Danilo da Cruz Centeno

Leaf metabolite profile of the Brazilian resurrection plant Barbacenia purpurea Hook. (Velloziaceae) shows two time-dependent responses during desiccation and recovering

Barbacenia purpurea is a resurrection species endemic to rock outcrops, in Rio de Janeiro, Brazil. It tolerates great temperature variations, which are associated to periods of up to 30 days without precipitation. Using a metabolomic approach, we analyzed, under winter and summer conditions, changes in the leaf metabolite profile (MP) of potted plants of B. purpurea submitted to daily watered and water deficit for at least 20 days and subsequent slow rehydration for 5 days. Leaves were collected at different time points and had their MP analyzed by GC/MS, HPAEC, and UHPLC techniques, allowing the identification of more than 60 different compounds, including organic and amino acids, sugars, and polyols, among others. In the winter experiment, results suggest the presence of two time-dependent responses in B. purpurea under water stress. The first one starts with the increase in the content of caffeoyl-quinic acids, substances with strong antioxidant activity, until the 16th day of water suppression. When RWC reached less than 80 and 70%, in winter and summer respectively, it was observed an increase in polyols and monosaccharides, followed by an increment in the content of RFO, suggesting osmotic adjustment. Amino acids, such as GABA and asparagine, also increased due to 16 days of water suppression. During rehydration, the levels of the mentioned compounds became similar to those found at the beginning of the experiment and when compared to daily watered plants. We conclude that the tolerance of B. purpurea to dehydration involves the perception of water deficit intensity, which seems to result in different strategies to overcome the gradient of water availability imposed along a certain period of stress mainly during winter. Data from summer experiment indicate that the metabolism of B. pupurea was already primed for drought stress. The accumulation of phenolics in summer seemed to be more temperature and irradiance-dependent than on the RWC.

Do recalcitrant seeds really exist

In the 70s, seeds were divided into two categories: recalcitrant and orthodox. In the 80s, it was necessary to create an intermediate category; from the 90s onwards, a gradient between orthodox and recalcitrant categories has been considered by several authors. Currently, the terms orthodox and recalcitrant are appropriate just for technological purposes, not for scientific studies. It seems that the differences between recalcitrant and orthodox seeds lie only on the maturity stage in which they are detached from the mother plant, the recalcitrant ones in a very immature stage. This implies that little progress should be expected to expand the storability of these recalcitrant seeds with the application of any treatment after harvesting. Efforts shall be focused on amplifying the maturation period of these seeds by keeping them linked to the mother plant until the maturation process has been completed.

Metabolic and structural changes during early maturation of Inga vera seeds are consistent with the lack of a desiccation phase

Inga vera, native to South America, is an important leguminous species used for ecological restoration of riparian forests and its seeds are among the most recalcitrant ones described up to date. In this work, we analysed the metabolic profile, cell ultrastructure as well as cell wall polysaccharides of I. vera seeds in order to better understand its maturation, which allows embryo germination without a quiescent phase. Increased amounts of citric, glutamic, pyroglutamic, and aspartic acids from stages I to II (120 and 129 days after flowering (DAF)) corroborate the hypothesis of high metabolism, shifting from fermentative to aerobic respiration at seed maturity. This phase was characterized by an extensive vacuolization of embryonic cells, which also indicate high metabolic activity. The proportion of arabinose in the cell walls of embryonic axis (approx. 20%) was lower than those found in some orthodox seeds (nearly 40%), suggesting that arabinose-containing polysaccharides, which are thought to provide more flexibility to the cell wall during natural drying, are less abundant in I. vera seeds. Taken together, our results provide evidence that the major changes occurred during early stages of seed maturation of I. vera, indicating that the rapid temporary metabolic shift observed between stages I and II may be related to the lack of desiccation phase, moving directly to germination.

Contrasting strategies used by lichen microalgae to cope with desiccation-rehydration stress revealed by metabolite profiling and cell wall analysis.

Most lichens in general, and their phycobionts in particular, are desiccation tolerant, but their mechanisms of desiccation tolerance (DT) remain obscure. The physiological responses and cell wall features of two putatively contrasting lichen-forming microalgae, Trebouxia sp. TR9 (TR9), isolated from Ramalina farinacea (adapted to frequent desiccation-rehydration cycles), and Coccomyxa solorina-saccatae (Csol), obtained from Solorina saccata (growing in usually humid limestone crevices, subjected to seasonal dry periods) was characterized. Microalgal cultures were desiccated under 25%-30% RH and then rehydrated. Under these conditions, RWC and ψw decreased faster and simultaneously during dehydration in Csol, whereas TR9 maintained its ψw until 70% RWC. The metabolic profile indicated that polyols played a key role in DT of both microalgae. However, TR9 constitutively accumulated higher amounts of polyols, whereas Csol induced the polyol synthesis under desiccation-rehydration. Csol also accumulated ascorbic acid, while TR9 synthesized protective raffinose-family oligosaccharides (RFOs) and increased its content of phenolics. Additionally, TR9 exhibited thicker and qualitatively different cell wall and extracellular polymeric layer compared with Csol, indicating higher water retention capability. The findings were consistent with the notion that lichen microalgae would have evolved distinct strategies to cope with desiccation-rehydration stress in correspondence with the water regime of their respective habitats.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility

Summary Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl‐CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p‐coumarate, changes in two‐dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40–60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Free cyclitol, soluble carbohydrate and protein contents in Vigna unguiculata and Phaseolus vulgaris bean sprouts.

Seeds sprouts have been used as a good source of basic nutrients and nutraceutical compounds. The high nutritional value of seeds derives from the deposition of compounds during development. However some of these molecules are used in metabolic processes like germination, which leads to a considerable variation in their concentrations once these events are completed. In this work, we investigate the levels of inositols (myo-inositol, D-pinitol and ononitol), soluble carbohydrates and proteins in cotyledons of Phaseolus vulgaris and Vigna unguiculata sprouts. Sprouting increased myo-inositol and glucose content and reduction of raffinose and ononitol was observed. The protein levels increased in P. vulgaris and decreased in V. unguiculata sprouting. The level of sucrose was maintained in both sprouts. D-Pinitol was detected only in quiescent seeds. Our results suggested that bean sprout is an important source of proteins, sucrose, glucose and myo-inositol. Additionally, bean sprouts have low levels of raffinose, an antinutritional compound.

Overexpression of BdMATE Gene Improves Aluminum Tolerance in Setaria viridis

Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop. Setaria viridis is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, Setaria viridis was used as a model plant to overexpress a newly identified MATE gene from Brachypodium distachyon (BdMATE), closely related to SbMATE, for aluminum tolerance assays. Transgenic S. viridis plants overexpressing a BdMATE presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that BdMATE gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.

Chemical analysis and toxicity of seaweed extracts with inhibitory activity against tropical fruit anthracnose fungi

BACKGROUND Banana and papaya are among the most important crops in the tropics, with a value amounting to millions of dollars per year. However, these fruits suffer significant losses due to anthracnose, a fungal disease. It is well known that certain seaweed extracts possess antifungal activity, but no published data appear to exist on the practical application of this property. In the present study, five organic Brazilian seaweed extracts were screened for their activity against banana and papaya anthracnose fungi. Furthermore, cytotoxic and mutagenic effects of the extracts were evaluated by the brine shrimp lethality assay and the Allium cepa root-tip mutagenicity test respectively, while their major components were identified by gas chromatography/mass spectrometry. RESULTS Strong fungus-inhibitory effects of Ochtodes secundiramea and Laurencia dendroidea extracts were observed on both papaya (100 and 98% respectively) and banana (89 and 78% respectively). This impressive activity could be associated with halogenated terpenes, the major components of both extracts. Only Hypnea musciformis extract showed cytotoxic and mutagenic effects. CONCLUSION The results of this study suggest the potential use of seaweed extracts as a source of antifungal agents with low toxicity to control anthracnose in papaya and banana during storage.

Exogenous nitric oxide-induced germination associated with pinitol content in Hymenaea courbaril low vigour seeds

Abstract Nitric oxide (NO), a membrane-permeable free radical, has been reported as a potent signalling molecule in animals and plants. It is commonly accepted that NO induces dormancy release and promotes seed germination. However, few studies were carried out with seeds of tropical arboreal species, which present distinct characteristics compared to temperate model seeds, in order to confirm a broad role of NO in seed germination. To our knowledge, this is the first work to investigate the role of exogenous NO on the germination of seeds accumulating cell wall polysaccharides as the main carbon reserve. The aim of this study was to evaluate the effect of NO donors on germination and primary metabolism of seeds from Hymenaea courbaril L., a leguminous tree species from Neotropical rainforests. Seeds with distinct vigour rates, namely lower vigour seeds (LVS) and higher vigour seeds (HVS), were imbibed in distilled water (control) and NO donor solutions, and selected parameters such as germination rate and normal seedlings, root growth, and metabolic profile were evaluated. Our results show that NO donors reduced the average time of germination and improved normal seedling development of LVS, without affecting the final germination rate. This treatment also induced changes in the relative proportion of soluble sugars and cyclitols identified by gas chromatography–mass spectrometry analysis. Taken together, our data suggest that exposure to the NO donor partially revert deterioration on naturally ageing seeds, possibly through the modulation of pinitol content, and open new perspectives to enhance the existing knowledge on the role of NO on seed viability.

Imaging of glyphosate uptake and identification of early microscopic markers in leaves of C3 and C4 glyphosate-resistant and -susceptible species

Details of glyphosate uptake are not fully elucidated, and although this herbicide promotes important alterations in the plant phenotype few hours after its application (early responses), a detailed description of the presumable changes in plant anatomy is still poorly assessed by now. Due to glyphosate effects over leaf permeability, the use of an inert fluorescent tracer may allow the observation of the uptake event in situ. In addition, microscopic markers might put a light on the recognition of glyphosate-resistant (GR) and -susceptible (GS) species or varieties, which may vary in C3 and C4 species due to their putative distinct leaf anatomy. Here we aimed (i) to provide a new technique to track the route of glyphosate formulation towards leaf tissues using a fluorescent tracer, and (ii) to describe the early specific microscopic alterations in GR and GS -C3 or -C4 caused by the glyphosate formulation. Roundup Transorb® was applied in seedlings cultivated in a greenhouse and response alterations in leaf anatomy were described. Lucifer Yellow CH (LYCH) was applied over the same region where glyphosate formulation was previously applied to track the alterations in leaf permeability caused by this herbicide. LYCH successfully tracked the glyphosate formulation uptake, reaching the vascular bundles of GS species, and becoming retained in leaf tissues of GR species. All species exhibited a decrease in chlorophyll content at the site of glyphosate application regardless of their photosynthetic metabolism or susceptibility. GS species showed alterations in chloroplast morphology and activity of non-enzymatic antioxidants (carotenoids and flavonoids), in addition to symptoms indicating a process of accelerated cell senescence. A specific type of cell necrosis (hypersensitive response) was observed in GR-C4 species as a way to prevent the translocation of this herbicide, while GR-C3 species accumulated phenolic compounds inside the vacuole, probably sequestrating and inactivating the glyphosate action. This study provides a reliable tool to track glyphosate formulation uptake in situ and is the first attempt to the identification of early specific microscopic markers caused by glyphosate formulation.