Biochemical responses in leaf tissues of alkaloid producing Psychotria brachyceras under multiple stresses.

Under natural conditions plants are generally subjected to complex scenarios of combined or sequential environmental stresses. Among the various components of plant biochemistry modulated by abiotic variables, a pivotal role is played by antioxidant systems, including specialized metabolites and their interaction with central pathways. To help address this knowledge gap, a comparative analysis of metabolic changes in leaf tissues of the alkaloid accumulating plant Psychotria brachyceras Müll Arg. under individual, sequential, and combined stress conditions was carried out. Osmotic and heat stresses were evaluated. Protective systems (accumulation of the major antioxidant alkaloid brachycerine, proline, carotenoids, total soluble protein, and activity of the enzymes ascorbate peroxidase and superoxide dismutase) were measured in conjunction with stress indicators (total chlorophyll, ChA/ChB ratio, lipid peroxidation, H2O2 content and electrolyte leakage). Metabolic responses had a complex profile in sequential and combined stresses compared to single ones, being also modified over time. Different stress application schemes affected alkaloid accumulation in distinct ways, exhibiting similar profile to proline and carotenoids, constituting a complementary triad of antioxidants. These complementary non-enzymatic antioxidant systems appeared to be essential for mitigating stress damage and re-establishing cellular homeostasis. The data herein provides clues that may aid the development of a key component framework of stress responses and their appropriate balance to modulate tolerance and yield of target specialized metabolites.

Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.

Clonal Eucalyptus propagation is essential for various industry sectors. It requires cuttings to successfully develop adventitious roots (ARs). Environmental conditions are influential on AR development and may be altered to modulate the productivity of hard-to-root clones. The current knowledge gap in research on the physiological patterns underlying commercial-scale propagation results hinders the design of novel strategies. This study aimed to identify patterns of variation in AR-relevant parameters in contrasting seasons and species with distinct rooting performances. E. dunnii and E. ×urograndis (hard- (hardR) and easy-to-root (easyR), respectively) mini-stumps were subjected to light modulation treatments and to mini-tunnel use (MT) for a year. The treatment impact on the branching and rooting rates was recorded. The carbohydrate content, AR-related gene expression, and mineral nutrition profiles of cuttings from the control (Ctrl) and treated mini-stumps were analyzed. Light treatments were often detrimental to overall productivity, while MTs had a positive effect during summer, when it altered the cutting leaf nutrient profiles. Species and seasonality played large roles in all the assessed parameters. E. ×urograndis was particularly susceptible to seasonality, and its overall superior performance correlated with changes in its gene expression profile from excision to AR formation. These patterns indicate fundamental differences between easyR and hardR clones that contribute to the design of data-driven management strategies aiming to enhance propagation protocols.

Non-target molecular network and putative genes of flavonoid biosynthesis in Erythrina velutina Willd., a Brazilian semiarid native woody plant.

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

Histochemical Localization of Phenolic Compounds and Reactive Oxygen Species in Eucalypt Microcuttings.

Among the main features of plant specialized metabolism are cell- and tissue- specific expression and responsiveness to oxidative stress conditions. Although quantitative techniques have significantly improved over time, allowing higher levels of resolution in plant metabolic studies, such analyses are often expensive and/or require relatively large amounts of starting material. The following protocols offer a relatively simple way to survey specialized and related metabolites (total phenolics, flavonoids, lignins), as well as reactive oxygen species (superoxide and hydrogen peroxide), using light and fluorescence microscopy. Also, a step-by-step guide on how to quantify stained areas is provided. Through the association of qualitative and quantitative data, general patterns of molecule distribution across plant tissues may be inferred, allowing for the solving of biological questions, and contributing to hypothesis refinement. Eucalyptus microcuttings were chosen as plant material to exemplify how these protocols can provide useful data for the understanding of complex developmental processes, such as adventitious root formation, which may be influenced by specialized metabolites and redox conditions.

Antifungal activity of essential oil from Eucalyptus staigeriana against Alternaria alternata causing of leaf spot and black rot in table grapes.

Alternaria alternata causes leaf spot and black rot diseases in leaves and grapes of grapevines, respectively, and leads to huge economic losses in table grapes production. As natural antifungal agents, essential oils (EOs), which are generally recognized as safe substances, shows strong antifungal activity against fungal phytopathogens. The aim of this study was to determine the chemical composition of Eucalyptus staigeriana EO and its in vitro and in vivo effects against A. alternata. The major compounds of E. staigeriana EO were citral (34.32%, of which 21.83% geranial and 12.49% neral), limonene (20.60%) and 1,8-cineole (12.33%). E. staigeriana EO exhibited the highest inhibitory activity on mycelial growth and conidial germination at 1 µL mL-1. Moreover, the EO was able to reduce the incidence and severity of leaf spot disease in leaves and black rot disease in table grapes caused by A. alternata. These results represent a possible alternative to reduce the use of synthetic molecules for the control of diseases in postharvest of table grapes and in vineyard.

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves.

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets for modulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga.

Microcutting Redox Profile and Anatomy in Eucalyptus spp. With Distinct Adventitious Rooting Competence.

Adventitious root (AR) development takes place in an intricate cellular environment. Reactive oxygen species (ROS) and antioxidant defenses, triggered by wounding in cuttings, can modulate this process. A comparative assessment of biochemical and anatomical parameters at critical rooting stages in hard- (Eucalyptus globulus Labill.) and easy- (Eucalyptus grandis W.Hill ex Maiden) to-root species was carried out. Microcuttings from seedlings were inoculated in auxin-free AR induction medium and, after 96 h, transferred to AR formation medium for a period of 24 h. Samples were collected upon excision (Texc) and at the 5th day post excision (Tform). Delayed xylem development, with less lignification, was recorded in E. globulus, when compared to E. grandis, suggesting lower activity of the cambium layer, an important site for AR development. Superoxide was more densely present around the vascular cylinder at both sampled times, and in greater quantity in E. globulus than E. grandis, declining with time in the former. Hydrogen peroxide was localized primarily along cell walls, more intensely in the primary xylem and phloem, and increased significantly at Tform in E. globulus. Ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) activities were generally higher in E. grandis and varied as a function of time in E. globulus. Soluble guaiacol peroxidase (GPRX) activity increased from Texc to Tform in both species, whereas cell wall-bound GPRX activity increased with time in E. grandis, surpassing E. globulus. Flavonoid content increased with time in E. grandis and was higher than E. globulus at Tform. Principal component analysis showed that species- and time-derived differences contributed to almost 80% of the variance. Overall, data indicate that E. grandis shows higher cambium activity and tighter modulation of redox conditions than E. globulus. These features may influence ROS-based signaling and phytohormone homeostasis of cuttings, thereby impacting on AR development. Besides being players in the realm of AR developmental differences, the specific features herein identified could become potential tools for early clone selection and AR modulation aiming at improved clonal propagation of this forest crop.

Rooting of eucalypt cuttings as a problem-solving oriented model in plant biology.

Species of Eucalyptus are some of the most planted trees in the world, providing fiber, cellulose, energy, and wood for construction and furniture in renewable fashion, with the added advantage of fixing large amounts of atmospheric carbon. The efficiency of eucalypts in forestry relies mostly on the clonal propagation of selected genotypes both as pure species and interspecific hybrids. The formation of new roots from cambium tissues at the base of cuttings, referred to as adventitious rooting (AR), is essential for accomplishing clonal propagation successfully. AR is a highly complex, multi-level regulated developmental process, affected by a number of endogenous and environmental factors. In several cases, highly desirable genotypes from an industrial point of view carry along the undesirable trait of difficulty-to-root (recalcitrance). Understanding the bases of this phenotype is needed to identify ways to overcome recalcitrance and allow efficient clonal propagation. Herein, an overview of the state-of-the-art on the basis of AR recalcitrance in eucalypts addressed at various levels of regulation (transcript, protein, metabolite and phenotype), and OMICs techniques is presented. In addition, a focus is also provided on the gaps that need to be filled in order to advance in this strategic biological problem for global forestry industry relying on eucalypts.