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.

Synthesis and Biological Evaluation of the Novel Growth Inhibitor Streptol Glucoside, Isolated from an Obligate Plant Symbiont.

The plant Psychotria kirkii hosts an obligatory bacterial symbiont, Candidatus Burkholderia kirkii, in nodules on their leaves. Recently, a glucosylated derivative of (+)-streptol, (+)-streptol glucoside, was isolated from the nodulated leaves and was found to possess a plant growth inhibitory activity. To establish a structure-activity relationship study, a convergent strategy was developed to obtain several pseudosugars from a single synthetic precursor. Furthermore, the glucosylation of streptol was investigated in detail and conditions affording specifically the α or ß glucosidic anomer were identified. Although (+)-streptol was the most active compound, its concentration in P. kirkii plant leaves extract was approximately ten-fold lower than that of (+)-streptol glucoside. These results provide compelling evidence that the glucosylation of (+)-streptol protects the plant host against the growth inhibitory effect of the compound, which might constitute a molecular cornerstone for this successful plant-bacteria symbiosis.

Superoxide generated in the chloroplast stroma causes photoinhibition of photosystem I in the shade-establishing tree species Psychotria henryi.

Our previous studies indicated that high light induced significant photoinhibition of photosystem I (PSI) in the shade-establishing tree species Psychotria henryi. However, the underlying mechanism has not been fully clarified. In the present study, in order to investigate the mechanism of PSI photoinhibition in P. henryi, we treated detached leaves with constant high light in the presence of methyl viologen (MV) or a soluble α-tocopherol analog, 2,2,5,7,8-pentamethyl-6-chromanol (PMC). We found that MV significantly depressed photochemical quantum yields in PSI and PSII when compared to PMC. On condition that no PSI photoinhibition happened, although cyclic electron flow (CEF) was abolished in the MV-treated samples, P700 oxidation ratio was maintain at higher levels than the PMC-treated samples. In the presence of PMC, PSI photoinhibition little changed but PSII photoinhibition was significantly alleviated. Importantly, PSI photoinhibition was largely accelerated in the presence of MV, which stimulates the production of superoxide and subsequently other reactive oxygen species at the chloroplast stroma by accepting electrons from PSI. Furthermore, MV largely aggravated PSII photoinhibition when compared to control. These results suggest that high P700 oxidation ratio cannot prevent PSI photoinhibition in P. henryi. Furthermore, the superoxide produced in the chloroplast stroma is critical for PSI photoinhibition in the higher plant P. henryi, which is opposite to the mechanism underlying PSI photoinhibition in Arabidopsis thaliana and spinach. These findings highlight a new mechanism of PSI photoinhibition in higher plants.

The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation.

Nickel is an economically important metal and phytotechnologies are being developed to limit the impact of nickel mining on the environment. More than 300 plant species are known to hyperaccumulate nickel. However, our knowledge of the mechanisms involved in nickel accumulation in plants is very limited because it has not yet been possible to study these hyperaccumulators at the genomic level. Here, we used next-generation sequencing technologies to sequence the transcriptome of the nickel hyperaccumulator Psychotria gabriellae of the Rubiaceae family, and used yeast and Arabidopsis as heterologous systems to study the activity of identified metal transporters. We characterized the activity of three metal transporters from the NRAMP and IREG/FPN families. In particular, we showed that PgIREG1 is able to confer nickel tolerance when expressed in yeast and in transgenic plants, where it localizes in the tonoplast. In addition, PgIREG1 shows higher expression in P. gabriellae than in the related non-accumulator species Psychotria semperflorens. Our results designate PgIREG1 as a candidate gene for nickel tolerance and hyperaccumulation in P. gabriellae. These results also show how next-generation sequencing technologies can be used to access the transcriptome of non-model nickel hyperaccumulators to identify the underlying molecular mechanisms.

Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions.

Several plant species of the genus Psychotria (Rubiaceae) harbor Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is considered to be obligatory because plants devoid of symbionts fail to develop into mature individuals. The genome of 'Candidatus Burkholderia kirkii' has been sequenced recently and has revealed evidence of reductive genome evolution, as shown by the proliferation of insertion sequences and the presence of numerous pseudogenes. We employed shotgun proteomics to investigate the expression of 'Ca. B. kirkii' proteins in the leaf nodule. Drawing from this dataset and refined comparative genomics analyses, we designed a new pseudogene prediction algorithm and improved the genome annotation. We also found conclusive evidence that nodule bacteria allocate vast resources to synthesis of secondary metabolites, possibly of the C7N aminocyclitol family. Expression of a putative 2-epi-5-valiolone synthase, a key enzyme of the C7N aminocyclitol synthesis, is high in the nodule population but downregulated in bacteria residing in the shoot apex, suggesting that production of secondary metabolites is particularly important in the leaf nodule.

Cyclotide Isolation from Psychotria brachyceras and Psychotria leiocarpa.

Cyclotides are small circular peptides carrying an array of interesting biological activities and also showing interesting features for storage and bioavailability. Here, an optimized method to isolate cyclotides from two species of Psychotria, P. brachyceras and P. leiocarpa, that can be integrally performed (to isolate cyclotides) or used in part (to obtain cyclotide-rich extracts), is described. In general this protocol can be applied for cyclotide isolation from any species, taking into account potential minor adaptations for the particularities of specific cases.

Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts.

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-D-glucose analogue (+)-streptol, systemically supplied by mature Ca. Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria-Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.

Metabolic engineering of cell cultures versus whole plant complexity in production of bioactive monoterpene indole alkaloids: recent progress related to old dilemma.

Monoterpene indole alkaloids (MIAs) are a large class of plant alkaloids with significant pharmacological interest. The sustained production of MIAs at high yields is an important goal in biotechnology. Intensive effort has been expended toward the isolation, cloning, characterization and transgenic modulation of genes involved in MIA biosynthesis and in the control of the expression of these biosynthesis-related genes. At the same time, considerable progress has been made in the detailed description of the subcellular-, cellular-, tissue- and organ-specific expressions of portions of the biosynthetic pathways leading to the production of MIAs, revealing a complex picture of the transport of biosynthetic intermediates among membrane compartments, cells and tissues. The identification of the particular environmental and ontogenetic requirements for maximum alkaloid yield in MIA-producing plants has been useful in improving the supply of bioactive molecules. The search for new bioactive MIAs, particularly in tropical and subtropical regions, is continuously increasing the arsenal for therapeutic, industrially and agriculturally useful molecules. In this review we focus on recent progress in the production of MIAs in transgenic cell cultures and organs (with emphasis on Catharanthus roseus and Rauvolfia serpentina alkaloids), advances in the understanding of in planta spatial-temporal expression of MIA metabolic pathways, and on the identification of factors capable of modulating bioactive alkaloid accumulation in nontransgenic differentiated cultures and plants (with emphasis on new MIAs from Psychotria species). The combined use of metabolic engineering and physiological modulation in transgenic and wild-type plants, although not fully exploited to date, is likely to provide the sustainable and rational supply of bioactive MIAs needed for human well being.

The use of chemometrics to study multifunctional indole alkaloids from Psychotria nemorosa (Palicourea comb. nov.). Part I: Extraction and fractionation optimization based on metabolic profiling.

Extraction methods evaluation to access plants metabolome is usually performed visually, lacking a truthful method of data handling. In the present study the major aim was developing reliable time- and solvent-saving extraction and fractionation methods to access alkaloid profiling of Psychotria nemorosa leaves. Ultrasound assisted extraction was selected as extraction method. Determined from a Fractional Factorial Design (FFD) approach, yield, sum of peak areas, and peak numbers were rather meaningless responses. However, Euclidean distance calculations between the UPLC-DAD metabolic profiles and the blank injection evidenced the extracts are highly diverse. Coupled with the calculation and plotting of effects per time point, it was possible to indicate thermolabile peaks. After screening, time and temperature were selected for optimization, while plant:solvent ratio was set at 1:50 (m/v), number of extractions at one and particle size at ≤180µm. From Central Composite Design (CCD) results modeling heights of important peaks, previously indicated by the FFD metabolic profile analysis, time was set at 65min and temperature at 45°C, thus avoiding degradation. For the fractionation step, a solid phase extraction method was optimized by a Box-Behnken Design (BBD) approach using the sum of peak areas as response. Sample concentration was consequently set at 150mg/mL, % acetonitrile in dichloromethane at 40% as eluting solvent, and eluting volume at 30mL. Summarized, the Euclidean distance and the metabolite profiles provided significant responses for accessing P. nemorosa alkaloids, allowing developing reliable extraction and fractionation methods, avoiding degradation and decreasing the required time and solvent volume.

Regeneration of Psychotria umbellata and production of the analgesic indole alkaloid umbellatine.

Psychotria umbellata Vell. (Rubiaceae), a Brazilian coastal woody species, produces umbellatine (also known as psychollatine), an analgesic indole alkaloid. An in vitro embryogenic regeneration protocol capable of yielding alkaloid-accumulating plants was developed. Rhizogenic calli, which were obtained from stem segments derived from rooted apical cuttings, were cultured on Murashige and Skoog's (MS) medium containing either 1 mg l(-1) NAA (naphthalene acetic acid) and no kinetin, or 5 mg l(-1) NAA + 1 mg l(-1) kinetin. Calli did not accumulate umbellatine. Segments of rhizogenic callus were cultured on complete MS medium with various concentrations of kinetin and sucrose. Plant regeneration was best in the light with 0.25 mg l(-1) of kinetin and 1.5% sucrose. After 3 months of acclimatization in soil mixture, plant survival was 81%. Leaves of 10-month-old regenerated plants yielded umbellatine concentrations equivalent to those of adult forest-grown plants.

Shoot accumulation kinetics and effects on herbivores of the wound-induced antioxidant indole alkaloid brachycerine of Psychotria brachyceras.

A major shoot-specific monoterpene indole alkaloid produced by Psychotria brachyceras, brachycerine, is regulated by either wounding or jasmonate application. Highest concentrations of the alkaloid are found in inflorescences, suggesting a defence role. Brachycerine has antimutagenic and antioxidant properties, capable of quenching singlet oxygen, hydroxyl radical, and superoxide. This study aimed at characterizing the putative role of brachycerine in P. brachyceras responses to wounding and herbivory. Damage to leaves increased the content of brachycerine locally. Wounding did not affect phenolics content in P brachyceras leaves, and no tannins were detected in the species. In generalist herbivore bioassays, neither brachycerine nor P. brachyceras extracts showed toxic effects. In vivo hydrogen peroxide staining assay showed less wound-generated peroxide accumulation in alkaloid treated tissues. This pattern was confirmed in quantitative assays measuring tissue hydrogen peroxide concentrations. Data indicate that brachycerine is not a herbivore deterrent, but rather an indirect chemical defence, modulating oxidative stress caused by mechanical damage.

Distribution of the cardiotoxin pavettamine in the coffee family (Rubiaceae) and its significance for gousiekte, a fatal poisoning of ruminants.

Gousiekte, a cardiac syndrome of ruminants in southern Africa, is caused by the ingestion of plants containing the polyamine pavettamine. All the six known gousiekte-causing plants are members of the Rubiaceae or coffee family and house endosymbiotic Burkholderia bacteria in their leaves. It was therefore hypothesized that these bacteria could be involved in the production of the toxin. The pavettamine level in the leaves of 82 taxa from 14 genera was determined. Included in the analyses were various nodulated and non-nodulated members of the Rubiaceae. This led to the discovery of other pavettamine producing Rubiaceae, namely Psychotria kirkii and Psychotria viridiflora. Our analysis showed that many plant species containing bacterial nodules in their leaves do not produce pavettamine. It is consequently unlikely that the endosymbiont alone can be accredited for the synthesis of the toxin. Until now the inconsistent toxicity of the gousiekte-causing plants have hindered studies that aimed at a better understanding of the disease. In vitro dedifferentiated plant cell cultures are a useful tool for the study of molecular processes. Plant callus cultures were obtained from pavettamine-positive species. Mass spectrometric analysis shows that these calli do not produce pavettamine but can produce common plant polyamines.

Accumulation of brachycerine, an antioxidant glucosidic indole alkaloid, is induced by abscisic acid, heavy metal, and osmotic stress in leaves of Psychotria brachyceras.

Psychotria brachyceras Muell. Arg. produces the antioxidant monoterpene indole alkaloid (MIA) brachycerine, which, besides retaining a glucose residue, has its terpenoid moiety derived not from secologanin, but probably from epiloganin, representing a new subclass of MIAs. In this work we showed that osmotic stress agents, such as sodium chloride, sorbitol and polyethylene glycol (PEG), induced brachycerine accumulation in leaf disks of P. brachyceras. Other oxidative stress inducers, such as exposure to aluminum and silver, also increased brachycerine content. Abscisic acid (ABA) treatment was shown to increase brachycerine yield, suggesting its involvement in brachycerine induction during osmotic stress. Ascorbate peroxidase activity was induced in PEG-treated leaf disks, whereas superoxide dismutase (SOD) activity remained unaltered. Assays with specific inhibitors of the cytosolic mevalonate (MVA) and plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways showed that the terpenoid moiety of brachycerine derived predominantly from the MEP pathway. These results suggest a potential involvement of brachycerine in plant defense against osmotic/oxidative stress damage, possibly contributing to detoxification of hydroxyl radical and superoxide anion as a SOD-like molecule.