Enhanced specialized metabolite, trichome density, and biosynthetic gene expression in Stevia rebaudiana (Bertoni) Bertoni plants inoculated with endophytic bacteria Enterobacter hormaechei.

Stevia rebaudiana (Bertoni) Bertoni is a plant of economic interest in the food and pharmaceutical industries due its steviol glycosides (SG), which are rich in metabolites that are 300 times sweeter than sucrose. In addition, S. rebaudiana plants contain phenolic compounds and flavonoids with antioxidant activity. Endophytic bacteria promote the growth and development and modulate the metabolism of the host plant. However, little is known regarding the role of endophytic bacteria in the growth; synthesis of SG, flavonoids and phenolic compounds; and the relationship between trichome development and specialized metabolites in S. rebaudiana, which was the subject of this study. The 12 bacteria tested did not increase the growth of S. rebaudiana plants; however, the content of SG increased with inoculation with the bacteria Enterobacter hormaechei H2A3 and E. hormaechei H5A2. The SG content in leaves paralleled an increase in the density of glandular, short, and large trichome. The image analysis of S. rebaudiana leaves showed the presence of SG, phenolic compounds, and flavonoids principally in glandular and short trichomes. The increase in the transcript levels of the KO, KAH, UGT74G1, and UGT76G1 genes was related to the SG concentration in plants of S. rebaudiana inoculated with E. hormaechei H2A3 and E. hormaechei H5A2. In conclusion, inoculation with the stimulating endophytes E. hormaechei H2A3 and E. hormaechei H5A2 increased SG synthesis, flavonoid content and flavonoid accumulation in the trichomes of S. rebaudiana plants.

Production of indole-3-acetic acid by Bacillus circulans E9 in a low-cost medium in a bioreactor.

Bacillus circulans E9 (now known as Niallia circulans) promotes plant growth-producing indole-3-acetic acid (IAA), showing potential for use as a biofertilizer. In this work, the use of a low-cost medium containing industrial substrates, soybean, pea flour, Solulys, Pharmamedia, yeast extract, and sodium chloride (NaCl), was evaluated as a substitute for microbiological Luria Broth (LB) medium for the growth of B. circulans E9 and the production of IAA. In Erlenmeyer flasks with pea fluor medium (PYM), the maximum production of IAA was 7.81 ± 0.16 µg mL-1, while in microbiological LB medium, it was 3.73 ± 0.15 µg mL-1. In addition, an oxygen transfer rate (OTR) of 1.04 kg O2 m-3 d-1 allowed the highest bacterial growth (19.3 ± 2.18 × 1010 CFU mL-1) and IAA production (10.7 µg mL-1). Consequently, the OTR value from the flask experiments was used to define the conditions for the operation of a 1 L stirred tank bioreactor. The growth and IAA production of B. circulans cultured in a bioreactor with PYM medium were higher (8 and 1.6 times, respectively) than those of bacteria cultured in Erlenmeyer flasks. IAA produced in a bioreactor by B. circulans was shown to induce the root system in Arabidopsis thaliana, similar to synthetic IAA. The results of this study demonstrate that PYM medium may be able to be used for the mass production of B. circulans E9 in bioreactors, increasing both bacterial growth and IAA production. This low-cost medium has the potential to be employed to grow other IAA-producing bacterial species.

Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations.

In plants, phosphorus (P) uptake occurs via arbuscular mycorrhizal (AM) symbiosis and through plant roots. The phosphate concentration is known to affect colonization by AM fungi, and the effect depends on the plant species. Stevia rebaudiana plants are valuable sources of sweetener compounds called steviol glycosides (SGs), and the principal components of SGs are stevioside and rebaudioside A. However, a detailed analysis describing the effect of the phosphate concentration on the colonization of AM fungi in the roots and the relationship of these factors to the accumulation of SGs and photochemical performance has not been performed; such an analysis was the aim of this study. The results indicated that low phosphate concentrations (20 and 200 µM KH2PO4) induced a high percentage of colonization by Rhizophagus irregularis in the roots of S. rebaudiana, while high phosphate concentrations (500 and 1,000 µM KH2PO4) reduced colonization. The morphology of the colonization structure is a typical Arum-type mycorrhiza, and a mycorrhiza-specific phosphate transporter was identified. Colonization with low phosphate concentrations improved plant growth, chlorophyll and carotenoid concentration, and photochemical performance. The transcription of the genes that encode kaurene oxidase and glucosyltransferase (UGT74G1) was upregulated in colonized plants at 200 µM KH2PO4, which was consistent with the observed patterns of stevioside accumulation. In contrast, at 200 µM KH2PO4, the transcription of UGT76G1 and the accumulation of rebaudioside A were higher in noncolonized plants than in colonized plants. These results indicate that a low phosphate concentration improves mycorrhizal colonization and modulates the stevioside and rebaudioside A concentration by regulating the transcription of the genes that encode kaurene oxidase and glucosyltransferases, which are involved in stevioside and rebaudioside A synthesis in S. rebaudiana.

Autophagy mediates hydrotropic response in Arabidopsis thaliana roots.

This work shows that autophagy plays a key role in the hydrotropic curvature of Arabidopsis thaliana roots. An analysis of GFP-ATG8a transgenic plants showed that autophagosomes accumulated in the root curvature 2 h after the transfer of seedlings to Normal Medium-Water Stress Medium (NM-WSM). Autophagy flux was required for root bending. Remarkably, several atg mutants did not show hydrotropic curvature in NM-WSM or the splitting-agar system. Hyper, an H2O2 sensor showed that H2O2 preferentially accumulated in the root curvature at a similar rate as the autophagosomes did during hydrotropic response. Peroxidase and ROBH activity inhibition affected, negatively or positively root curvature. This data suggested H2O2 balance was required for root bending. Malondialdehyde, a metabolite used as an indicator of oxidative stress, accumulated at the same rate during the development of the curvature in NM-WSM. These results suggest that autophagy is required for the hydrotropic response in NM-WSM. We discuss the possible regulatory role of H2O2 on autophagy during the hydrotropic response that might relieve oxidative stress provoked by water stress. NM-WSM is water stress system suitable for studying hydrotropic responses on a short-term basis.

Cell Culture of Bursera linanoe in a Stirred Tank Bioreactor for Production of Linalool and Linalyl Acetate.

Bursera linanoe cell suspension cultures were initiated from callus grown in Murashige and Skoog medium supplemented with naphthalene acetic acid (3.0 mg L⁻¹) and 6-benzylaminopurine (0.5 mg L⁻¹). In flasks, B. linanoe cell cultures grew over a 9 day period, reaching a maximum biomass of 11.16 g DW L⁻¹. Throughout the growth phase, cell viability was constant at 60 - 70%. In contrast, B. linanoe cells growing in a bioreactor achieved a maximum biomass of 22.26 g DW L⁻¹ (after 7 days), and cell viability was constant at 75 - 85%. Production of linalool and linalyl acetate in the bioreactor (3.02 and 2.40 mg g⁻¹ DW, respectively) was significantly greater than that achieved from cells in flask cultures (1.05 and 0.97 mg g⁻¹ DW, respectively). B. linanoe cell suspension culture has potential as an alternative method for the production of essential oils.

Robust root growth in altered hydrotropic response1 (ahr1) mutant of Arabidopsis is maintained by high rate of cell production at low water potential gradient.

Hydrotropism is the directional root growth response determined by water stimulus. In a water potential gradient system (WPGS) the roots of the Arabidopsis wild type have a diminished root growth compared to normal medium (NM). In contrast, the altered hydrotropic response1 (ahr1) mutant roots maintain their robust growth in the same WPGS. The aims of this work were to ascertain how ahr1 roots could sustain growth in the WPGS, with a special focus on the integration of cellular processes involved in the signaling that determines root growth during abiotic stress and their relation to hydrotropism. Cellular analysis of the root apical meristem of ahr1 mutant contrary to the wild type showed an absence of changes in the meristem length, the elongation zone length, the length of fully elongated cells, and the cell cycle duration. The robust and steady root growth of ahr1 seedlings in the WPGS is explained by the mutant capacity to maintain cell production and cell elongation at the same level as in the NM. Analysis of auxin response at a transcriptional level showed that roots of the ahr1 mutant had a lower auxin response when grown in the WPGS, compared to wild type, indicating that auxin signaling participates in attenuation of root growth under water stress conditions. Also, wild type plants exhibited a high increase in proline content while ahr1 mutants showed minimum changes in the Normal Medium→Water Stress Medium (NM→WSM), a lower water potential gradient system than the WPGS. Accordingly, in this condition, gene expression of Δ1-6 Pyrroline-5-Carboxylate Synthetase1 (P5CS1) involved in proline synthesis strongly increased in wild type but not in ahr1 seedlings. The ahr1 phenotype shows unique features since the mutant root cells continue to proliferate and grow in the presence of a progressively negative water potential gradient at a level comparable to wild type growing in the NM. As such, it represents an exceptional resource for understanding hydrotropism.

Las proteínas arabinogalactanos en cultivos de células vegetales / Arabinogalactan proteins in plant cell cultures / As proteínas arabinogalactanos em cultivos de células vegetais

Las proteínas arabinogalactanos (AGPs) son macromoléculas que se encuentran prácticamente en todos los órganos de las plantas, siendo asociadas con varios aspectos del crecimiento y desarrollo vegetal. Estas moléculas se caracterizan bioquímicamente por contener carbohidratos y proteínas en relación 9:1. El carbohidrato está compuesto principalmente por arabinogalactanos tipo II; mientras que la parte proteica está organizada en dominios que definen a las AGPs como clásicas o no clásicas. Las primeras se caracterizan además por presentar una secuencia C-terminal que predice la incorporación de un grupo glicosilfosfatidilinositol (GFI), que permite su unión a la membrana plasmática. En cultivos de células vegetales se reportan varias especies que liberan AGPs al medio de cultivo. Se presenta una revisión de las características bioquímicas de las AGPs liberadas al medio y de las propuestas sobre los mecanismos bioquímicos y celulares por los cuales las AGPs participan en la diferenciación y crecimiento de las células vegetales. Los cultivos de células liberan al medio de cultivo AGPs clásicas y no clásicas, y se propone que podrían provenir de la membrana plasmática o la pared celular. Las AGPs intervienen en el control del crecimiento celular, además de estar relacionadas con la embriogénesis somática y la organogénesis, procesos de diferenciación celular importantes en los sistemas de micropropagación de plantas. El mecanismo bioquímico por el cual las AGPs participan en el crecimiento celular y la diferenciación implica que éstas, o los productos de su degradación, quizás actúen como moléculas de señalización.

The arabinogalactan proteins (AGPs) are macromolecules found in practically all plant organs, being associated with several aspects of the plant growth and development. These molecules contain carbohydrates and proteins in a 9:1 relation. The carbohydrate moiety is composed mainly of type II arabinogalactans, whereas the protein has particular amino acid domains that allow classifying the AGPs into two groups, classical and non-classical. In addition, the former are characterized by a C-terminal tail that predicts the incorporation of a glycosylphosphatidylinositol group (GPI) that allows the attachment of the AGPs to the plasma membrane. Plant cell cultures of several species release AGPs into the culture medium. The biochemical characteristics of the AGPs released into the medium, and the proposed biochemical and cellular mechanisms by which AGPs participate in plant cell differentiation and growth are reviewed. The plant cells release classical as well as non-classical AGPs into the culture medium. The origin of these AGPs could likely be the plasma membrane or the cell wall. They are involved in the control of cellular growth and differentiation processes, aspects that have fundamental importance in the induction of somatic embryogenesis and organogenesis, key steps in plant micropropagation programs. The biochemical mechanism by which the AGPs participate in cell growth and differentiation implies that the AGPs or their degradation products participate like signal molecules.

As proteínas arabinogalactanos (AGPs) são macromoléculas que se encontram praticamente em todos os órgãos das plantas, sendo associadas com vários aspectos do crescimento e desenvolvimento vegetal. Estas moléculas se caracterizam bioquímicamente por conter carboidratos e proteínas em relação 9:1. O carboidrato está composto principalmente por arabinogalactanos tipo II; enquanto que a parte protéica está organizada em domínios que definem as AGPs como clássicas ou não clássicas. As primeiras se caracterizam, além disso, por apresentar uma sequência C-terminal que prediz a incorporação de um grupo glicosilfosfatidilinositol (GFI), que permite sua união à membrana plasmática. Em cultivos de células vegetais se relatam várias espécies que liberam AGPs ao meio de cultivo. Apresenta-se uma revisão das características bioquímicas das AGPs liberadas ao meio e das propostas sobre os mecanismos bioquímicos e celulares pelos quais as AGPs participam na diferenciação e crescimento das células vegetais. Os cultivos de células liberam ao meio de cultivo AGPs clássicas e não clássicas, e se propõe que poderiam provir da membrana plasmática ou da parede celular. As AGPs intervêm no controle do crescimento celular, além de estar relacionadas com a embriogênese somática e a organogênese, processos de diferenciação celular importantes nos sistemas de micropropagação de plantas. O mecanismo bioquímico pelo qual as AGPs participam no crescimento celular e a diferenciação, implica que estas, ou os produtos de sua degradação, talvez atuem como moléculas de sinalização.

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.

A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress.

Mechanical wounding, infiltration with P. syringae or A. tumefaciens, and exposure to an H(2)O(2)-generating system (Glc/Glc oxidase) induce betacyanin synthesis in red beet (Beta vulgaris) leaves. These conditions also induced the expression of BvGT, a gene encoding a glucosyltransferase (GT) from Beta vulgaris. BvGT has a high similarity to Dorotheanthus bellidiformis betanidin-5 GT involved in betacyanin synthesis. Furthermore, the transient expression of a BvGT antisense construct resulted in the reduction of BvGT transcript accumulation and betanin synthesis, suggesting a role for this gene product in betacyanin glucosylation. In addition, the NADPH oxidase inhibitor, diphenylene iodonium (DPI), inhibited the accumulation of the BvGT transcript in response to infiltration with Agrobacterium tumefaciens. Hence, this result suggests that ROS produced by a plasma membrane NADPH oxidase may act as a signal to induce BvGT expression, necessary for betanin synthesis after wounding and bacterial infiltration.