysis revealed 31 MsGH3 genes in the alfalfa genome. These genes were classified phylogenetically into the GH3-I, GH3-II, and GH3-III subgroups. Additionally, the data analysis showed that most MsGH3 genes are tissue specific and responsive to environmental stress-related hormones. Furthermore, the analysis of cis elements and potential biological functions revealed that the MsGH3 genes play potential roles in various stress responses. Notably, qRT-PCR results following exposure to high temperature, drought, and salt treatments demonstrated that most of the MsGH3 family genes, especially MsGH3-12, MsGH3-13, and MsGH3-15, play important roles in stress responses. These findings provide invaluable insight for future practical analyses and genetic improvement of alfalfa abiotic stress tolerance.