Lourdes Macías-Rodríguez

Trichoderma virens, a Plant Beneficial Fungus, Enhances Biomass Production and Promotes Lateral Root Growth through an Auxin-Dependent Mechanism in Arabidopsis

Trichoderma species belong to a class of free-living fungi beneficial to plants that are common in the rhizosphere. We investigated the role of auxin in regulating the growth and development of Arabidopsis (Arabidopsis thaliana) seedlings in response to inoculation with Trichoderma virens and Trichoderma atroviride by developing a plant-fungus interaction system. Wild-type Arabidopsis seedlings inoculated with either T. virens or T. atroviride showed characteristic auxin-related phenotypes, including increased biomass production and stimulated lateral root development. Mutations in genes involved in auxin transport or signaling, AUX1, BIG, EIR1, and AXR1, were found to reduce the growth-promoting and root developmental effects of T. virens inoculation. When grown under axenic conditions, T. virens produced the auxin-related compounds indole-3-acetic acid, indole-3-acetaldehyde, and indole-3-ethanol. A comparative analysis of all three indolic compounds provided detailed information about the structure-activity relationship based on their efficacy at modulating root system architecture, activation of auxin-regulated gene expression, and rescue of the root hair-defective phenotype of the rhd6 auxin response Arabidopsis mutant. Our results highlight the important role of auxin signaling for plant growth promotion by T. virens.

The role of microbial signals in plant growth and development.

Plant growth and development involves a tight coordination of the spatial and temporal organization of cell division, cell expansion and cell differentiation. Orchestration of these events requires the exchange of signaling molecules between the root and shoot, which can be affected by both biotic and abiotic factors. The interactions that occur between plants and their associated microorganisms have long been of interest, as knowledge of these processes could lead to the development of novel agricultural applications. Plants produce a wide range of organic compounds including sugars, organic acids and vitamins, which can be used as nutrients or signals by microbial populations. On the other hand, microorganisms release phytohormones, small molecules or volatile compounds, which may act directly or indirectly to activate plant immunity or regulate plant growth and morphogenesis. In this review, we focus on recent developments in the identification of signals from free-living bacteria and fungi that interact with plants in a beneficial way. Evidence has accumulated indicating that classic plant signals such as auxins and cytokinins can be produced by microorganisms to efficiently colonize the root and modulate root system architecture. Other classes of signals, including N-acyl-L-homoserine lactones, which are used by bacteria for cell-to-cell communication, can be perceived by plants to modulate gene expression, metabolism and growth. Finally, we discuss the role played by volatile organic compounds released by certain plant growth-promoting rhizobacteria in plant immunity and developmental processes. The picture that emerges is one in which plants and microbes communicate themselves through transkingdom signaling systems involving classic and novel signals.

Bacillus megaterium Rhizobacteria Promote Growth and Alter Root-System Architecture Through an Auxin- and Ethylene-Independent Signaling Mechanism in Arabidopsis thaliana

Soil microorganisms are critical players in plant-soil interactions at the rhizosphere. We have identified a Bacillus megaterium strain that promoted growth and development of bean (Phaseolus vulgaris) and Arabidopsis thaliana plants. We used Arabidopsis thaliana as a model to characterize the effects of inoculation with B. megaterium on plant-growth promotion and postembryonic root development. B. megaterium inoculation caused an inhibition in primary-root growth followed by an increase in lateral-root number, lateral-root growth, and root-hair length. Detailed cellular analyses revealed that primary root-growth inhibition was caused both by a reduction in cell elongation and by reduction of cell proliferation in the root meristem. To study the contribution of auxin and ethylene signaling pathways in the alterations in root-system architecture elicited by B. megaterium, a suite of plant hormone mutants of Arabidopsis, including aux1-7, axr4-1, eir1, etr1, ein2, and rhd6, defective in either auxin or ethylene signaling, were evaluated for their responses to inoculation with this bacteria. When inoculated, all mutant lines tested showed increased biomass production. Moreover, aux1-7 and eir1, which sustain limited root-hair and lateral-root formation when grown in uninoculated medium, were found to increase the number of lateral roots and to develop long root hairs when inoculated with B. megaterium. The ethylene-signaling mutants etr1 and ein2 showed an induction in lateral-root formation and root-hair growth in response to bacterial inoculation. Taken together, our results suggest that plant-growth promotion and root-architectural alterations by B. megaterium may involve auxin- and-ethylene independent mechanisms.

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.

Trichoderma-induced plant immunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance against necrotrophic fungi Botrytis cinerea.

Filamentous fungi belonging to the genus Trichoderma have long been recognized as agents for the biocontrol of plant diseases. In this work, we investigated the mechanisms involved in the defense responses of Arabidopsis thaliana seedlings elicited by co-culture with Trichoderma virens and Trichoderma atroviride. Interaction of plant roots with fungal mycelium induced growth and defense responses, indicating that both processes are not inherently antagonist. Expression studies of the pathogenesis-related reporter markers pPr1a:uidA and pLox2:uidA in response to T. virens or T. atroviride provided evidence that the defense signaling pathway activated by these fungi involves salicylic acid (SA) and/or jasmonic acid (JA) depending on the amount of conidia inoculated. Moreover, we found that Arabidopsis seedlings colonized by Trichoderma accumulated hydrogen peroxide and camalexin in leaves. When grown under axenic conditions, T. virens produced indole-3-carboxaldehyde (ICAld) a tryptophan-derived compound with activity in plant development. In Arabidopsis seedlings whose roots are in contact with T. virens or T. atroviride, and challenged with Botrytis cinerea in leaves, disease severity was significantly reduced compared to axenically grown seedlings. Our results indicate that the defense responses elicited by Trichoderma in Arabidopsis are complex and involve the canonical defense hormones SA and JA as well as camalexin, which may be important factors in boosting plant immunity.

A volatile organic compound analysis from Arthrobacter agilis identifies dimethylhexadecylamine, an amino-containing lipid modulating bacterial growth and Medicago sativa morphogenesis in vitro

Plant growth promoting rhizobacteria (PGPR) stimulate plant growth and development by different mechanisms, including the production of different classes of signaling molecules, which may directly affect plant morphogenesis. Here, we report the effects of inoculation of Arthrobacter agilis UMCV2, a PGPR isolated from the rhizosphere of maize plants on growth and development of Medicago sativa seedlings. A. agilis UMCV2 inoculation promoted growth in M. sativa plants as revealed by increased stem length, root length and plant biomass. Inoculation of A. agilis using divided Petri plates decreased taproot growth and increased lateral root formation in plants grown in separate compartments suggesting a role of volatile organic compounds (VOCs) produced by this bacterium in root development. The analysis of VOCs produced by A. agilis UMCV2 identified N,N-dimethyl-hexadecanamine (dimethylhexadecylamine), an amino lipid structurally related to bacterial quorum-sensing signals, which modulated A. agilis UMCV2 growth and plant development in a dose-dependent way. Taken together, our results indicate that bacterial VOCs can be perceived by legume plants to modulate growth and morphogenetic processes and identify a novel signaling molecule potentially involved in plant-rhizobacterial interactions.

Serotonin, a Tryptophan-Derived Signal Conserved in Plants and Animals, Regulates Root System Architecture Probably Acting as a Natural Auxin Inhibitor in Arabidopsis thaliana

Serotonin (5-hydroxytryptamine) is a well-known neurotransmitter in mammals and is widely distributed in plants. This compound is synthesized from tryptophan and shares structural similarity with IAA. To date, little is known about the morphological, physiological and molecular responses of plants to serotonin. In this study, we characterized the effects of serotonin on growth and development in Arabidopsis thaliana seedlings. Gas chromatography-mass spectrometry (GC-MS) analysis showed that plants are able to take up serotonin from the growth medium, which coincided with greatly stimulated lateral root development at concentrations from 10 to 160 μM. In contrast, higher doses of serotonin repressed lateral root growth, primary root growth and root hair development, but stimulated adventitious root formation. To investigate the role of serotonin in modulating auxin responses, we performed experiments using transgenic Arabidopsis lines expressing the auxin-responsive marker constructs DR5:uidA, BA3:uidA and HS::AXR3NT-GUS, as well as a variety of Arabidopsis mutants defective at the AUX1, AXR1, AXR2 and AXR4 auxin-related loci. We found that serotonin strongly inhibited both DR5:uidA and BA3:uidA gene expression in primary and adventitious roots and in lateral root primordia. This compound also abolished the effects of IAA or naphthaleneacetic acid on auxin-regulated developmental and genetic responses, indicating an anti-auxin activity in the plant. Mutant analysis further showed that lateral root induction elicited by serotonin was independent of the AUX1 and AXR4 loci but required AXR1 and AXR2. Our results show that serotonin regulates root development probably by acting as a natural auxin inhibitor.

Trichoderma spp. Improve growth of Arabidopsis seedlings under salt stress through enhanced root development, osmolite production, and Na⁺ elimination through root exudates.

Salt stress is an important constraint to world agriculture. Here, we report on the potential of Trichoderma virens and T. atroviride to induce tolerance to salt in Arabidopsis seedlings. We first characterized the effect of several salt concentrations on shoot biomass production and root architecture of Arabidopsis seedlings. We found that salt repressed plant growth and root development in a dose-dependent manner by blocking auxin signaling. Analysis of the wild type and eir1, aux1-7, arf7arf19, and tir1abf2abf19 auxin-related mutants revealed a key role for indole-3-acetic acid (IAA) signaling in mediating salt tolerance. We also found that T. virens (Tv29.8) and T. atroviride (IMI 206040) promoted plant growth in both normal and saline conditions, which was related to the induction of lateral roots and root hairs through auxin signaling. Arabidopsis seedlings grown under saline conditions inoculated with Trichoderma spp. showed increased levels of abscissic acid, L-proline, and ascorbic acid, and enhanced elimination of Na⁺ through root exudates. Our data show the critical role of auxin signaling and root architecture to salt tolerance in Arabidopsis and suggest that these fungi may enhance the plant IAA level as well as the antioxidant and osmoprotective status of plants under salt stress.

The volatile 6‐pentyl‐2H‐pyran‐2‐one from Trichoderma atroviride regulates Arabidopsis thaliana root morphogenesis via auxin signaling and ETHYLENE INSENSITIVE 2 functioning

Plants interact with root microbes via chemical signaling, which modulates competence or symbiosis. Although several volatile organic compounds (VOCs) from fungi may affect plant growth and development, the signal transduction pathways mediating VOC sensing are not fully understood. 6-pentyl-2H-pyran-2-one (6-PP) is a major VOC biosynthesized by Trichoderma spp. which is probably involved in plant-fungus cross-kingdom signaling. Using microscopy and confocal imaging, the effects of 6-PP on root morphogenesis were found to be correlated with DR5:GFP, DR5:VENUS, H2B::GFP, PIN1::PIN1::GFP, PIN2::PIN2::GFP, PIN3::PIN3::GFP and PIN7::PIN7::GFP gene expression. A genetic screen for primary root growth resistance to 6-PP in wild-type seedlings and auxin- and ethylene-related mutants allowed identification of genes controlling root architectural responses to this metabolite. Trichoderma atroviride produced 6-PP, which promoted plant growth and regulated root architecture, inhibiting primary root growth and inducing lateral root formation. 6-PP modulated expression of PIN auxin-transport proteins in a specific and dose-dependent manner in primary roots. TIR1, AFB2 and AFB3 auxin receptors and ARF7 and ARF19 transcription factors influenced the lateral root response to 6-PP, whereas EIN2 modulated 6-PP sensing in primary roots. These results indicate that root responses to 6-PP involve components of auxin transport and signaling and the ethylene-response modulator EIN2.

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the bakers yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 bakers yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae bakers yeast used in tequila production.