Homero Reyes-De la Cruz

Plant growth-promoting rhizobacteria modulate root-system architecture in Arabidopsis thaliana through volatile organic compound emission

Extensive communication occurs between plants and microorganisms during different stages of plant development in which signaling molecules from the two partners play an important role. Volatile organic compounds (VOCs) emission by certain plant-growth promoting rhizobacteria (PGPR) has been found to be involved in plant growth. However, little is known about the role of bacterial VOCs in plant developmental processes. In this work, we investigated the effects of inoculation with twelve bacterial strains isolated from the rhizosphere of lemon plants (Citrus aurantifolia) on growth and development of Arabidopsis thaliana seedlings. Several bacterial strains showed a plant growth promoting effect stimulating biomass production, which was related to differential modulation of root-system architecture. The isolates L263, L266, and L272a stimulated primary root growth and lateral root development, while L254, L265a and L265b did not significantly alter primary root growth but strongly promoted lateral root formation. VOC emission analysis by SPME-GC-MS identified aldehydes, ketones and alcohols as the most abundant compounds common to most rhizobacteria. Other VOCs, including 1-octen-3-ol and butyrolactone were strain specific. Characterization of L254, L266 and L272a bacterial isolates by 16S rDNA analysis revealed the identity of these strains as Bacillus cereus, Bacillus simplex and Bacillus sp, respectively. Taken together, our data suggest that rhizospheric bacterial strains can modulate both plant growth promotion and root-system architecture by differential VOC emission.

The Pseudomonas aeruginosa liuE gene encodes the 3-hydroxy-3-methylglutaryl coenzyme A lyase, involved in leucine and acyclic terpene catabolism

The enzymes involved in the catabolism of leucine are encoded by the liu gene cluster in Pseudomonas aeruginosa PAO1. A mutant in the liuE gene (ORF PA2011) of P. aeruginosa was unable to utilize both leucine/isovalerate and acyclic terpenes as the carbon source. The liuE mutant grown in culture medium with citronellol accumulated metabolites of the acyclic terpene pathway, suggesting an involvement of liuE in both leucine/isovalerate and acyclic terpene catabolic pathways. The LiuE protein was expressed as a His-tagged recombinant polypeptide purified by affinity chromatography in Escherichia coli. LiuE showed a mass of 33 kDa under denaturing and 79 kDa under nondenaturing conditions. Protein sequence alignment and fingerprint sequencing suggested that liuE encodes 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HMG-CoA lyase), which catalyzes the cleavage of HMG-CoA to acetyl-CoA and acetoacetate. LiuE showed HMG-CoA lyase optimal activity at a pH of 7.0 and 37 degrees C, an apparent K(m) of 100 microM for HMG-CoA and a V(max) of 21 micromol min(-1) mg(-1). These results demonstrate that the liuE gene of P. aeruginosa encodes for the HMG-CoA lyase, an essential enzyme for growth in both leucine and acyclic terpenes.

Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings

Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins.

Oligogalacturonides inhibit growth and induce changes in S6K phosphorylation in maize ( Zea mays L. var. Chalqueño)

Cell growth is regulated by the target of rapamycin (TOR) signaling pathway, which integrates environmental cues in eukaryotes. In plants the final organ size is determined by the number and cell size. Several proteins involved in the TOR signaling pathway are conserved in plants, although a differential regulation has been proposed because insensitivity to rapamycin by Arabidopsis thaliana was reported. Reports about the role of auxin in the activation of maize S6 ribosomal protein kinase (S6K), a downstream substrate of TOR, have been published indicating a central role of the TOR pathway in the regulation of plant growth. However, in addition to phytohormones, there are a variety of plant growth regulators, including cell wall fragments called oligogalacturonides (OGs). Here we report the effect of OGs on maize growth and development, as well as on S6K activation. We found that oligogalacturonides inhibit coleoptile growth and modify root architecture of maize seedlings. Western blot analyses indicated a modulation of maize S6K activity from seedlings and embryonic axes in response to OGs treatment. These results show that oligosaccharides regulate growth and development through the modulation of TOR signaling pathway in maize.

Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides.

In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gβ (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gβ and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gβ and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gβ), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis.

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0–G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60–250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~103–105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K–Akt–mTOR–S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

Insulin-induced changes in metabolism-related proteins during maize germination

Insulin regulates a wide range of metabolic processes in mammals, such as homeostasis and the breakdown of glucose. Recently, the existence of an insulin-related growth factor in maize (ZmIGF) and a possible receptor for this growth factor has been reported. This peptide exerts effects on plant growth and promotes germination by activating the target of rapamycin (TOR) signaling pathways, which is similar to the insulin response in mammals. In this study, we analyzed the insulin response in maize embryos using a proteomic approach. Our results indicated that insulin modulates the expression of proteins involved in processes, such as storage protein degradation, protein processing, redox and desiccation stress, and glucose metabolism. The involvement of TOR signaling pathways was analyzed using the TOR inhibitor, rapamycin. The results showed that the modulation of these proteins by insulin is independent of the TOR pathway. These results indicated that insulin promotes changes in metabolism-related proteins to ensure successful germination in maize.

Biological effects of carbon nanotubes generated in forest wildfire ecosystems rich in resinous trees on native plants

Carbon nanotubes (CNTs) have a broad range of applications and are generally considered human-engineered nanomaterials. However, carbon nanostructures have been found in ice cores and oil wells, suggesting that nature may provide appropriate conditions for CNT synthesis. During forest wildfires, materials such as turpentine and conifer tissues containing iron under high temperatures may create chemical conditions favorable for CNT generation, similar to those in synthetic methods. Here, we show evidence of naturally occurring multiwalled carbon nanotubes (MWCNTs) produced from Pinus oocarpa and Pinus pseudostrobus, following a forest wildfire. The MWCNTs showed an average of 10 walls, with internal diameters of ∼2.5 nm and outer diameters of ∼14.5 nm. To verify whether MWCNT generation during forest wildfires has a biological effect on some characteristic plant species of these ecosystems, germination and development of seedlings were conducted. Results show that the utilization of comparable synthetic MWCNTs increased seed germination rates and the development of Lupinus elegans and Eysenhardtia polystachya, two plants species found in the burned forest ecosystem. The finding provides evidence that supports the generation and possible ecological functions of MWCNTs in nature.

Volatile compound profile conferred to tequila beverage by maturation in recycled and regenerated white oak barrels from Quercus alba

The main goal of the ageing process of tequila is to improve its organoleptic properties. This process is mainly performed in white oak barrels from trees of the Quercus species, whose wood confers a wide variety of aromas, flavors, and colors to the beverage, providing the desirable organoleptic character to this beverage. In this work, the effects of tequila maturation in recycled and regenerated oak barrels were evaluated by determining minor volatile compounds (VOCs) obtained by microdistillation of the liquid–liquid extraction of tequila samples. GC–MS analysis shows that in silver, aged, and extra-aged tequila types, ~62 VOCs were identified. The principal component analysis of VOC content for each type of tequila showed that they could be classified as four patterns (α, β, γ, and δ groups), proposed to establish their VOC content associated with the maturation process in recycled and regenerated oak barrels from Quercus alba. The α group, constituted by 21 VOCs, did not show significant differences in the concentration of compounds between the silver, aged, and extra-aged types of tequila, β group, constituted by 16 VOCs, which decreased or went missing by maturation process of silver tequila, the γ and δ groups, constituted by 15 and 10 VOCs, correlate with aged and extra-aged tequila, respectively. Findings indicate that the γ and δ groups are associated with the maturation process of tequila, suggesting that recycled and regenerated oak barrels of Q. alba is a procedure suitable for ageing of tequila, providing important VOC content to the beverage.