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.

Modulation of early gene expression responses to water deprivation stress by the E3 ubiquitin ligase ATL80: implications for retrograde signaling interplay.

BACKGROUND: Primary response genes play a pivotal role in translating short-lived stress signals into sustained adaptive responses. In this study, we investigated the involvement of ATL80, an E3 ubiquitin ligase, in the dynamics of gene expression following water deprivation stress. We observed that ATL80 is rapidly activated within minutes of water deprivation stress perception, reaching peak expression around 60 min before gradually declining. ATL80, despite its post-translational regulation role, emerged as a key player in modulating early gene expression responses to water deprivation stress. RESULTS: The impact of ATL80 on gene expression was assessed using a time-course microarray analysis (0, 15, 30, 60, and 120 min), revealing a burst of differentially expressed genes, many of which were associated with various stress responses. In addition, the diversity of early modulation of gene expression in response to water deprivation stress was significantly abolished in the atl80 mutant compared to wild-type plants. A subset of 73 genes that exhibited a similar expression pattern to ATL80 was identified. Among them, several are linked to stress responses, including ERF/AP2 and WRKY transcription factors, calcium signaling genes, MAP kinases, and signaling peptides. Promoter analysis predicts enrichment of binding sites for CAMTA1 and CAMTA5, which are known regulators of rapid stress responses. Furthermore, we have identified a group of differentially expressed ERF/AP2 transcription factors, proteins associated with folding and refolding, as well as pinpointed core module genes which are known to play roles in retrograde signaling pathways that cross-referenced with the early ATL80 transcriptome. CONCLUSIONS: Based on these findings, we propose that ATL80 may target one or more components within the retrograde signaling pathways for degradation. In essence, ATL80 serves as a bridge connecting these signaling pathways and effectively functions as an alarm signal.

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.

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.

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.