Chemical diversity of Brittonodoxa subpinnata, a Brazilian native species of moss.

Plants should be probably thought of as the most formidable chemical laboratory that can be exploited for the production of an incredible number of molecules with remarkable structural and chemical diversity that cannot be matched by any synthetic libraries of small molecules. The bryophytes chemistry has been neglected for too long, but in the last ten years, this scenery is changing, with several studies being made using extracts from bryophytes, aimed at the characterization of interesting metabolites, with their metabolome screened. The main objective of this study was to analyze the metabolome of Brittonodoxa subpinnata, a native Brazilian moss species, which occurs in the two Brazilian hotspots. GC-MS and LC-MS2 were performed. All extracts were analyzed using the molecular networking approach. The four extracts of B. subpinnata (polar, non-polar, soluble, and insoluble) resulted in 928 features detected within the established parameters. 189 (20.4%) compounds were annotated, with sugars, fatty acids, flavonoids, and biflavonoids as the major constituents. Sucrose was the sugar with the highest quantity; palmitic acid the major fatty acid but with great presence of very long-chain fatty acids rarely found in higher plants, glycosylated flavonoids were the major flavonoids, and biflavonoids majorly composed by units of flavones and flavanones, exclusively found in the cell wall. Despite the high percentage, this work leaves a significant gap for future works using other structure elucidation techniques, such as NMR.

Stress memory of physiological, biochemical and metabolomic responses in two different rice genotypes under drought stress: The scale matters.

Drought is a pivotal cause for crop yield reductions. When subjected to recurrent external stimuli, plants can develop memory of stress responses that, eventually, enables improved plant tolerance to environmental changes. In addition, despite causal relationships, these responses may vary according to hierarchical levels of observation. Thus, this study aims to check the responses of recurrent and non-recurrent stresses in two rice genotypes observing their drought memory responses at different levels of organization, that is, on a physiological, biochemical and metabolomic scale and for end in global PCA. For this, seventy variables were measured on the scales described in order to obtain a large number of observations. The memory responses were evident in almost all scales observed. The lowland genotype, especially plants not subjected to recurrent water shortage, showed higher damage to the photosynthetic apparatus under drought conditions, although it has exhibited more evident memory response effect after rehydration. On the other hand, the upland genotype appears to be more tolerant to drought insofar lower biochemical damage was observed. Specific behaviors of each genotype at biochemical and metabolomics levels and similar behavior at physiological level were observed. This study demonstrates the importance of observation at different hierarchical levels.

Metabolic and physiological alterations indicate that the tropical broadleaf tree Eugenia uniflora L. is sensitive to ozone.

Eugenia uniflora L. is an important fruit tree native to tropical South America that adapts to different habitats, thanks to its metabolic diversity and ability to adjust the leaf antioxidant metabolism. We hypothesized that this metabolic diversity would also enable E. uniflora to avoid oxidative damage and tolerate the enhanced ozone (O3) concentrations that have been registered in the (sub)tropics. We investigated whether carbohydrates, polyphenols and antioxidants are altered and markers of oxidative damage (ROS accumulation, alterations in leaf gas exchange, growth and biomass production) are detected in plants exposed to two levels of O3 (ambient air and twice elevated ozone level in a O3-FACE system for 75 days). Phytotoxic O3 dose above a threshold of 0 nmol m-2 s-1 (POD0) and accumulated exposure above 40 ppb (AOT40) were 3.6 mmol m-2 and 14.898 ppb h at ambient, and 4.7 mmol m-2 and 43.881 ppb h at elevated O3. Twenty-seven primary metabolites and 16 phenolic compounds were detected in the leaves. Contrary to the proposed hypothesis that tropical broadleaf trees are relatively O3 tolerant, we concluded that E. uniflora plants are sensitive to elevated O3 concentrations. Experimental POD0 values were lower than the critical levels for visible foliar O3, because of low stomatal conductance. In spite of this low stomatal O3 uptake, we found classic O3 injury, e.g. reduction in carbohydrates and fatty acids concentrations; non-significant changes in the polyphenol profile; inefficient antioxidant responses; increased contents of ROS and indicators of lipid peroxidation; reductions in stomatal conductance, net photosynthesis, root/shoot ratio and height growth. However, we also found some compensation mechanisms, e.g. increased leaf concentration of polyols for protecting the membranes, and increased leaf number for compensating the decline of photosynthetic rate. These results help filling the knowledge gap about tropical tree responses to O3.

The Regulation of Floral Colour Change in Pleroma raddianum (DC.) Gardner.

Floral colour change is a widespread phenomenon in angiosperms, but poorly understood from the genetic and chemical point of view. This article investigates this phenomenon in Pleroma raddianum, a Brazilian endemic species whose flowers change from white to purple. To this end, flavonoid compounds and their biosynthetic gene expression were profiled. By using accurate techniques (Ultra Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UPLC-HRMS)), thirty phenolic compounds were quantified. Five key genes of the flavonoid biosynthetic pathway were partially cloned, sequenced, and the mRNA levels were analysed (RT-qPCR) during flower development. Primary metabolism was also investigated by gas chromatography coupled to mass spectrometry (GC-EIMS), where carbohydrates and organic acids were identified. Collectively, the obtained results suggest that the flower colour change in P. raddianum is determined by petunidin and malvidin whose accumulation coincides with the transcriptional upregulation of early and late biosynthetic genes of the flavonoid pathway, mainly CHS and ANS, respectively. An alteration in sugars, organic acids and phenolic co-pigments is observed together with the colour change. Additionally, an increment in the content of Fe3+ ions in the petals, from the pink to purple stage, seemed to influence the saturation of the colour.

Metabolomics of Myrcia bella Populations in Brazilian Savanna Reveals Strong Influence of Environmental Factors on Its Specialized Metabolism.

Environmental conditions influence specialized plant metabolism. However, many studies aiming to understand these modulations have been conducted with model plants and/or under controlled conditions, thus not reflecting the complex interaction between plants and environment. To fully grasp these interactions, we investigated the specialized metabolism and genetic diversity of a native plant in its natural environment. We chose Myrcia bella due to its medicinal interest and occurrence in Brazilian savanna regions with diverse climate and soil conditions. An LC-HRMS-based metabolomics approach was applied to analyze 271 samples harvested across seven regions during the dry and rainy season. Genetic diversity was assessed in a subset of 40 samples using amplified fragment length polymorphism. Meteorological factors including rainfall, temperature, radiation, humidity, and soil nutrient and mineral composition were recorded in each region and correlated with chemical variation through multivariate analysis (MVDA). Marker compounds were selected using a statistically informed molecular network and annotated by dereplication against an in silico database of natural products. The integrated results evidenced different chemotypes, with variation in flavonoid and tannin content mainly linked to soil conditions. Different levels of genetic diversity and distance of populations were found to be correlated with the identified chemotypes. These observations and the proposed analytical workflow contribute to the global understanding of the impact of abiotic factors and genotype on the accumulation of given metabolites and, therefore, could be valuable to guide further medicinal exploration of native species.

Beyond the limits of photoperception: constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato.

Photoreceptor engineering has recently emerged as a means for improving agronomically beneficial traits in crop species. Despite the central role played by the red/far-red photoreceptor phytochromes (PHYs) in controlling fruit physiology, the applicability of PHY engineering for increasing fleshy fruit nutritional content remains poorly exploited. In this study, we demonstrated that the fruit-specific overexpression of a constitutively active GAF domain Tyr252 -to-His PHYB2 mutant version (PHYB2Y252H ) significantly enhances the accumulation of multiple health-promoting antioxidants in tomato fruits, without negative collateral consequences on vegetative development. Compared with the native PHYB2 overexpression, PHYB2Y252H -overexpressing lines exhibited more extensive increments in transcript abundance of genes associated with fruit plastid development, chlorophyll biosynthesis and metabolic pathways responsible for the accumulation of antioxidant compounds. Accordingly, PHYB2Y252H -overexpressing fruits developed more chloroplasts containing voluminous grana at the green stage and overaccumulated carotenoids, tocopherols, flavonoids and ascorbate in ripe fruits compared with both wild-type and PHYB2-overexpressing lines. The impacts of PHYB2 or PHYB2Y252H overexpression on fruit primary metabolism were limited to a slight promotion in lipid biosynthesis and reduction in sugar accumulation. Altogether, these findings indicate that mutation-based adjustments in PHY properties represent a valuable photobiotechnological tool for tomato biofortification, highlighting the potential of photoreceptor engineering for improving quality traits in fleshy fruits.

Acylated Flavonoid Glycosides are the Main Pigments that Determine the Flower Colour of the Brazilian Native Tree Tibouchina pulchra (Cham.) Cogn.

Tibouchina pulchra (Cham.) Cogn. is a plant native to Brazil whose genus and family (Melastomataceae) are poorly studied with regards to its metabolite profile. Phenolic pigments of pink flowers were studied by ultra-performance liquid chromatography with a photodiode array detector and electrospray ionization quadrupole time-of-flight mass spectrometry. Therein, twenty-three flavonoids were identified with eight flavonols isolated by preparative high-performance liquid chromatography and analysed by one- and two-dimensional nuclear magnetic resonance. Kaempferol derivatives were the main flavonols, encompassing almost half of the detected compounds with different substitution patterns, such as glucoside, pentosides, galloyl-glucoside, p-coumaroyl-glucoside, and glucuronide. Concerning the anthocyanins, petunidin p-coumaroyl-hexoside acetylpentoside and malvidin p-coumaroyl-hexoside acetylpentoside were identified and agreed with previous reports on acylated anthocyanins from Melastomataceae. A new kaempferol glucoside was identified as kaempferol-(2''-O-methyl)-4'-O-α-d-glucopyranoside. Moreover, twelve compounds were described for the first time in the genus with five being new to the family, contributing to the chemical characterisation of these taxa.

Does Phoradendron perrottetii (mistletoe) alter polyphenols levels of Tapirira guianensis (host plant)?

The present study aimed to investigate the reciprocal effects of Phoradendron perrottetii (mistletoe) and T. guianensis (host plant) regarding their polyphenol composition. Taking into account that tannins are important molecules in plant defense and their biosynthesis tends to be enhanced when a species is exposed to stress, we address the following questions: (1) Are the tannins found in our model species important in the interaction between host and mistletoe? (2) Does the presence of mistletoe induce changes in the content of tannins and other polyphenols in the host plant? (3) Do we find differences between the tannin sub-groups in the responses of the host plant to mistletoe? (4) Could the observed differences reflect the relative importance of one tannin group over another as chemical defense against the mistletoe? Using a polyphenol and tannin group-specific MRM methods we quantified four different tannin sub-groups together with flavonoid and quinic acid derivatives by ultra-performance liquid chromatography tandem mass spectrometry together with the oxidative and protein precipitation activities of leaves and branches of Tapirira guianensis and Phoradendron perrottetii. We selected leaves and branches of six non-parasitized trees of T. guianensis. Leaves and branches of nine individuals of T. guianensis parasitized by P. perrottetii were also sampled. For each parasitized tree, we sampled an infested branch and its leaves, as well as a non-infested branch and its leaves. Infested branches were divided into three groups: gall (the host-parasite interface), proximal, and distal region. Both proanthocyanidins and ellagitanins seem to be important for plant-plant parasitism interaction: host infested tissues (gall and surrounding regions) have clearly less tannin contents than healthy tissues. Mistletoe showed high levels of quinic acid derivatives and flavonoids that could be important during hastorium formation and intrusion on host tissues, suggesting a defense mechanism that could promote oxidative stress together with an inhibition of mistletoe seed germination, consequently avoiding secondary infestations. Polyphenol detected in T. guianensis-P. perrottetii interaction could play different role as plant-mistletoe strategies of survival.

The passion fruit liana (Passiflora edulis Sims, Passifloraceae) is tolerant to ozone.

Passiflora edulis Sims is a liana species of high economic interest and is an interesting model plant for understanding ozone action on disturbed vegetation. In this work we hypothesized that P. edulis has adaptive responses to oxidative stress that enable it to tolerate ozone damage based on its capacity to grow under a diversity of environmental conditions and to dominate disturbed areas. We exposed seedlings to three levels of ozone in a Free-Air Controlled Exposure (FACE) system (22, 41 and 58 ppb h AOT40 and 13.52, 17.24 and 20.62 mmol m-2 POD0, over 97 days) for identifying its tolerance mechanisms. Anatomical (leaf blade structure and fluorescence emission of chloroplast metabolites), physiological (leaf gas exchange, growth rate and biomass production) and biochemical (pigments, total sugars, starch, enzymatic and non-enzymatic antioxidant metabolites, reactive oxygen species and lipid peroxidation derivatives) responses were assessed. Ozone caused decreased total number of leaves, hyperplasia and hypertrophy of the mesophyll cells, and accelerated leaf senescence. However, O3 did not affect carbohydrates content, net photosynthetic rate, or total biomass production, indicating that the carboxylation efficiency and associated physiological processes were not affected. In addition, P. edulis showed higher leaf contents of ascorbic acid, glutathione (as well high ratio between their reduced and total forms), carotenoids, and flavonoids located in the chloroplast outer envelope membrane. Our results indicate that P. edulis is an O3-tolerant species due to morphological acclimation responses and an effective antioxidant defense system represented by non-enzymatic antioxidants, which maintained the cellular redox balance under ozone.

Genetic Diversity and Structure of Populations of Annona crassiflora Mart. of Brazilian Savanna and Its Association with Chemical Variability.

Annona crassiflora Mart. is a native tree from Brazilian savanna. Isoquinoline alkaloids are characteristic of species of Annonaceae. This work aimed to assess the magnitude of genetic diversity among different populations of A. crassiflora using AFLP markers, and verify the existence of any correlation between the AFLP data and previous reported alkaloid composition. A. crassiflora from eight populations in the states of São Paulo, Goiás, Minas Gerais, and Distrito Federal were analyzed. The data suggest a low, moderate, and high level of genetic diversity from different populations of A. crassiflora. Concentration of alkaloids was significantly correlated with AFLP data, suggesting interaction between chemical and molecular markers in A. crassiflora. The data of association between the chemical and genetic differentiation of A. crassiflora may be useful to establish cultivation areas allowing the definition of strategies to preserve their genetic diversity with an interest in specific chemotypes for genetic improvement programs focused on sustainable utilization of this specie.

Anthocyanins and tannins in ozone-fumigated guava trees.

Psidium guajava "Paluma", a tropical tree species, is known to be an efficient ozone indicator in tropical countries. When exposed to ozone, this species displays a characteristic leaf injury identified by inter-veinal red stippling on adaxial leaf surfaces. Following 30 days of three ozone treatments consisting of carbon filtered air (CF - AOT40=17 ppb h), ambient non-filtered air (NF - AOT40=542 ppb h) and ambient non-filtered air+40 ppb ozone (NF+O(3) - AOT40=7802 ppb h), the amounts of residual anthocyanins and tannins present in 10 P. guajava ("Paluma") saplings were quantified. Higher amounts of anthocyanins were found in the NF+O(3) treatment (1.6%) when compared to the CF (0.97%) and NF (1.30%) (p<0.05), and of total tannins in the NF+O(3) treatment (0.16%) compared to the CF (0.14%). Condensed tannins showed the same tendency as enhanced amounts. Regression analyses using amounts of tannins and anthocyanins, AOT40 and the leaf injury index (LII), showed a correlation between the leaf injury index and quantities of anthocyanins and total tannins. These results are in accordance with the association between the incidence of red-stippled leaves and ozone polluted environments.

Effects of initial climatic conditions on growth and accumulation of fluoride and nitrogen in leaves of two tropical tree species exposed to industrial air pollution.

Saplings of Tibouchina pulchra and Psidium guajava, cultivated under standardized soil conditions, were placed in two sites at Cubatão (state of São Paulo, southeast Brazil) to study the effects of air pollution on growth, biomass allocation and foliar nitrogen and fluoride concentrations. Thirty-six potted plants were maintained over two periods of one year (Jul/00 to Jun/01; Dec/00 to Nov/01) at each of two experimental sites with distinct levels of air pollution: Pilões River Valley (PV) with vegetation virtually unaffected by air pollution; and Mogi River Valley (MV) severely affected by pollutants released mainly by chemical, fertilizer, iron and steel industries. For both species, saplings growing at MV showed alterations of growth and biomass allocation, as well as increased leaf concentrations of nitrogen and fluoride. Comparing both experimental periods, the one starting in winter (the driest season in Southeastern Brazil) seemed to affect the saplings more severely, the differences of the measured parameters between MV and PV being higher than in the second period. Multivariate analysis revealed two groups of data: one representing the MV and the other the PV saplings. For both species, saplings growing at MV showed differences in chemical composition, growth and biomass allocation, compared with the PV saplings. The results suggested that seasonal conditions of the first months of sapling exposure (summer or winter) modulate the intensity of responses to pollution stress.