Hydrogen peroxide modulates the expression of the target of rapamycin (TOR) and cell division in Arabidopsis thaliana.

Hydrogen peroxide (H2O2) is naturally produced by plant cells during normal development and serves as a messenger that regulates cell metabolism. Despite its importance, the relationship between hydrogen peroxide and the target of rapamycin (TOR) pathway, as well as its impact on cell division, has been poorly analyzed. In this study, we explore the interaction of H2O2 with TOR, a serine/threonine protein kinase that plays a central role in controlling cell growth, size, and metabolism in Arabidopsis thaliana. By applying two concentrations of H2O2 exogenously (0.5 and 1 mM), we could correlate developmental traits, such as primary root growth, lateral root formation, and fresh weight, with the expression of the cell cycle gene CYCB1;1, as well as TOR expression. When assessing the expression of the ribosome biogenesis-related gene RPS27B, an increase of 94.34% was noted following exposure to 1 mM H2O2 treatment. This increase was suppressed by the TOR inhibitor torin 2. The elimination of H2O2 accumulation with ascorbic acid (AA) resulted in decreased cell division as well as TOR expression. The potential molecular mechanisms associated with the effects of H2O2 on the cell cycle and TOR expression in roots are discussed in the context of the results.

Biosynthetic Gene Clusters in Sequenced Genomes of Four Contrasting Rhizobacteria in Phytopathogen Inhibition and Interaction with Capsicum annuum Roots.

Through screening of rhizobacteria, species that effectively suppress phytopathogens and/or promote plant growth are found. Genome sequencing is a crucial step in obtaining a complete characterization of microorganisms for biotechnological applications. This study aimed to sequence the genomes of four rhizobacteria that differ in their inhibition of four root pathogens and in their interaction with chili pepper roots to identify the species and analyze differences in the biosynthetic gene clusters (BGCs) for antibiotic metabolites and to determine possible phenotype-genotype correlations. Results from sequencing and genome alignment identified two bacteria as Paenibacillus polymyxa, one as Kocuria polaris, and one that was previously sequenced as Bacillus velezensis. Analysis with antiSMASH and PRISM tools showed that B. velezensis 2A-2B, the strain with the best performance of referred characteristics, had 13 BGCs, including those related to surfactin, fengycin, and macrolactin, not shared with the other bacteria, whereas P. polymyxa 2A-2A and 3A-25AI, with up to 31 BGCs, showed lower pathogen inhibition and plant hostility; K. polaris showed the least antifungal capacity. P. polymyxa and B. velezensis had the highest number of BGCs for nonribosomal peptides and polyketides. In conclusion, the 13 BGCs in the genome of B. velezensis 2A-2B that were not present in the other bacteria could explain its effective antifungal capacity and could also contribute to its friendly interaction with chili pepper roots. The high number of other BGCs for nonribosomal peptides and polyketide shared by the four bacteria contributed much less to phenotypic differences. IMPORTANCE To advance the characterization of a microorganism as a biocontrol agent against phytopathogens, it is highly recommended to analyze the potential of the profile of secondary metabolites as antibiotics that it produces to counteract pathogens. Some specific metabolites have positive impacts in plants. By analyzing sequenced genomes with bioinformatic tools, such as antiSMASH and PRISM, outstanding bacterial strains with high potential to inhibit phytopathogens and/or promote plant growth can be quickly selected to confirm and expand our knowledge of BGCs of great value in phytopathology.

Comparison of the Rhizobacteria Serratia sp. H6 and Enterobacter sp. L7 on Arabidopsis thaliana Growth Promotion.

The genera Serratia and Enterobacter belong to the Enterobacteriaceae family and several members have been described as plant growth-promoting rhizobacteria (PGPR). However, how these bacteria influence growth and development is unclear. We performed in vitro interaction assays between either Serratia sp. H6 or Enterobacter sp. L7 with Arabidopsis thaliana seedlings to analyze their effects on plant growth. In experiments of co-cultivation distant from the root tip, Enterobacter sp. decreased root length, markedly increased lateral root number, and slightly increased plant biomass by 33%, 230%, and 69%, respectively, and relative to the control. The volatile organic compounds (VOCs) emitted from Serratia sp. H6 but not those from Enterobacter sp. L7 promoted Arabidopsis growth. A blend of volatile compounds from the two bacteria had effects on plant growth that were similar to those observed for volatile compounds from H6 only. At several densities, the direct contact of roots with Serratia sp. H6 had phytostimulant properties but Enterobacter sp. L7 had clear deleterious effects. Together, these results suggest that direct contact and VOCs of Serratia sp. H6 were the main mechanisms to promote plant growth of A. thaliana, while diffusible compounds of Enterobacter sp. L7 were predominant in their PGPR activity.

Bacterial cyclodipeptides elicit Arabidopsis thaliana immune responses reducing the pathogenic effects of Pseudomonas aeruginosa PAO1 strains on plant development.

Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens.

Participation of Acyl-Coenzyme A Synthetase FadD4 of Pseudomonas aeruginosa PAO1 in Acyclic Terpene/Fatty Acid Assimilation and Virulence by Lipid A Modification.

The pathogenic bacterium Pseudomonas aeruginosa possesses high metabolic versatility, with its effectiveness to cause infections likely due to its well-regulated genetic content. P. aeruginosa PAO1 has at least six fadD paralogous genes, which have been implicated in fatty acid (FA) degradation and pathogenicity. In this study, we used mutagenesis and a functional approach in P. aeruginosa PAO1 to determine the roles of the fadD4 gene in acyclic terpene (AT) and FA assimilation and on pathogenicity. The results indicate that fadD4 encodes a terpenoyl-CoA synthetase utilized for AT and FA assimilation. Additionally, mutations in fadD paralogs led to the modification of the quorum-sensing las/rhl systems, as well as the content of virulence factors pyocyanin, biofilm, rhamnolipids, lipopolysaccharides (LPS), and polyhydroxyalkanoates. In a Caenorhabditis elegans in vivo pathogenicity model, culture supernatants from the 24-h-grown fadD4 single mutant increased lethality compared to the PAO1 wild-type (WT) strain; however, the double mutants fadD1/fadD2, fadD1/fadD4, and fadD2/fadD4 and single mutant fadD2 increased worm survival. A correlation analysis indicated an interaction between worm death by the PAO1 strain, the fadD4 mutation, and the virulence factor LPS. Fatty acid methyl ester (FAME) analysis of LPS revealed that a proportion of the LPS and FA on lipid A were modified by the fadD4 mutation, suggesting that FadD4 is also involved in the synthesis/degradation and modification of the lipid A component of LPS. LPS isolated from the fadD4 mutant and double mutants fadD1/fadD4 and fadD2/fadD4 showed a differential behavior to induce an increase in body temperature in rats injected with LPS compared to the WT strain or from the fadD1 and fadD2 mutants. In agreement, LPS isolated from the fadD4 mutant and double mutants fadD1/fadD2 and fadD2/fadD4 increased the induction of IL-8 in rat sera, but IL1-ß cytokine levels decreased in the double mutants fadD1/fadD2 and fadD1/fadD4. The results indicate that the fadD genes are implicated in the degree of pathogenicity of P. aeruginosa PAO1 induced by LPS-lipid A, suggesting that FadD4 contributes to the removal of acyl-linked FA from LPS, rendering modification in its immunogenic response associated to Toll-like receptor TLR4. The genetic redundancy of fadD is important for bacterial adaptability and pathogenicity over the host.

Azospirillum brasilense Sp245 triggers cytokinin signaling in root tips and improves biomass accumulation in Arabidopsis through canonical cytokinin receptors.

The plant growth promoting rhizobacterium Azospirillum brasilense Sp245 enhances biomass production in cereals and horticultural species and is an interesting model to study the physiology of the phytostimulation program. Although auxin production by Azospirillum appears to be critical for root architectural readjustments, the role of cytokinins in the growth promoting effects of Azospirillum remains unclear. Here, Arabidopsis thaliana seedlings were co-cultivated in vitro with A. brasilense Sp245 to assess whether direct contact of roots with bacterial colonies or exposure to the bacterial volatiles using divided Petri plates would affect biomass production and root organogenesis. Both interaction types increased root and shoot fresh weight but had contrasting effects on primary root length, lateral root formation and root hair development. Cell proliferation in root meristems analyzed with the CYCB1;1::GUS reporter decreased over time with direct contact, but was augmented by plant exposure to volatiles. Noteworthy, the expression of the cytokinin-inducible reporters TCS::GFP and ARR5::GUS increased in root tips in response to bacterial contact, without being affected by the volatiles. In A. thaliana having single (cre1-12, ahk2-2, ahk3-3), double (cre1-12/ahk2-2, cre1-12/ahk3-3, ahk2-2/ahk3-3) or triple (cre1-12/ahk2-2/ahk3-3) mutations in canonical cytokinin receptors, only the triple mutant had a marked effect on plant growth in response to A. brasilense. These results show that different mechanisms are elicited by A. brasilense, which influence the cytokinin-signaling pathway.

Growth promotion in Arabidopsis thaliana by bacterial cyclodipeptides involves the TOR/S6K pathway activation.

Cyclodipeptides (CDPs) are the smallest peptidic molecules that can be produced by diverse organisms such as bacteria, fungi, and animals. They have multiple biological effects. In this paper, we examined the CDPs produced by the bacteria Pseudomonas aeruginosa PAO1, which are known as opportunistic pathogens of humans and plants on TARGET OF RAPAMYCIN (TOR) signaling pathways, and regulation of root system architecture. This bacterium produces the bioactive CDPs: cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In a previous report, these molecules were found to modulate basic cellular programs not only via auxin mechanisms but also by promoting the phosphorylation of the S6 ribosomal protein kinase (S6K), a downstream substrate of the TOR kinase. In the present work, we found that the inoculation of Arabidopsis plants with P. aeruginosa PAO1, the non-pathogenic P. aeruginosa ΔlasI/Δrhll strain (JM2), or by direct exposure of plants to CDPs influenced growth and promoted root branching depending upon the treatment imposed, while genetic evidence using Arabidopsis lines with enhanced or decreased TOR levels indicated a critical role of this pathway in the bacterial phytostimulation.

The nature of the interaction Azospirillum-Arabidopsis determine the molecular and morphological changes in root and plant growth promotion.

Plant growth promoting rhizobacteria influence host functional and adaptive traits via complex mechanisms that are just started to be clarified. Azospirillum brasilense acts as a probiotic bacterium, but detailed information about its molecular mechanisms of phytostimulation is scarce. Three interaction systems were established to analyze the impact of A. brasilense Sp245 on the phenotype of Arabidopsis seedlings, and underlying molecular responses were assessed under the following growth conditions: (1) direct contact of roots with the bacterium, (2) chemical communication via diffusible compounds produced by the bacterium, (3) signaling via volatiles. A. brasilense Sp245 improved shoot and root biomass and lateral root production in the three interaction systems assayed. Cell division, quiescent center, and differentiation protein reporters pCYCB1;1::GUS, WOX5::GFP, and pAtEXP7::GUS had a variable expression in roots depending of the nature of interaction. pCYCB1;1::GUS and WOX5::GFP increased with volatile compounds, whereas pAtEXP7::GUS expression was enhanced towards the root tip in plants with direct contact with the bacterium. The auxin reporter DR5::GUS was highly expressed with diffusible and volatile compounds, and accordingly, auxin signaling mutants pin3, slr1, arf7arf19, and tir1afb2afb3 showed differential phytostimulant responses when compared with the wild type. By contrast, ethylene signaling was not determinant to mediate root changes in response to the different interactions, as observed using the ethylene-related mutants etr1, ein2, and ein3. Our data highlight the diverse effects by which A. brasilense Sp245 improves plant growth and root architectural traits and define a critical role of auxin but not ethylene in mediating root response to bacterization.

Azospirillum brasilense Sp245 lipopolysaccharides induce target of rapamycin signaling and growth in Arabidopsis thaliana.

The Target of Rapamycin (TOR) protein kinase plays a pivotal role in metabolism and gene expression, which enables cell proliferation, growth and development. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell surface of Gram-negative bacteria and mediate plant-bacteria interactions. In this study, we examined whether LPS from Azospirillum brasilense Sp245 affect Arabidopsis thaliana growth via a mechanism involving TOR. A. thaliana plants were treated with LPS and plant growth and development were analyzed in mature plants. Morphological and molecular changes as well as TOR expression and activity were analyzed in root tissues. LPS increased total fresh weight, root length and TOR::GUS expression in the root meristem. Phosphorylation of S6k protein, a downstream target of TOR, increased following LPS treatment, which correlated with increased or decreased expression of CycB1;1::GUS protein upon treatment with LPS or TOR inhibitor AZD-8055, respectively. Long term LPS treatment further increased the rosette size as well as the number of stems and siliques per plant, indicating an overall phytostimulant effect for these signaling molecules. Taken together, the results suggest that A. brasilense LPS play probiotic roles in plants influencing TOR-mediated processes.

Bacterial Compound N,N-Dimethylhexadecylamine Modulates Expression of Iron Deficiency and Defense Response Genes in Medicago truncatula Independently of the Jasmonic Acid Pathway.

Plants face a variety of biotic and abiotic stresses including attack by microbial phytopathogens and nutrient deficiencies. Some bacterial volatile organic compounds (VOCs) activate defense and iron-deficiency responses in plants. To establish a relationship between defense and iron deficiency through VOCs, we identified key genes in the defense and iron-deprivation responses of the legume model Medicago truncatula and evaluated the effect of the rhizobacterial VOC N,N-dimethylhexadecylamine (DMHDA) on the gene expression in these pathways by RT-qPCR. DMHDA increased M. truncatula growth 1.5-fold under both iron-sufficient and iron-deficient conditions compared with untreated plants, whereas salicylic acid and jasmonic acid decreased growth. Iron-deficiency induced iron uptake and defense gene expression. Moreover, the effect was greater in combination with DMHDA. Salicylic acid, Pseudomonas syringae, jasmonic acid, and Botrytis cinerea had inhibitory effects on growth and iron response gene expression but activated defense genes. Taken together, our results showed that the VOC DMHDA activates defense and iron-deprivation pathways while inducing a growth promoting effect unlike conventional phytohormones, highlighting that DMHDA does not mimic jasmonic acid but induces an alternative pathway. This is a novel aspect in the complex interactions between biotic and abiotic stresses.

TARGET OF RAPAMYCIN signaling plays a role in Arabidopsis growth promotion by Azospirillum brasilense Sp245.

Azospirillum brasilense colonizes plant roots and improves productivity, but the molecular mechanisms behind its phytostimulation properties remain mostly unknown. Here, we uncover an important role of TARGET OF RAPAMYCIN (TOR) signaling on the response of Arabidopsis thaliana to A. brasilense Sp245. The effect of the bacterium on TOR expression was analyzed in the transgenic line TOR/tor-1, which carries a translational fusion with the GUS reporter protein, and the activity of TOR was assayed thought the phosphorylation of its downstream signaling target S6K protein. Besides, the role of TOR on plant growth in inoculated plants was assessed using the ATP-competitive inhibitor AZD-8055. A decrease in growth of the primary root correlates with an improved branching and absorptive capacity via lateral root and root hair proliferation 6 days after transplant to different concentrations of the bacterium (103 or 105 CFU/mL). Bacterization increased the expression of TOR in shoot and root apexes and promoted phosphorylation of S6K 3 days after transplant. The TOR inhibitor AZD-8055 (1 µM) inhibited plant growth and cell division in root meristems and in lateral root primordia, interfering with the phytostimulation by A. brasilense. In addition, the role of auxin produced by the bacterium to stimulate TOR expression was explored. Noteworthy, the A. brasilense mutant FAJ009, impaired in auxin production, was unable to elicit TOR signaling to the level observed for the wild-type strain, showing the importance of this phyhormone to stimulate TOR signaling. Together, our findings establish an important role of TOR signaling for the probiotic traits elicited by A. brasilense in A. thaliana.

Early plant growth and biochemical responses induced by Azospirillum brasilense Sp245 lipopolysaccharides in wheat (Triticum aestivum L.) seedlings are attenuated by procyanidin B2.

This study analyzes the effects of procyanidin B2 on early wheat plant growth and plant biochemical responses promoted by lipopolysaccharides (LPS) derived from the rhizobacteria Azospirillum brasilense Sp245. Measurements of leaf, root length, fresh weight, and dry weight showed in vitro plant growth stimulation 4 days after treatment with A. brasilense as well as LPS. Superoxide anion (O2·-) and hydrogen peroxide (H2O2) levels increased in seedling roots treated with LPS (100 µg mL-1). The chlorophyll content in leaf decreased while the starch content increased 24 h after treatment in seedling roots. The LPS treatment induced a high increase in total peroxidase (POX) (EC 1.11.1.7) activity and ionically bound cell wall POX content in roots, when compared to respective controls. Early plant growth and biochemical responses observed in wheat seedlings treated with LPS were inhibited by the addition of procyanidin B2 (5 µg mL-1), a B type proanthocyanidin (PAC), plant-derived polyphenolic compound with binding properties of LPS. All results suggest first that the ionically bound cell wall POX enzymes could be a molecular target of A. brasilense LPS, and second that the recognition or association of LPS by plant cells is required to activate plant responses. This last event could play a critical role during plant growth regulation by A. brasilense LPS.

Oxidative and antioxidative responses in the wheat-Azospirillum brasilense interaction.

Azospirillum is a plant growth-promoting rhizobacteria (PGPR) able to enhance the growth of wheat. The aim of this study was to test the effect of Azospirillum brasilense cell wall components on superoxide (O2·(-)) production in wheat roots and the effect of oxidative stress on A. brasilense viability. We found that inoculation with A. brasilense reduced O2·(-) levels by approx. 30 % in wheat roots. Inoculation of wheat with papain-treated A. brasilense, a Cys protease, notably increased O2·(-) production in all root tissues, as was observed by the nitro blue tetrazolium (NBT) reduction. However, a 24-h treatment with rhizobacteria lipopolysaccharides (50 and 100 µg/mL) alone did not affect the pattern of O2·(-) production. Analysis of the effect of plant cell wall components on A. brasilense oxidative enzyme activity showed no changes in catalase activity but a decrease in superoxide dismutase activity in response to polygalacturonic acid treatment. Furthermore, A. brasilense growth was only affected by high concentrations of H2O2 or paraquat, but not by sodium nitroprusside. Our results suggest that rhizobacterial cell wall components play an important role in controlling plant cell responses and developing tolerance of A. brasilense to oxidative stress produced by the plant.

Avocado roots treated with salicylic acid produce phenol-2,4-bis (1,1-dimethylethyl), a compound with antifungal activity.

We demonstrated the ability of salicylic acid (SA) to induce a compound in avocado roots that strengthens their defense against Phytophthora cinnamomi. The SA content of avocado roots, before and after the application of exogenous SA, was determined by High-Performance Liquid Chromatography (HPLC). After 4h of SA feeding, the endogenous level in the roots increased to 223 µg g(-1) FW, which was 15 times the amount found in control roots. The methanolic extract obtained from SA-treated avocado roots inhibited the radial growth of P. cinnamomi. A thin layer chromatographic bioassay with the methanolic extract and spores of Aspergillus showed a distinct inhibition zone. The compound responsible for the inhibition was identified as phenol-2,4-bis (1,1-dimethylethyl) by gas chromatography and mass spectrometry. At a concentration of 100 µg/mL, the substance reduced germinative tube length in Aspergillus and radial growth of P. cinnamomi. A commercial preparation of phenol-2,4-bis (1,1-dimethylethyl) caused the same effects on mycelium morphology and radial growth as our isolate, confirming the presence of this compound in the root extracts. This is the first report of the induction of this compound in plants by SA, and the results suggest that it plays an important role in the defense response of avocado.

Identification and characterization of superoxide dismutase in Phytophthora cinnamomi.

Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O(2) (-)), which was added exogenously. Our results suggest that the metabolism of O(2) (-) has an important role in the development of P. cinnamomi.