Evolutionary history of the GH3 family of acyl adenylases in rosids

GH3 amino acid conjugases have been identified in many plant and bacterial species. The evolution of GH3 genes in plant species is explored using the sequenced rosids Arabidopsis, papaya, poplar, and grape. Analysis of the sequenced non-rosid eudicots monkey flower and columbine, the monocots maize and rice, as well as spikemoss and moss is included to provide further insight into the origin of GH3 clades. Comparison of co-linear genes in regions surrounding GH3 genes between species helps reconstruct the evolutionary history of the family. Combining analysis of synteny with phylogenetics, gene expression and functional data redefines the Group III GH3 genes, of which AtGH3.12/PBS3, a regulator of stress-induced salicylic acid metabolism and plant defense, is a member. Contrary to previous reports that restrict PBS3 to Arabidopsis and its close relatives, PBS3 syntelogs are identified in poplar, grape, columbine, maize and rice suggesting descent from a common ancestral chromosome dating to before the eudicot/monocot split. In addition, the clade containing PBS3 has undergone a unique expansion in Arabidopsis, with expression patterns for these genes consistent with specialized and evolving stress-responsive functions.     

The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis

The pbs3-1 mutant, identified in a screen for Arabidopsis (Arabidopsis thaliana) mutants exhibiting enhanced susceptibility to the avirulent Pseudomonas syringae pathogen DC3000 (avrPphB), also exhibits enhanced susceptibility to virulent P. syringae strains, suggesting it may impact basal disease resistance. Because induced salicylic acid (SA) is a critical mediator of basal resistance responses, free and glucose-conjugated SA levels were measured and expression of the SA-dependent pathogenesis-related (PR) marker, PR1, was assessed. Surprisingly, whereas accumulation of the SA glucoside and expression of PR1 were dramatically reduced in the pbs3-1 mutant in response to P. syringae (avrRpt2) infection, free SA was elevated. However, in response to exogenous SA, the conversion of free SA to SA glucoside and the induced expression of PR1 were similar in pbs3-1 and wild-type plants. Through positional cloning, complementation, and sequencing, we determined that the pbs3-1 mutant contains two point mutations in the C-terminal region of the protein encoded by At5g13320, resulting in nonconserved amino acid changes in highly conserved residues. Additional analyses with Arabidopsis containing T-DNA insertion (pbs3-2) and transposon insertion (pbs3-3) mutations in At5g13320 confirmed our findings with pbs3-1. PBS3 (also referred to as GH3.12) is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes. Characterized GH3 family members, such as JAR1, act as phytohormone-amino acid synthetases. Thus, our results suggest that amino acid conjugation plays a critical role in SA metabolism and induced defense responses, with PBS3 acting upstream of SA, directly on SA, or on a competitive inhibitor of SA.     

Strigolactones: new plant hormones in action

Main conclusion

The key step in the mode of action of strigolactones is the enzymatic detachment of the D-ring. The thus formed hydroxy butenolide induces conformational changes of the receptor pocket which trigger a cascade of reactions in the signal transduction.

Abstract

Strigolactones (SLs) constitute a new class of plant hormones which are of increasing importance in plant science. For the last 60 years, they have been known as germination stimulants for parasitic plants. Recently, several new bio-properties of SLs have been discovered such as the branching factor for arbuscular mycorrhizal fungi, regulation of plant architecture (inhibition of bud outgrowth and of shoot branching) and the response to abiotic factors, etc. To broaden horizons and encourage new ideas for identifying and synthesising new and structurally simple SLs, this review is focused on molecular aspects of this new class of plant hormones. Special attention has been given to structural features, the mode of action of these phytohormones in various biological actions, the design of SL analogs and their applications.

     

Inhibition of plant glutamine synthetases by substituted phosphinothricins

Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS₁ and chloroplastic GS₂ competitively versusl-glutamate, with K_i values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low K_i values and slow enzyme turnover, as described by reactivation characteristics. Both the GS₁ and GS₂ isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series.     

Salicylic acid:The roles in plant immunity and crosstalk with other hormones

Land plants use diverse hormones to coordinate their growth, development and responses against biotic and abiotic stresses. Salicylic acid(SA) is an essential hormone in plant immunity, with its levels and signaling tightly regulated to ensure a balanced immune output. Over the past three decades,molecular genetic analyses performed primarily in Arabidopsis have elucidated the biosynthesis and signal transduction pathways of key plant hormones, including abscisic acid, jasmonic acid, ethylene, auxin, cytokinin, brassinosteroids, and gibberellin. Crosstalk between different hormones has become a major focus in plant biology with the goal of obtaining a full picture of the plant hormone signaling network. This review highlights the roles of SA in plant immunity and summarizes our current understanding of the pairwise interactions of SA with other major plant hormones. The complexity of these interactions is discussed, with the hope of stimulating research to address existing knowledge gaps in hormone crosstalk, particularly in the context of balancing plant growth and defense.     

Rice GH3 gene family: Regulators of growth and development

Auxin is an indispensable hormone throughout the lifetime of nearly all plant species. Several aspects of plant growth and development are rigidly governed by auxin, from micro to macro hierarchies; auxin also has a close relationship with plant-pathogen interactions. Undoubtedly, precise auxin levels are vitally important to plants, which have many effective mechanisms to maintain auxin homeostasis. One mechanism is conjugating amino acid to excessive indole-3-acetic acid (IAA; main form of auxin) through some GH3 family proteins to inactivate it. Our previous study demonstrated that GH3-2 mediated broad-spectrum resistance in rice (Oryza sativa L.) by suppressing pathogen-induced IAA accumulation and downregulating auxin signaling. Here, we further investigated the expression pattern of GH3-2 and other GH3 family paralogs in the life cycle of rice and presented the possible function of GH3-2 on rice root development by histochemical analysis of GH3-2 promoter:GUS reporter transgenic plants.Key words: auxin, GH3 gene, indole-3-acetic acid, Oryza sativa, rootThe phytohormone auxin regulates tropism and organ development and influences phyllotaxis, vascular canalization and root patterning by exerting its effect on cell division, elongation and differentiation in plants.^1,2 Indole-3-acetic acid (IAA) is the most widespread form of auxin in most plants. Supraoptimal or insufficient concentration of auxin will cause plants to exhibit abnormal phenotypes. 3-9 Auxin homeostasis is partly sustained by the GH3 gene family, a supervisor of the fluctuation of auxin. Most GH3 genes contain auxin-responsive cis-acting elements (AuxRE) in their promoter regions and react rapidly and transiently to auxin signaling.¹ Nineteen GH3 paralogs have been discovered in Arabidopsis.¹⁰ According to the phylogenetic relationship and acyl acid substrate preference, these genes are classified into three groups (I, II and III), which catalyze the formation of jasmonates, salicylic acid, 4-substituted benzoates or IAA acyl acid amido conjugates.^11,12 The rice GH3 gene family includes 13 paralogs, 4 belonging to group I (GH3-3, -5, -6 and -12) and 9 to group II (GH3-1, -2, -4, -7, -8, -9, -10, -11 and -13); group III GH3 is absent in rice.¹⁰ Rice GH3-1, -2, -8 and -13 paralogs have been biochemically confirmed to have IAA-amido synthetase activity by in vivo or in vitro assays.^6–9 It is believed that other GH3 group II paralogs in rice may also possess this enzymatic activity. But why does rice have such a functionally redundant group of GH3 proteins, which disobeys the economic principle? The explanation could be based on the different temporal and spatial expression of the genes encoding these proteins.     

Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57

The glycoside hydrolase family 57 (GH57) contains five well-established enzyme specificities: α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and α-galactosidase. Around 700 GH57 members originate from Bacteria and Archaea, a substantial number being produced by thermophiles. An intriguing feature of family GH57 is that only slightly more than 2 % of its members (i.e., less than 20 enzymes) have already been biochemically characterized. The main goal of the present bioinformatics study was to retrieve from databases, and analyze in detail, sequences having clear features of the five GH57 enzyme specificities mentioned above. Of the 367 GH57 sequences, 56 were evaluated as α-amylases, 99 as amylopullulanases, 158 as branching enzymes, 46 as 4-α-glucanotransferases and 8 as α-galactosidases. Based on the analysis of collected sequences, sequence logos were created for each specificity and unique sequence features were identified within the logos. These features were proposed to define the so-called sequence fingerprints of GH57 enzyme specificities. Domain arrangements characteristic of the individual enzyme specificities as well as evolutionary relationships within the family GH57 are also discussed. The results of this study could find use in rational protein design of family GH57 amylolytic enzymes and also in the possibility of assigning a GH57 specificity to a hypothetical GH57 member prior to its biochemical characterization.     

Aspergillus niger CSR3 regulates plant endogenous hormones and secondary metabolites by producing gibberellins and indoleacetic acid

In this study, an endophytic fungus, Aspergillus niger CSR3, was isolated from Cannabis sativa. The culture filtrate (CF) was initially screened for growth-promoting activities such as the presence of siderophores, phosphate solubilization, and the production of indole acetic acid (IAA) and gibberellins and was further assayed for its ability to promote the growth of mutant waito-C rice. Nearly all plant growth attributes examined (root–shoot length, biomass, and chlorophyll content) were significantly enhanced by treatment with CSR3. This growth promotion action was due to the presence of various types of gibberellins (GAs) and IAA in the endophyte CF. Moreover, the presence of GA pathway genes (P50-1, P450-3, P450-4, ggs2, and des) was confirmed by means of semi-quantitative RT-PCR. Finally, the application of CSR3 spore suspension with uniconazole and yucasin on maize seedlings revealed that, similar to exogenous IAA and GA₃, CSR3 has the potential to alleviate the inhibitory effect of these inhibitors.     

Evidence for conversion of human salivary alpha-amylase family A to family B by an enzyme action.

SDS-PAGE showed that human salivary alpha-amylase family A (HSA-A) was converted to family B (HSA-B) in human saliva. This conversion did not occur in the supernatant of saliva which had been centrifuged at 105,000 x g for 60 min. An enzyme which catalyzed the conversion existed in the insoluble fraction of human saliva. The enzyme was solubilized with nonionic or zwitterionic detergents, and showed the maximum activity around pH 6. It was stable between pH 4 and 10, and at a temperature lower than 40 degrees C. The enzyme reduced the molecular weight of HSA-A (62,000) to the same molecular weight (58,000) as that of HSA-B without forming any intermediate. It also changed the PAGE pattern of multiple forms of HSA-A to the same pattern as that of HSA-B. It was not inhibited by protease inhibitors, and it did not destroy the reactivity of HSA-A with anti-human salivary alpha-amylase antiserum. The enzyme diminished the reactivity of HSA-A with concanavalin A. These results indicate that HSA-A was converted to HSA-B through the release of sugar chains by the action of the enzyme in the insoluble fraction of human saliva.     

Interaction of the plant hormone, indole-3-acetic acid, with phosphatidylcholine model membranes: effects of acyl chain length

The interaction between the plant hormone, indole-3-acetic acid (IAA), and phosphatidylcholines (PC) of varying acyl chain length has been studied by monitoring the IAA-induced changes in 1H-NMR chemical shifts of lipid headgroup -+N(CH3)3 protons. For PCs in both micellar and vesicle bilayer systems these shifts increase with chain length although for the latter the magnitude of the shifts decreases with an increase in chain unsaturation. In systems composed of mixtures of pure PCs the headgroup -+N(CH3)3 resonance for each phospholipid is shifted by IAA to different extents, indicating that IAA is able to distinguish between individual PCs in mixtures. In di-C12PC and di-C14PC, but not di-C10PC vesicle systems, the -+N(CH3)3 resonance is split into two components reflecting differences in packing of the inside and outside lamellae. This splitting is altered by IAA indicating that IAA interacts differently with the inside and outside PC molecules.     

Inhibition of fatty acid synthetases by the antibiotic cerulenin

The antibiotic cerulenin is a potent and apparently non-competitive inhibitor of fatty acid synthetase systems isolated from various microorganisms and from rat liver. Cerulenin specifically blocks the activity of β-keto acyl thioester synthetase (condensing enzyme). This effect may account for the inhibition of overall fatty acid synthesis by the antibiotic.     

Ribonucleic acid synthesis during the early action of thyroid hormones

1. The effect on RNA synthesis in rat liver of thyroidectomy and the administration of thyroid hormone, especially during its physiological latent period, was studied by determining: (a) the activity of DNA-dependent RNA polymerase in isolated nuclei; (b) the rate of synthesis of nuclear and cytoplasmic RNA in vivo; (c) polyribosomal sedimentation profiles; (d) the response of microsomes and ribonucleoprotein particles to polyuridylic acid; (e) the effect of inhibitors of RNA and protein synthesis on the biological activity of hormones. 2. The DNA-dependent RNA-polymerase activity of isolated rat-liver nuclei was lowered by thyroidectomy and stimulated by the administration of tri-iodo-l-thyronine or l-thyroxine (2-25mug./100g. body wt.) to both normal and thyroidectomized rats. In thyroidectomized rats, the activity of the Mg(2+)-activated RNA-polymerase reaction (for which the product is mainly ribosomal type of RNA) was stimulated at 10-12hr. after a single injection of tri-iodothyronine, reaching a peak value of 60-90% stimulation at 45hr. after hormone administration. The Mn(2+)/ammonium sulphate-activated RNA-polymerase reaction (for which the RNA product is more DNA-like) was not affected for 24hr. after hormone administration but stimulated by 30-40% at 45hr. The response of both RNA-polymerase reactions to the hormone in vivo paralleled the physiological response but the enzyme was not stimulated by the addition in vitro of the hormone to isolated nuclei. 3. Within 3-4hr. after tri-iodothyronine administration to thyroidectomized rats, the specific activity of rapidly labelled nuclear RNA, after a 10min. pulse of [6-(14)C]orotic acid, was 30-40% greater than the control values, the stimulation reaching 100 and 200% at 11 and 16hr. respectively after hormone administration. Longer exposures to [6-(14)C]orotic acid and [(32)P]phosphate showed that the hormone accelerated the synthesis of mitochondrial, microsomal (or ribosomal) and soluble RNA. The greater part of the labelled nuclear RNA was of the ribosomal type. The hormone-induced increases in the incorporation of radioactive precursors into RNA were not preceded, but followed, by enhanced uptake of the precursor. There was no change, per g. of liver, of DNA, nuclear RNA or soluble RNA, but there was a 40-60% increase in the amount of ribosomal RNA between 35 and 45hr. after a single injection of tri-iodothyronine to thyroidectomized rats. 4. Coinciding with the increase in ribosomal RNA after hormone administration was an increase in the average size and amount of polyribosomes. The newly formed ribonucleoprotein particles, or messenger RNA attached to them, or both, were more firmly bound to microsomal membranes after hormone treatment. 5. Polyuridylic acid caused a bigger stimulation of incorporation of [(14)C]phenyl-alanine by ribonucleoprotein particles, but not by microsomes, from thyroidectomized rats as compared with preparations from normal animals. The response of ribonucleoprotein particles to polyuridylic acid was lowered after tri-iodothyronine treatment of thyroidectomized rats. 6. Actinomycin D, 5-fluorouracil, puromycin and cycloheximide caused a 70-100% inhibition of the stimulatory effect of l-thyroxine and tri-iodo-l-thyronine on basal metabolic rate and growth rate in both normal and thyroidectomized animals. Administration of actinomycin D also abolished the stimulation of RNA polymerase by tri-iodothyronine. 7. It is concluded that regulation of nuclear and ribosomal RNA synthesis is an essential step leading to the biological action of thyroid hormones and that the formation of new ribosomes is an important aspect of the control of cytoplasmic protein synthesis by these hormones.