Gretchen Hagen 3 Research Articles

Natural variation in the transcription factor REPLUMLESS contributes to both disease resistance and plant growth in Arabidopsis

When attacked by pathogens, plants need to reallocate energy from growth to defense to fend off the invaders, frequently incurring growth penalties. This phenomenon is known as the growth–defense tradeoff and is orchestrated by a hardwired transcriptional network. Altering key factors involved in this network has the potential to increase disease resistance without growth or yield loss, but the mechanisms underlying such changes require further investigation. By conducting a genome-wide association study (GWAS) of leaves infected by the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, we discovered that the Arabidopsis transcription factor REPLUMLESS (RPL) is necessary for bacterial resistance. More importantly, RPL functions in promoting both disease resistance and growth. Transcriptome analysis revealed a cluster of genes in the GRETCHEN HAGEN 3 (GH3) family that were significantly upregulated in rpl mutants, leading to the accumulation of indole-3-acetic acid-aspartic acid (IAA-Asp). Consistent with this observation, transcripts of virulence effector genes were activated by IAA-Asp accumulated in the rpl mutants. We found that RPL protein could directly bind to GH3 promoters and repress their expression. RPL also repressed flavonol synthesis by directly repressing CHI expression and thus activated the auxin transport pathway, which promotes plant growth. Therefore, RPL plays an important role in plant immunity and functions in the auxin pathway to optimize Arabidopsis growth and defense.

Genome-wide identification of CmaGH3 family genes, and expression analysis in response to cold and hormonal stresses in Cucurbita maxima

Auxin crosstalk with other plant hormones in a series of responses to environmental stresses, is a process that contributes vitally to plant growth and development. Gretchen Hagen 3 (GH3) family genes play an imperative role in the maintenance of hormone homeostasis and rapid response to biotic/abiotic stresses. Nevertheless, knowledge about GH3s in Cucurbita maxima has not been fully determined. In the present study, a total of 14 GH3 genes were identified and characterized, and their expression profiles were evaluated using real-time PCR. Phylogenetic tree and interspecies collinearity analyses facilitated the evolutionary relationships in C. maxima with other species and prediction of CmaGH3 genes function from genetic and genomic perspectives, respectively. After cold stress, the temporal expression of the CmaGH3 genes in two inbred strains (cold-tolerant inbred line: Cma 7, cold-susceptible inbred line: Cma 14) indicated that CmaGH3–4/6/8/10 were as potential candidate genes for cold tolerance breeding. After phytohormone treatments, CmaGH3–10 and CmaGH3–12 were obviously involved in the response of IAA, ABA and GA stresses. These results indicate that CmaGH3–10 gene may not only be involved in the resistance of pumpkin to cold stress, but also closely associated with auxin homeostasis. In addition, CmaGH3–10 contained 16 SNP sites, four of which are nonsynonymous mutation sites. The polymorphism mutations of CmaGH3–10 will be useful for genetic and breeding study in pumpkin.

Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit.

Summary Indole‐3‐acetic acid (IAA) controls a plethora of developmental processes. Thus, regulation of its concentration is of great relevance for plant performance. Cellular IAA concentration depends on its transport, biosynthesis and the various pathways for IAA inactivation, including oxidation and conjugation.Group II members of the GRETCHEN HAGEN 3 (GH3) gene family code for acyl acid amido synthetases catalysing the conjugation of IAA to amino acids. However, the high degree of functional redundancy among them has hampered thorough analysis of their roles in plant development.In this work, we generated an Arabidopsis gh3.1,2,3,4,5,6,9,17 (gh3oct) mutant to knock out the group II GH3 pathway. The gh3oct plants had an elaborated root architecture, showed an increased tolerance to different osmotic stresses, including an IAA‐dependent tolerance to salinity, and were more tolerant to water deficit. Indole‐3‐acetic acid metabolite quantification in gh3oct plants suggested the existence of additional GH3‐like enzymes in IAA metabolism. Moreover, our data suggested that 2‐oxindole‐3‐acetic acid production depends, at least in part, on the GH3 pathway. Targeted stress‐hormone analysis further suggested involvement of abscisic acid in the differential response to salinity of gh3oct plants.Taken together, our data provide new insights into the roles of group II GH3s in IAA metabolism and hormone‐regulated plant development.

Dual Roles of OsGH3.2 in Modulating Rice Root Morphology and Affecting Arbuscular Mycorrhizal Symbiosis.

Several angiosperm GRETCHEN HAGEN 3 (GH3) genes, including tomato SlGH3.4 and rice OsGH3.2 are induced during arbuscular mycorrhizal (AM) symbiosis, but their functions remain largely unclear. Recently, tomato SlGH3.4 was suggested to negatively regulate arbuscule incidence via decreasing auxin levels in colonized cells. In this study, by acquiring rice OsGH3.2pro:β-glucuronidase (GUS) transgenic plants and generating Osgh3.2 mutants via CRISPR/Cas9 technique, the roles of OsGH3.2 in modulating rice root morphology and affecting AM symbiosis were investigated through time course experiments. Unlike SlGH3.4, OsGH3.2 showed asymbiotic expression in rice young lateral roots, and its mutation resulted in a “shallow” root architecture. Such root morphological change was also observed under symbiotic condition and it likely promoted AM fungal colonization, as the mutants exhibited higher colonization levels and arbuscule incidence than wild-type at early stages. Similar to SlGH3.4, OsGH3.2 showed symbiotic expression in cortical cells that have formed mature arbuscules. At late stages of symbiosis, Osgh3.2 mutants showed elongated cortical cells and larger arbuscules than wild-type, indicating elevated auxin level in the colonized cells. Together, these results revealed both asymbiotic and symbiotic roles of OsGH3.2 in modulating rice root architecture and controlling auxin levels in arbusculated cells, which further affected colonization rate and arbuscule phenotype.

AUXIN RESPONSE FACTOR 16 (StARF16) regulates defense gene StNPR1 upon infection with necrotrophic pathogen in potato.

We demonstrate a new regulatory mechanism in the jasmonic acid (JA) and salicylic acid (SA)mediated crosstalk in potato defense response, wherein, miR160 target StARF16 (a geneinvolved in growth and development) binds to the promoter of StNPR1 (a defense gene) andnegatively regulates its expression to suppress the SA pathway. Overall, our study establishes theimportance of StARF16 in regulation of StNPR1 during JA mediated defense response uponnecrotrophic pathogen interaction. Plants employ antagonistic crosstalk between salicylic acid (SA) and jasmonic acid (JA) to effectively defend them from pathogens. During biotrophic pathogen attack, SA pathway activates and suppresses the JA pathway via NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1). However, upon necrotrophic pathogen attack, how JA-mediated defense response suppresses the SA pathway, is still not well-understood. Recently StARF10 (AUXIN RESPONSE FACTOR), a miR160 target, has been shown to regulate SA and binds to the promoter of StGH3.6 (GRETCHEN HAGEN3), a gene proposed to maintain the balance between the free SA and auxin in plants. In the current study, we investigated the role of StARF16 (a miR160 target) in the regulation of the defense gene StNPR1 in potato upon activation of the JA pathway. We observed that a negative correlation exists between StNPR1 and StARF16 upon infection with the pathogen. The results were further confirmed through the exogenous application of SA and JA. Using yeast one-hybrid assay, we demonstrated that StARF16 binds to the StNPR1 promoter through putative ARF binding sites. Additionally, through protoplast transfection and chromatin immunoprecipitation experiments, we showed that StARF16 could bind to the StNPR1 promoter and regulate its expression. Co-transfection assays using promoter deletion constructs established that ARF binding sites are present in the 2.6 kb sequence upstream to the StNPR1 gene and play a key role in its regulation during infection. In summary, we demonstrate the importance of StARF16 in the regulation of StNPR1, and thus SA pathway, during JA-mediated defense response upon necrotrophic pathogen interaction.

Expression of Genes Related to Plant Hormone Signal Transduction in Jerusalem Artichoke (Helianthus tuberosus L.) Seedlings under Salt Stress

Background: Jerusalem artichoke (Helianthus tuberosus L.) is moderately tolerant to salinity stress and has high economic value. The salt tolerance mechanisms of Jerusalem artichoke are still unclear. Especially in the early stage of Jerusalem artichoke exposure to salt stress, gene transcription is likely to undergo large changes. Previous studies have hinted at the importance of temporal expression analysis in plant transcriptome research. Elucidating these changes may be of great significance to understanding the salt tolerance mechanisms of it. Results: We obtained high-quality transcriptome from leaves and roots of Jerusalem artichoke exposed to salinity (300 mM NaCl) for 0 h (hour), 6 h, 12 h, 24 h, and 48 h, with 150 and 129 unigenes and 9023 DEGs (differentially expressed genes). The RNA-seq data were clustered into time-dependent groups (nine clusters each in leaves and roots); gene functions were distributed evenly among them. KEGG enrichment analysis showed the genes related to plant hormone signal transduction were enriched in almost all treatment comparisons. Under salt stress, genes belonging to PYL (abscisic acid receptor PYR/PYL family), PP2C (Type 2C protein phosphatases), GH3 (Gretchen Hagen3), ETR (ethylene receptor), EIN2/3 (ethylene-insensitive protein 2/3), JAZ (genes such as jasmonate ZIM-domain gene), and MYC2 (Transcription factor MYC2) had extremely similar expression patterns. The results of qRT-PCR of 12 randomly selected and function known genes confirmed the accuracy of RNA-seq. Conclusions: Under the influence of high salinity (300 mM) environment, Jerusalem artichoke suffer serious damage in a short period of time. Based on the expression of genes on the time scale, we found that the distribution of gene functions in time is relatively even. Upregulation of the phytohormone signal transduction had a crucial role in the response of Jerusalem artichoke seedlings to salt stress, and the genes of abscisic acid, auxin, ethylene, and jasmonic acid had the most obvious change pattern. Research emphasized the regulatory role of hormones under high salt shocks and provided an explorable direction for the study of plant salt tolerance mechanisms.

Insight into VvGH3 genes evolutional relationship from monocotyledons and dicotyledons reveals that VvGH3-9 negatively regulates the drought tolerance in transgenic Arabidopsis

The Gretchen Hagen3 (GH3) gene family is necessary for growth and development in plants and is regulated by osmotic stress and various hormones. Although it has been reported in many plants, the evolutionary relationship of GH3 in grape has not been systematically analyzed from the perspective of monocotyledonous and dicotyledonous. This study identified and analyzed 188 GH3 genes, which were distinctly divided into 9 subgroups, and found these subgroups have obviously been clustered between monocotyledonous and dicotyledonous. VvGH3-x genes had higher synteny with apple and Arabidopsis than that of rice, and the average Ka/Ks value in monocotyledons was higher than that of dicotyledons. The codon usage index showed that monocotyledons preferred to use G3s, C3s, and GC3s, while dicotyledons preferred to use A3s and T3s. The GH3 genes of grape exhibited different expression patterns in various tissues, different abiotic stresses, and hormonal treatments. The subcellular localization showed that VvGH3-9 was expressed in the nucleus and cytoplasm. Additionally, under 20% PEG treatment, the IAA and ABA contents, relative expression levels of VvGH3-9, relative electrical conductivity (REC), as well as MDA were obviously increased in VvGH3-9 overexpression lines at 72 h. In contrast, compared to WT, the contents of proline and H2O2, the activities of POD, SOD, and CAT, and the relative expression levels of drought responsive genes were significantly decreased in overexpressing lines. Collectively, this study provided helpful insight for the evolution of GH3 genes and presented some possibilities to study the functions of GH3 genes in monocotyledons and dicotyledons.

MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance.

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

Local conjugation of auxin by the GH3 amido synthetases is required for normal development of roots and flowers in Arabidopsis

Gretchen Hagen 3 (GH3) amido synthetases conjugate amino acids to a carboxyl group of small molecules including hormones auxin, jasmonate, and salicylic acid. The Arabidopsis genome harbors 19 GH3 genes, whose exact roles in plant development have been difficult to define because of genetic redundancy among the GH3 genes. Here we use CRISPR/Cas9 gene editing technology to delete the Arabidopsis group II GH3 genes, which are able to conjugate indole-3-acetic acid (IAA) to amino acids. We show that plants lacking the eight group II GH3 genes (gh3 octuple mutants) accumulate free IAA and fail to produce IAA-Asp and IAA-Glu conjugates. Consequently, gh3 octuple mutants have extremely short roots, long and dense root hairs, and long hypocotyls. Our characterization of gh3 septuple mutants, which provide sensitized backgrounds, reveals that GH3.17 and GH3.9 play prominent roles in root elongation and seed production, respectively. We show that GH3 functions correlate with their expression patterns, suggesting that local deactivation of auxin also contributes to maintaining auxin homeostasis. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions.

Mitochondrial proteomics reveals new insights into embryogenic competence acquisition in Carica papaya L. callus

Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (EC) and nonembryogenic calli (NEC) of using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide (H2O2) contents. EC and NEC presented remarkable differences in anatomical and histochemical characteristics, with EC showing a higher reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate that mitochondrial metabolism affects the embryogenic competence of C. papaya callus. The EC presented higher participation of alternative oxidase (AOX) enzymes, higher total cell respiration and presented a stronger accumulation of mitochondrial stress response proteins. Differential accumulation of auxin-responsive Gretchen Hagen 3 (GH3) family proteins in EC was related to a decrease in the content of free 2,4-dichlorophenoxyacetic acid (2,4-D). EC also showed higher endogenous H2O2 contents. H2O2 is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos. SignificanceTo further advance the understanding of somatic embryogenesis, we performed a broad characterization of embryogenic and nonembryogenic callus, through global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. Based on these results, we propose a working model for the competence of papaya callus. This model suggests that GH3 proteins play an important role in the regulation of auxins. In addition, embryogenic callus showed a greater abundance of stress response proteins and folding proteins. Embryogenic callus respiration occurs predominantly via AOX, and the inhibition of its activity is capable of inhibiting callus differentiation. Although the embryogenic callus presented greater total respiration and a greater abundance of oxidative phosphorylation proteins, they had less COX participation and less coupling efficiency, indicating less ATP production.

Genome-Wide Analysis, Modeling, and Identification of Amino Acid Binding Motifs Suggest the Involvement of GH3 Genes during Somatic Embryogenesis of Coffea canephora.

Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.

De novo transcriptome analysis provides insights into formation of in vitro adventitious root from leaf explants of Arnebia euchroma

BackgroundAdventitious root formation is considered a major developmental step during the propagation of difficult to root plants, especially in horticultural crops. Recently, adventitious roots induced through plant tissue culture methods have also been used for production of phytochemicals such as flavonoids, anthocyanins and anthraquinones. It is rather well understood which horticultural species will easily form adventitious roots, but the factors affecting this process at molecular level or regulating the induction process in in vitro conditions are far less known. The present study was conducted to identify transcripts involved in in vitro induction and formation of adventitious roots using Arnebia euchroma leaves at different time points (intact leaf (control), 3 h, 12 h, 24 h, 3 d, 7 d, 10 d and 15 d). A. euchroma is an endangered medicinal Himalayan herb whose root contains red naphthoquinone pigments. These phytoconstituents are widely used as an herbal ingredient in Asian traditional medicine as well as natural colouring agent in food and cosmetics.ResultsA total of 137.93 to 293.76 million raw reads were generated and assembled to 54,587 transcripts with average length of 1512.27 bps and N50 of 2193 bps, respectively. In addition, 50,107 differentially expressed genes were identified and found to be involved in plant hormone signal transduction, cell wall modification and wound induced mitogen activated protein kinase signalling. The data exhibited dominance of auxin responsive (AUXIN RESPONSE FACTOR8, IAA13, GRETCHEN HAGEN3.1) and sucrose translocation (BETA-31 FRUCTOFURANOSIDASE and MONOSACCHARIDE-SENSING protein1) genes during induction phase. In the initiation phase, the expression of LATERAL ORGAN BOUNDARIES DOMAIN16, EXPANSIN-B15, ENDOGLUCANASE25 and LEUCINE-rich repeat EXTENSION-like proteins was increased. During the expression phase, the same transcripts, with exception of LATERAL ORGAN BOUNDARIES DOMAIN16 were identified. Overall, the transcriptomic analysis revealed a similar patterns of genes, however, their expression level varied in subsequent phases of in vitro adventitious root formation in A. euchroma.ConclusionThe results presented here will be helpful in understanding key regulators of in vitro adventitious root development in Arnebia species, which may be deployed in the future for phytochemical production at a commercial scale.

Overexpression of SgGH3.1 from Fine-Stem Stylo (Stylosanthes guianensis var. intermedia) Enhances Chilling and Cold Tolerance in Arabidopsis thaliana.

Stylosanthes (stylo) species are commercially significant tropical and subtropical forage and pasture legumes that are vulnerable to chilling and frost. However, little is known about the molecular mechanisms behind stylos’ responses to low temperature stress. Gretchen-Hagen 3 (GH3) proteins have been extensively investigated in many plant species for their roles in auxin homeostasis and abiotic stress responses, but none have been reported in stylos. SgGH3.1, a cold-responsive gene identified in a whole transcriptome profiling study of fine-stem stylo (S. guianensis var. intermedia) was further investigated for its involvement in cold stress tolerance. SgGH3.1 shared a high percentage of identity with 14 leguminous GH3 proteins, ranging from 79% to 93%. Phylogenetic analysis classified SgGH3.1 into Group Ⅱ of GH3 family, which have been proven to involve with auxins conjugation. Expression profiling revealed that SgGH3.1 responded rapidly to cold stress in stylo leaves. Overexpression of SgGH3.1 in Arabidopsis thaliana altered sensitivity to exogenous IAA, up-regulated transcription of AtCBF1-3 genes, activated physiological responses against cold stress, and enhanced chilling and cold tolerances. This is the first report of a GH3 gene in stylos, which not only validated its function in IAA homeostasis and cold responses, but also gave insight into breeding of cold-tolerant stylos.

Genome-wide identification, characterization, and expression profiles of auxin responsive GH3 gene family in Salvia miltiorrhiza involved in MeJA treatment

GH3 (Gretchen Hagen 3) is a gene family involved in the response to auxin and plays a role in regulation of plant growth, development and stress responses. The GH3 gene family has been well investigated in genome wide in various plants containing Arabidopsis, rice, maize, etc. However, the study on the GH3 family and its roles involved in JA-signal pathway in Salvia miltiorrhiza is lacking. In this study, we performed a systematic identification of the SmGH3 gene family in genome wide and detected 11 members on 8 S. miltiorrhiza scaffolds. Phylogenetic analyses revealed that SmGH3 proteins could be clustered in two major categories with groups 1 and 2 of GH3 family of Arabidopsis. Diversified cis-elements in the promoter of SmGH3 were predicted as essential players in regulating SmGH3 expression patterns by using PlantCARE database. Gene structure and motif analyses indicated that most SmGH3 genes had relatively conserved exon/intron arrangements and motif compositions. RNA-seq analysis and RT-qPCR showed that 3 SmGH3s (SmGH3.2, SmGH3.6, and SmGH3.10) were up-regulated in S. miltiorrhiza treated by MeJA. Moreover, tissue-specific expression patterns of each SmGH3 genes in different tissues suggested that the various members of GH3 genes conducted their role in the different tissues of S. miltiorrhiza. These results would provide a comprehensive understanding of the GH3 gene family in S. miltiorrhiza and lay a foundation for exploration of their functional divergence and genetic manipulation.

Genome-wide identification of GH3 genes in Brassica oleracea and identification of a promoter region for anther-specific expression of a GH3 gene

BackgroundThe Gretchen Hagen 3 (GH3) genes encode acyl acid amido synthetases, many of which have been shown to modulate the amount of active plant hormones or their precursors. GH3 genes, especially Group III subgroup 6 GH3 genes, and their expression patterns in economically important B. oleracea var. oleracea have not been systematically identified.ResultsAs a first step to understand regulation and molecular functions of Group III subgroup 6 GH3 genes, 34 GH3 genes including four subgroup 6 genes were identified in B. oleracea var. oleracea. Synteny found around subgroup 6 GH3 genes in B. oleracea var. oleracea and Arabidopsis thaliana indicated that these genes are evolutionarily related. Although expression of four subgroup 6 GH3 genes in B. oleracea var. oleracea is not induced by auxin, gibberellic acid, or jasmonic acid, the genes show different organ-dependent expression patterns. Among subgroup 6 GH3 genes in B. oleracea var. oleracea, only BoGH3.13–1 is expressed in anthers when microspores, polarized microspores, and bicellular pollens are present, similar to two out of four syntenic A. thaliana subgroup 6 GH3 genes. Detailed analyses of promoter activities further showed that BoGH3.13–1 is expressed in tapetal cells and pollens in anther, and also expressed in leaf primordia and floral abscission zones.ConclusionsSixty-two base pairs (bp) region (− 340 ~ − 279 bp upstream from start codon) and about 450 bp region (− 1489 to − 1017 bp) in BoGH3.13–1 promoter are important for expressions in anther and expressions in leaf primordia and floral abscission zones, respectively. The identified anther-specific promoter region can be used to develop male sterile transgenic Brassica plants.

Silencing MdGH3-2/12 in apple reduces cadmium resistance via the regulation of AM colonization

Arbuscular mycorrhizal fungi (AMF) can form a symbiotic relationship with most terrestrial plant roots, promote plant growth, and heavy metal (HM) tolerance and thus plays a crucial role in phytoremediation. However, research on the relationship between colonization level and HM tolerance is limited. In this study, apple (Malus domestica) Gretchen Hagen3 genes MdGH3-2/12 silencing plants were treated with four AMF and Cd combination treatments to determine AMF colonization levels, biomass, Cd accumulation, photosynthesis, fluorescence, reactive oxygen species (ROS) and antioxidant substance accumulation, and Cd uptake, transport and detoxification gene expression levels. Results indicate the greater sensitivity of transgenic plants under AMF inoculation and Cd treatment compared with wild type (WT) via lower AMF colonization levels, biomass accumulation, photosynthetic parameters, and the accumulation and clearance homeostasis of ROS, as well as lower detoxification expression levels and higher Cd uptake and transport expression levels. Our study essentially demonstrates that MdGH3-2/12 plays an important role in Cd stress tolerance by regulating AM colonization in apple.

Crystal structure of the acyl acid amido synthetase GH3-8 from Oryza sativa

The Gretchen Hagen 3 (GH3) family of acyl acid amido synthetases regulate the levels and activities of plant hormones containing carboxyl groups, thereby modulating diverse physiological responses. While structure-function relationships have been elucidated for dicotyledonous GH3s, the catalytic mechanism of monocotyledonous GH3 remains elusive. Rice (Oryza sativa) is a representative monocot, and its yield is controlled by the natural growth hormone IAA (indole-3-acetic acid). OsGH3-8 is a model GH3 enzyme that conjugates excess IAA to amino acids in an ATP-dependent manner, ensuring auxin homeostasis and regulating disease resistance, growth and development. Here, we report the crystal structure of OsGH3-8 protein in complex with AMP to uncover the molecular and structural basis for the activity of monocotyledonous GH3-8. Structural and sequence comparisons with other GH3 proteins reveal that the AMP/ATP binding sites are highly conserved. Molecular docking studies with IAA, the GH3-inhibitor Adenosine-5’-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP), and Aspartate provide important information for substrate binding and selectivity of OsGH3-8. Moreover, the observation that AIEP nearly occupies the entire binding site for AMP, IAA and amino acid, offers a ready explanation for the inhibitory effect of AIEP. Taken together, the present study provides vital insights into the molecular mechanisms of monocot GH3 function, and will help to shape the future designs of effective inhibitors.

Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. – A new perspective on formation of high-molecular weight conjugates of auxin

Gretchen Hagen 3 (GH3) acyl acid amidosynthetases are encoded by early auxin-responsive genes and catalyze an ATP-dependent biosynthesis of IAA-amino acid conjugates. An amide conjugate of IAA, indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp), is a predominant form of bound auxin in immature seeds of pea. However, there is some evidence that IAA is also able to form high molecular weight amide conjugates with proteins in pea and other plant species. In this short study we report that recombinant PsGH3 IAA-amino acid synthetase, which exhibits a preference for the formation of IAA-Asp, can also conjugate IAA with the protein fraction from immature seeds of pea (S-10 fraction). We studied [14C]IAA incorporation to the S-10 protein fraction by two assays: TLC method and protein precipitation by trichloroacetic acid (TCA). In both cases, radioactivity of [14C]IAA in the protein fraction increases in comparison to the control (without PsGH3), about 9.3- and 3.17-fold, respectively. l-Asp, as a preferred substrate in the IAA conjugation catalyzed by PsGH3, down-regulates [14C]IAA conjugation to the proteins as shown by the TLC assay (∼2.8-fold decrease) and the TCA precipitation variant (∼2-fold decrease). Moreover, l-Trp that competes with Asp for the catalytic site of PsGH3 and inhibits activity of the enzyme, diminished radioactivity of [14C]IAA-proteins about 1.2- and 2.8-fold, respectively. Taking into account that amino group of an amino acid or a protein acts as an acceptor of the indole-3-acetyl moiety from IAA-AMP intermediate during GH3-dependent conjugation, we masked amine groups (α- and ε-NH2) of the S-10 protein fraction from pea seeds by reductive alkylation. The alkylated proteins revealed about 3- and 2.8-fold lower radioactivity of [14C]IAA than non-alkylated fraction for TLC and TCA precipitation variant, respectively. This is a first study demonstrating that formation of high molecular weight IAA conjugates with proteins is catalyzed by a GH3 acyl acid amidosynthetase.

Comprehensive transcriptomic analysis provides new insights into the mechanism of ray floret morphogenesis in chrysanthemum

BackgroundThe ray floret shapes referred to as petal types on the chrysanthemum (Chrysanthemum × morifolium Ramat.) capitulum is extremely abundant, which is one of the most important ornamental traits of chrysanthemum. However, the regulatory mechanisms of different ray floret shapes are still unknown. C. vestitum is a major origin species of cultivated chrysanthemum and has flat, spoon, and tubular type of ray florets which are the three basic petal types of chrysanthemum. Therefore, it is an ideal model material for studying ray floret morphogenesis in chrysanthemum. Here, using morphological, gene expression and transcriptomic analyses of different ray floret types of C. vestitum, we explored the developmental processes and underlying regulatory networks of ray florets.ResultsThe formation of the flat type was due to stagnation of its dorsal petal primordium, while the petal primordium of the tubular type had an intact ring shape. Morphological differences between the two ray floret types occurred during the initial stage with vigorous cell division. Analysis of genes related to flower development showed that CYCLOIDEA genes, including CYC2b, CYC2d, CYC2e, and CYC2f, were differentially expressed in different ray floret types, while the transcriptional levels of others, such as MADS-box genes, were not significantly different. Hormone-related genes, including SMALL AUXIN UPREGULATED RNA (SAUR), GRETCHEN HAGEN3 (GH3), GIBBERELLIN 2-BETA-DIOXYGENASE 1 (GA2OX1) and APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF), were identified from 1532 differentially expressed genes (DEGs) in pairwise comparisons among the flat, spoon, and tubular types, with significantly higher expression in the tubular type than that in the flat type and potential involvement in the morphogenesis of different ray floret types.ConclusionsOur findings, together with the gene interactional relationships reported for Arabidopsis thaliana, suggest that hormone-related genes are highly expressed in the tubular type, promoting petal cell division and leading to the formation of a complete ring of the petal primordium. These results provide novel insights into the morphological variation of ray floret of chrysanthemum.

Salicylic acid regulates adventitious root formation via competitive inhibition of the auxin conjugation enzyme CsGH3.5 in cucumber hypocotyls.

Exogenous SA treatment at appropriate concentrations promotes adventitious root formation in cucumber hypocotyls, via competitive inhibiting the IAA-Asp synthetase activity of CsGH3.5, and increasing the local free IAA level. Adventitious root formation is critical for the cutting propagation of horticultural plants. Indole-3-acetic acid (IAA) has been shown to play a central role in regulating this process, while for salicylic acid (SA), its exact effects and regulatory mechanism have not been elucidated. In this study, we showed that exogenous SA treatment at the concentrations of both 50 and 100µM promoted adventitious root formation at the base of the hypocotyl of cucumber seedlings. At these concentrations, SA could induce the expression of CYCLIN and Cyclin-dependent Kinase (CDK) genes during adventitious rooting. IAA was shown to be involved in SA-induced adventitious root formation in cucumber hypocotyls. Exposure to exogenous SA led to a slight increase in the free IAA content, and pre-treatment with the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) almost completely abolished the inducible effects of SA on adventitious root number. SA-induced IAA accumulation was also associated with the enhanced expression of Gretchen Hagen3.5 (CsGH3.5). The in vitro enzymatic assay indicated that CsGH3.5 has both IAA- and SA-amido synthetase activity and prefers aspartate (Asp) as the amino acid conjugate. The Asp concentration dictated the functional activity of CsGH3.5 on IAA. Both affinity and catalytic efficiency (Kcat/Km) increased when the Asp concentration increased from 0.3 to 1mM. In contrast, CsGH3.5 showed equal catalytic efficiency for SA at low and high Asp concentrations. Furthermore, SA functioned as a competitive inhibitor of the IAA-Asp synthetase activity of CsGH3.5. During adventitious formation, SA application indeed repressed the IAA-Asp levels in the rooting zone. These data show that SA plays an inducible role in adventitious root formation in cucumber through competitive inhibition of the auxin conjugation enzyme CsGH3.5. SA reduces the IAA conjugate levels, thereby increasing the local free IAA level and ultimately enhancing adventitious root formation.