Deciphering magnesium binding site and structure-function insights in a class II sesquiterpene cyclase.

Magnesium ions (Mg2+) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without cleaving the diphosphate group, instead initiating the reaction through protonation. In our recent research, we discovered a novel class II sesquiterpene cyclase in Streptomyces showdoensis. Notably, we determined its crystal structure and identified Mg2+ within its active site. This finding has shed light on the previously elusive question of Mg2+ binding in class II terpene cyclases. In this chapter, we outline our methods for discovering this novel enzyme, including steps for its purification, crystallization, and kinetic analysis.

Novel Bacillus and Prestia isolates from Dwarf century plant enhance crop yield and salinity tolerance.

Soil salinity is a major environmental stressor impacting global food production. Staple crops like wheat experience significant yield losses in saline environments. Bioprospecting for beneficial microbes associated with stress-resistant plants offers a promising strategy for sustainable agriculture. We isolated two novel endophytic bacteria, Bacillus cereus (ADJ1) and Priestia aryabhattai (ADJ6), from Agave desmettiana Jacobi. Both strains displayed potent plant growth-promoting (PGP) traits, such as producing high amounts of indole-3-acetic acid (9.46, 10.00 µgml-1), ammonia (64.67, 108.97 µmol ml-1), zinc solubilization (Index of 3.33, 4.22, respectively), ACC deaminase production and biofilm formation. ADJ6 additionally showed inorganic phosphate solubilization (PSI of 2.77), atmospheric nitrogen fixation, and hydrogen cyanide production. Wheat seeds primed with these endophytes exhibited enhanced germination, improved growth profiles, and significantly increased yields in field trials. Notably, both ADJ1 and ADJ6 tolerated high salinity (up to 1.03 M) and significantly improved wheat germination and seedling growth under saline stress, acting both independently and synergistically. This study reveals promising stress-tolerance traits within endophytic bacteria from A. desmettiana. Exploiting such under-explored plant microbiomes offers a sustainable approach to developing salt-tolerant crops, mitigating the impact of climate change-induced salinization on global food security.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.

Overexpression of acdS in petunia reduces ethylene production and improves tolerance to heat stress.

Petunia hybrida, widely grown as a bedding plant, has reduced growth and flower quality at temperatures above 30 °C (heat stress), primarily due to heat stress-induced ethylene (ET) production. The gene acdS encodes the 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) enzyme, which is known for its role in reducing ET production by breaking down the ET precursor, ACC, in plant tissues. This study investigated the impact of heat stress on both 'Mirage Rose' WT petunia and its acdS-overexpressing transgenic lines. Heat stress-induced growth inhibition was observed in WT plants but not in transgenic plants. The increased stress tolerance of transgenic plants over WT plants was associated with lower ET production, ROS accumulation, higher SPAD values, water content, and relative water content. Furthermore, higher sensitivity of the WT to heat stress than the transgenic plants was confirmed by analysing ET signalling genes, heat shock transcription factor genes, and antioxidant- and proline-related genes, more strongly induced in WT than in transgenic plants. Overall, this study suggests the potential application of acdS overexpression in other floriculture plants as a viable strategy for developing heat stress-tolerant varieties. This approach holds promise for advancing the floricultural industry by overcoming challenges related to heat-induced growth inhibition and loss of flower quality.

Chasmophyte associated stress tolerant bacteria confer drought resilience to chickpea through efficient nutrient mining and modulation of stress response.

In the present study, ten (10) selected bacteria isolated from chasmophytic wild Chenopodium were evaluated for alleviation of drought stress in chickpea. All the bacterial cultures were potential P, K and Zn solubilizer. About 50% of the bacteria could produce Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The bacteria showed wide range of tolerance towards pH, salinity, temperature and osmotic stress. Bacillus paralicheniformis L38, Pseudomonas sp. LN75, Enterobacter hormachei subsp. xiangfengensis LJ89, B. paramycoides L17 and Micrococcus luteus LA9 significantly improved growth and nutrient (N, P, K, Fe and Zn) content in chickpea under water stress during a green house experiment conducted following a completely randomized design (CRD). Application of Microbacterium imperiale LJ10, B. stercoris LN74, Pseudomonas sp. LN75, B. paralicheniformis L38 and E. hormachei subsp. xiangfengensis LJ89 reduced the antioxidant enzymes under water stress. During field experiments conducted following randomized block design (RBD), all the bacterial inoculations improved chickpea yield under water stress. Highest yield (1363 kg ha-1) was obtained in plants inoculated with Pseudomonas sp. LN75. Pseudomonas sp. LN75, B. paralicheniformis L38 and E. hormachei subsp. xiangfengensis LJ89 have potential as microbial stimulants to alleviate the water stress in chickpea. To the best of our knowledge this is the first report of using chasmophyte associated bacteria for alleviation of water stress in a crop plant.

The potential of enhanced phytoremediation to clean up multi-contaminated soil - insights from metatranscriptomics.

This study aimed to (i) investigate the potential for enhanced phytoremediation to remove contaminants from soil historically co-contaminated with petroleum hydrocarbons (PHs) and heavy metals (HMs) and (ii) analyze the expression of crucial bacterial genes and whole metatranscriptomics profiles for better understanding of soil processes during applied treatment. Phytoremediation was performed using Zea mays and supported by the Pseudomonas qingdaonensis ZCR6 strain and a natural biofertilizer: meat and bone meal (MBM). In previous investigations, mechanisms supporting plant growth and PH degradation were described in the ZCR6 strain. Here, ZCR6 survived in the soil throughout the experiment, but the efficacy of PH removal from all soils fertilized with MBM reached 32 % regardless of the bacterial inoculation. All experimental groups contained 2 % (w/w) MBM. The toxic effect of this amendment on plants was detected 30 days after germination, irrespective of ZCR6 inoculation. Among the 17 genes tested using the qPCR method, only expression of the acdS gene, encoding 1-aminocyclopropane-1-carboxylic acid deaminase, and the CYP153 gene, encoding cytochrome P450-type alkane hydroxylase, was detected in soils. Metatranscriptomic analysis of soils indicated increased expression of methane particulated ammonia monooxygenase subunit A (pmoA-amoA) by Nitrosomonadales bacteria in all soils enriched with MBM compared to the non-fertilized control. We suggest that the addition of 2 % (w/w) MBM caused the toxic effect on plants via the rapid release of ammonia, and this led to high pmoA-amoA expression. In parallel, due to its wide substrate specificity, enhanced bacterial hydrocarbon removal in MBM-treated soils was observed. The metatranscriptomic results indicate that MBM application should be considered to improve bioremediation of soils polluted with PHs rather than phytoremediation. However, lower concentrations of MBM could be considered for phytoremediation enhancement. From a broader perspective, these results indicated the superior capability of metatranscriptomics to investigate the microbial mechanisms driving various bioremediation techniques.

A Single Latent Plant Growth-Promoting Endophyte BH46 Enhances Houttuynia cordata Thunb. Yield and Quality.

Plant growth-promoting endophytes (PGPE) can effectively regulate plant growth and metabolism. The regulation is modulated by metabolic signals, and the resulting metabolites can have considerable effects on the plant yield and quality. Here, tissue culture Houttuynia cordata Thunb., was inoculated with Rhizobium sp. (BH46) to determine the effect of BH46 on H. cordata growth and metabolism, and elucidate associated regulatory mechanisms. The results revealed that BH46 metabolized indole-3-acetic acid and induced 1-aminocyclopropane-1-carboxylate deaminase to decrease ethylene metabolism. Host peroxidase synthesis MPK3/MPK6 genes were significantly downregulated, whereas eight genes associated with auxins, cytokinins, abscisic acid, jasmonic acid, and antioxidant enzymes were significantly upregulated. Eight genes associated with flavonoid biosynthesis were significantly upregulated, with the CPY75B1 gene regulating the production of rutin and quercitrin and the HCT gene directly regulating the production of chlorogenic acid. Therefore, BH46 influences metabolic signals in H. cordata to modulate its growth and metabolism, in turn, enhancing yield and quality of H. cordata.

Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Lifecycle of Botrytis cinerea.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed-Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

Growth-promoting bacteria and arbuscular mycorrhizal fungus enhance maize tolerance to saline stress.

Climate change intensifies soil salinization and jeopardizes the development of crops worldwide. The accumulation of salts in plant tissue activates the defense system and triggers ethylene production thus restricting cell division. We hypothesize that the inoculation of plant growth-promoting bacteria (PGPB) producing ACC (1-aminocyclopropane-1-carboxylate) deaminase favors the development of arbuscular mycorrhizal fungi (AMF), promoting the growth of maize plants under saline stress. We investigated the efficacy of individual inoculation of PGPB, which produce ACC deaminase, as well as the co-inoculation of PGPB with Rhizophagus clarus on maize plant growth subjected to saline stress. The isolates were acquired from the bulk and rhizospheric soil of Mimosa bimucronata (DC.) Kuntze in a temporary pond located in Pernambuco State, Brazil. In the first greenhouse experiment, 10 halophilic PGPB were inoculated into maize at 0, 40 and 80 mM of NaCl, and in the second experiment, the PGPB that showed the best performance were co-inoculated with R. clarus in maize under the same conditions as in the first experiment. Individual PGPB inoculation benefited the number of leaves, stem diameter, root and shoot dry mass, and the photosynthetic pigments. Inoculation with PGPB 28-10 Pseudarthrobacter enclensis, 24-1 P. enclensis and 52 P. chlorophenolicus increased the chlorophyll a content by 138%, 171%, and 324% at 0, 40 and 80 mM NaCl, respectively, comparing to the non-inoculated control. We also highlight that the inoculation of PGPB 28-10, 28-7 Arthrobacter sp. and 52 increased the content of chlorophyll b by 72%, 98%, and 280% and carotenoids by 82%, 98%, and 290% at 0, 40 and 80 mM of NaCl, respectively. Co-inoculation with PGPB 28-7, 46-1 Leclercia tamurae, 70 Artrobacter sp., and 79-1 Micrococcus endophyticus significantly increased the rate of mycorrhizal colonization by roughly 50%. Furthermore, co-inoculation promoted a decrease in the accumulation of Na and K extracted from plant tissue, with an increase in salt concentration, from 40 mM to 80 mM, also favoring the establishment and development of R. clarus. In addition, co-inoculation of these PGPB with R. clarus promoted maize growth and increased plant biomass through osmoregulation and protection of the photosynthetic apparatus. The tripartite symbiosis (plant-fungus-bacterium) is likely to reprogram metabolic pathways that improve maize growth and crop yield, suggesting that the AMF-PGPB consortium can minimize damages caused by saline stress.

Enhancing Pisum sativum growth and symbiosis under heat stress: the synergistic impact of co-inoculated bacterial consortia and ACC deaminase-lacking Rhizobium.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminase is a crucial bacterial trait, yet it is not widely distributed among rhizobia. Hence, employing a co-inoculation approach that combines selected plant growth-promoting bacteria with compatible rhizobial strains, especially those lacking ACC deaminase, presents a practical solution to alleviate the negative effects of diverse abiotic stresses on legume nodulation. Our objective was to explore the efficacy of three non-rhizobial endophytes, Phyllobacterium salinisoli (PH), Starkeya sp. (ST) and Pseudomonas turukhanskensis (PS), isolated from native legumes grown in Tunisian arid regions, in improving the growth of cool-season legume and fostering symbiosis with an ACC deaminase-lacking rhizobial strain under heat stress. Various combinations of these endophytes (ST + PS, ST + PH, PS + PH, and ST + PS + PH) were co-inoculated with Rhizobium leguminosarum 128C53 or its ΔacdS mutant derivative on Pisum sativum plants exposed to a two-week heat stress period.Our findings revealed that the absence of ACC deaminase activity negatively impacted both pea growth and symbiosis under heat stress. Nevertheless, these detrimental effects were successfully mitigated in plants co-inoculated with ΔacdS mutant strain and specific non-rhizobial endophytes consortia. Our results indicated that heat stress significantly altered the phenolic content of pea root exudates. Despite this, there was no impact on IAA production. Interestingly, these changes positively influenced biofilm formation in consortia containing the mutant strain, indicating synergistic bacteria-bacteria interactions. Additionally, no positive effects were observed when these endophytic consortia were combined with the wild-type strain. This study highlights the potential of non-rhizobial endophytes to improve symbiotic performance of rhizobial strains lacking genetic mechanisms to mitigate stress effects on their legume host, holding promising potential to enhance the growth and yield of targeted legumes by boosting symbiosis.

Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes.

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.

The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers.

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.

Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of epi-Isozizaene Synthase into α-Bisabolol Synthase.

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product epi-isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S-F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.

Multi-trait efficiency and interactivity of bacterial consortia used to enhance plant performance under water stress conditions.

Water stress is a major limiting factor for agricultural production under current and projected climate change scenarios. As a sustainable strategy, plant growth-promoting bacterial consortia have been used to reduce plant water stress. However, few studies have examined the effects of stress on multi-trait efficiency and interactivity of bacterial species. In this study, we used several in-vitro experiments, plant assays and greenhouse trials to investigate the effects of stress and bacterial consortia on 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activities, indole-3-acetic acid (IAA) production and plant growth-promoting traits (Phosphate-solubilization, starch hydrolysis, siderophores and ammonium production). We further assessed biofilm formation and the chemotactic behaviour in response to ACC. A total of fifteen ACCD rhizobacteria with multiple growth-promoting traits from the dominant plant species from the hyperseasonal Aripo Savannas were screened in this study. Five of the isolates were further analyzed based on their ACCD activities and were tested in single and dual consortium to assess their abilities in promoting growth under simulated drought stress (-0.35 MPa) and chemically induced ACC conditions (0.03 mM). Our findings showed that bacteria which produce high concentrations of IAA affected the isolates' ability to promote growth under stress, irrespective of microbial combination with ACCD activity above the minimal threshold of 20 nmol α-ketobutyrate mg-1 h-1. Biofilm production with co-culture interaction varied greatly across treatments, however, the general trend showed an increase in biofilm under stress induce conditions. The best performing co-culture, UWIGT-83 and UWIGT-120 (Burkholderia sp.) showed enhanced growth in germination assays and in greenhouse trials with Capsicum chinense (Moruga red hot peppers) under drought stress, when compared to non-inoculated treatments. The findings highlight the importance of testing interactivity of bacterial species with multiple growth promoting traits under stress conditions; and proposed the use of ACC growth media as a novel biofilm screening method for selecting potential stress plant growth-promoting bacteria. Better screening strategies for appropriate plant growth-promoting bacteria may narrow the inconsistency observed between laboratory and field trials.

Broad Chain-Length Specificity of the Alkane-Forming Enzymes NoCER1A and NoCER3A/B in Nymphaea odorata.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

Functional analysis of novel variants identified in cis in the PCCB gene in a patient with propionic acidemia.

Next-generation sequencing has improved the diagnosis of inborn errors of metabolism, allowing rapid confirmation of cases detected by clinical/biochemical studies or newborn screening. The challenge, however, remains for establishing the pathogenicity of the identified variants, especially for novel missense changes or small in-frame deletions. In this work we report a propionic acidemia patient exhibiting a severe neonatal form with coma and hyperammonaemia. Genetic analysis identified the previously described pathogenic PCCB variant p.R512C in the maternal allele and two novel PCCB variants in cis in the paternal allele, p.G246del and p.S322F. Expression analysis in a eukaryotic system confirmed the deleterious effect of the novel missense variant and of the one amino acid deletion, as they both exhibited reduced protein levels and reduced or null PCC activity compared to the wild-type construct. Accordingly, the double mutant resulted in no residual activity. This study increases the knowledge of the genotype-phenotype correlations in the rare disease propionic acidemia and highlights the necessity of functional analysis of novel variants to understand their contribution to disease severity and to accurately classify their pathogenic status. In conclusion, two novel PCCB pathogenic variants have been identified, expanding the current mutational spectrum of propionic acidemia.

Community structure and abundance of ACC deaminase containing bacteria in soils with 16S-PICRUSt2 inference or direct acdS gene sequencing.

Bacteria containing the enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD+) can reduce plant ethylene levels and increase root development and elongation resulting in increased resiliency to drought and other plant stressors. Although these bacteria are ubiquitous in the soil, non-culture-based methods for their enumeration and identification are not well developed. In this study we compare two culture-independent approaches for identifying ACCD+ bacteria. First, quantitative PCR (qPCR) and direct acdS sequencing with newly designed gene-specific primers; and second, phylogenetic construction of 16S rRNA amplicon libraries with the PICRUSt2 tool. Using soils from eastern Colorado, we showed complementary yet differing results in ACCD+ abundance and community structure responding to water availability. Across all sites, gene abundances estimated from qPCR with the acdS gene-specific primers and phylogenetic reconstruction using PICRUSt2 were significantly correlated. However, PICRUSt2 identified members of the Acidobacteria, Proteobacteria, and Bacteroidetes phyla (now known as Acidobacteriota, Pseudomonadota, and Bacteroidota according to the International Code of Nomenclature of Prokaryotes) as ACCD+ bacteria, whereas the acdS primers amplified only members of the Proteobacteria phyla. Despite these differences, both measures showed that bacterial abundance of ACCD+ decreased as soil water content decreased along a potential evapotranspiration (PET) gradient at three sites in eastern Colorado. One major advantage of using 16S sequencing and PICRUSt2 in metagenomic studies is the ability to get a potential functional profile of all known KEGG (Kyoto Encyclopedia of Genes and Genomes) enzymes within the bacterial community of a single soil sample. The 16S-PICRUSt2 method paints a broader picture of the biological and biochemical function of the soil microbiome compared to direct acdS sequencing; however, phylogenetic analysis based on 16S gene relatedness may not reflect that of the functional gene of interest.

Phyllosphere symbiont promotes plant growth through ACC deaminase production.

Plant growth promoting bacteria can confer resistance to various types of stress and increase agricultural yields. The mechanisms they employ are diverse. One of the most important genes associated with the increase in plant biomass and stress resistance is acdS, which encodes a 1-aminocyclopropane-1-carboxylate- or ACC-deaminase. The non-proteinogenic amino acid ACC is the precursor and means of long-distance transport of ethylene, a plant hormone associated with growth arrest. Expression of acdS reduces stress induced ethylene levels and the enzyme is abundant in rhizosphere colonizers. Whether ACC hydrolysis plays a role in the phyllosphere, both as assembly cue and in growth promotion, remains unclear. Here we show that Paraburkholderia dioscoreae Msb3, a yam phyllosphere symbiont, colonizes the tomato phyllosphere and promotes plant growth by action of its ACC deaminase. We found that acdS is required for improved plant growth but not for efficient leaf colonization. Strain Msb3 readily proliferates on the leaf surface of tomato, only occasionally spreading to the leaf endosphere through stomata. The strain can also colonize the soil or medium around the roots but only spreads into the root if the plant is wounded. Our results indicate that the degradation of ACC is not just an important trait of plant growth promoting rhizobacteria but also one of leaf dwelling phyllosphere bacteria. Manipulation of the leaf microbiota by means of spray inoculation may be more easily achieved than that of the soil. Therefore, the application of ACC deaminase containing bacteria to the phyllosphere may be a promising strategy to increasing plant stress resistance, pathogen control, and harvest yields.

Assessing the role of ACC deaminase-producing bacteria in alleviating salinity stress and enhancing zinc uptake in plants by altering the root architecture of French bean (Phaseolus vulgaris) plants.

MAIN CONCLUSION: The consortium inoculation with strains R1 and R4 modified the root system to boost seedling growth, increase the zinc content of French bean pods, and reduce salinity stress. The present study demonstrated the effect of two 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing plant growth-promoting rhizobacteria (Pantoea agglomerans R1 and Pseudomonas fragi R4) alone and consortia on the root system development, French bean growth, and zinc content as well as salinity stress tolerance. Both the strains were characterized for ACC utilization activity (426.23 and 380.54 nmol α-ketobutyrate mg protein-1 h-1), indole acetic acid (IAA) production, phosphate solubilization, ammonia, hydrogen cyanide (HCN), and siderophore production. The strains exhibited zinc solubilization in both plate and broth assays with zinc oxide and zinc carbonate as zinc sources as validated by atomic absorption spectroscopy (AAS). Single or combined inoculations with the selected strains significantly modulated the architectural and morphological traits of the root system of French bean plants. Furthermore, the application of R1and R4 consortia has enhanced zinc content in roots (60.83 mg kg-1), shoots (15.41 mg kg-1), and pods (30.04 mg kg-1) of French bean plants grown in ZnCO3 amended soil. In another set of pot experiments, the consortium bacterization has significantly enhanced length as well as fresh and dry biomass of roots and shoots of the French bean plant under saline stress conditions. Additionally, inoculation with ACC-degrading rhizobacterial strains has increased chlorophyll and carotenoid contents, osmoprotectant content, and antioxidative enzyme (catalase and peroxidase) activity in comparison to their counterparts exposed to salt treatments only. Current findings suggested ACC deaminase-producing rhizobacterial strains hold the potential to improve root architecture which in turn promotes plant growth under salt-stressed conditions as well as enhances micronutrient concentration in host plants.