Cuticular wax composition contributes to different strategies of foliar water uptake in six plant species from foggy rupestrian grassland in tropical mountains.

The cuticle is the outermost region of the epidermal cell wall of plant aerial organs. The cuticle acts as a two-way lipid barrier for water diffusion; therefore, it plays a vital role in foliar water uptake (FWU). We hypothesised that the chemical composition of the cuticular waxes influences the FWU strategy that plants adopt in a foggy tropical ecosystem. We analysed the leaf cuticular waxes of six plant species known by their different FWU strategies, in both qualitative and quantitative approaches, to test this hypothesis. We also investigated the fine structure of the plant cuticle by scanning electron microscopy. Neither the total wax loads nor the amounts of single wax compound classes correlated to the FWU. In contrast, the qualitative chemical composition of the cuticular waxes was related to the water absorption speed but not to the maximum water absorbed. The presence of wax crystals might interfere with the FWU. Our findings suggest that a complex three-dimensional network of the cuticular compounds contributes to different strategies of FWU in six plant species from foggy tropical mountaintops.

Apoplast-symplast compartmentalization and functional traits of iron and aluminum in promeristematic tissues of nematode induced galls on Miconia spp.

The nutritive tissues of galls induced by Ditylenchus gallaeformans (Nematoda) have promeristematic capacity, which may turn these galls into sinks of Al on their Melastomataceae Al-accumulating hosts. Such a sink of Al may affect gall growth and mineral nutrient intake. Based on the fact that galls are good models for plant developmental studies, we aimed to understand how Al-accumulating host plants in the Cerrado environment deal with Al toxicity in subcellular levels. Here, we used the ICP-OES method to check the variations on mineral nutrients, and the morin, hematoxylin, and Prussian blue stainings for Al and Fe histolocalization in galls induced on four Miconia species of the Brazilian Cerrado. We confirmed the new Al-accumulating feature for two Miconia species of the Cerrado environment. Furthermore, we found that Al accumulates in lesser concentrations in gall tissues than in non-galled tissues of the Miconia hosts. Staining methods indicated that the polyphenols avoid Al-binding to the apoplast and the nucleolus of the promeristematic cells, and mediated its binding to parenchyma cell walls. As well, we inferred that Fe3+ is transported by xylem and stored in gall parenchyma, where it is reduced to Fe2+, being available in gall nutritive cells. Our results demonstrated an Al compartmentalization between the apoplast and symplast of the inner cell layers in galls, as well as indicated the phenolics action against Al-toxicity and toward Fe availability for the diet of Ditylenchus gallaeformans.

Immunocytochemistry and Density Functional Theory evidence the competition of aluminum and calcium for pectin binding in Urochloa decumbens roots.

Root growth is reduced in soils with low pH [H+] and abundant soluble aluminum [Al3+], which can be a consequence of the interaction between Al3+ and cell wall composition. The competition between Al3+ and Ca2+ toward binding to pectin molecules was evaluated in roots of Urochloa decumbens, an African grass highly adapted to acidic Al-rich soils. Variations in the composition and distribution of pectins can change the extensibility, rigidity, porosity, and adhesive properties of plant cell walls, which were tested in seedlings of U. decumbens exposed to pH 3.5, 4.5 and 5.8 and to 0, 80, 160 and 320 µM of Al3+ for 80h. Root growth corroborated that U. decumbens is very tolerant to soil acidity, with effective reduction of root growth only at pH 3.5. Immunocytochemical approaches demonstrated variations in pectin composition induced both by Al3+ and by H+ in root tissues and zones. Based on the usual linkage between Ca2+ and pectins, Density Functional Theory (DFT) analyses indicated that Al3+ bound easier to pectins than Ca2+ did, leading to the formation of more Al3+-pectate complexes than Ca2+-pectate complexes, which resulted in higher rigidity of cell walls, and hampered cell extension.

Belowground microbes mitigate plant-plant competition.

Dimorphandra wilsonii, a Cerrado endemic Fabaceae tree, is threatened by land-use changes. The few remaining individuals occur in areas dominated by alien grasses like Urochloa decumbens. We tested the impact of nitrogen (N) availability and symbionts' presence on mitigating the effects of competition from U. decumbens. Dimorphandra wilsonii seedlings were 50-week pot-cultivated under limiting (3mM) or non-limiting (10mM) N, with or without U. decumbens, and inoculated or not with a N-fixer (Bradyrhizobium sp.) and an arbuscular mycorrhizal fungus (AMF - Glomus etunicatum), both forming symbioses in the field. Since D. wilsonii seedlings grew more and 'lost' fewer nutrients under the symbionts' presence, symbionts mitigated plant-plant competition. Under limiting N, inoculated D. wilsonii seedlings grew more (despite no nodulation), but N fixation was only suggested when inoculated D. wilsonii seedlings competed with U. decumbens. D. wilsonii13C, and substrate's carbon and respiration suggest that only the microbes performing key functions received plant carbon. Under non-limiting N, inoculated D. wilsonii seedlings became enriched in 13C, substrate accumulated carbon and microbial respiration increased, suggesting a more generalist microbial community. Data suggest inoculating D. wilsonii seeds/seedlings with AMF and N-fixers as a conservation measure. However, long-term field-studies need to confirm these conclusions.

Morphology, ultrastructure, and element uptake in Calophyllum brasiliense Cambess. (Calophyllaceae J. Agardh) seedlings under cadmium exposure.

Cadmium (Cd) is a metal known for its genotoxicity and cytotoxicity, much concerned for its potential environmental and human health impacts. This study evaluates the toxic effect of Cd in Calophyllum brasiliense plants. The plants were cultivated for 30 days in full nutrient solution in order to adapt, and for 15 days in nutrient solution without Cd or with 4, 8, 16, and 32 µmol Cd L-1. Anatomical analysis of the leaf showed no significant effects of Cd on epidermal thickness in abaxial and adaxial sides, palisade, and spongy parenchyma. Contrastingly, changes were noticed in the ultrastructural level in the leaf mesophyll cells as rupture of the membrane of chloroplasts and disorganization of the thylakoid membranes, in starch grains and in mitochondria with rupture of the membrane and invagination of the nuclear membrane. Electron dense materials into cells of the cortex and vascular bundle were also observed. In the cells of the root system, the observed ultrastructural changes were disruption of the cell wall and electron dense material deposition in the cortex cells and vascular region. Cd accumulated in roots with low translocation into shoot. Cd toxicity also affected the photosynthetic activity, inducing stomatal closure and photosynthetic assimilation reduction and the instantaneous carboxylation efficiency, drastically reducing the leaf transpiration. The nutrient content in the stem and root was variable, according to Cd increase in nutrient solution. Based on the experimental evidence, it can be concluded that C. brasiliense has potential to bioconcentrate high Cd levels in the root system.

Different salt concentrations induce alterations both in photosynthetic parameters and salt gland activity in leaves of the mangrove Avicennia schaueriana.

Mangrove sites are constantly under tidal regimes, where there is variation of dissolved oxygen and saline content in water. This study evaluated the effects of varying salinity in Avicennia schaueriana, an abundant species in the sea-mangrove-river interface. Seedlings of A. schaueriana were harvested at the riverbank and placed in different saline and nutrient solutions in a greenhouse in order to simulate a saline flow. After 7 acclimatization days, plants were exposed to the following saline concentrations: 0, 50, 170 and 250mM NaCl, for a period of 30 days. After this, leaf gas exchange, chlorophyll a fluorescence, Falker chlorophyll index, leaf ultrastructure and micromorphology were evaluated. The highest saline concentration affected mainly the CO2 assimilation, internal CO2 concentration and the intrinsic efficiency of water use. The chlorophyll a fluorescence and Falker index were not significantly affected by any of the saline conditions, however chloroplast was damaged. Concomitantly, leaf salt glands clearly evidenced a higher salt secretion, when plants were submitted to the greatest saline concentration. The results indicated that A. schaueriana tolerates different salt concentration in the mangrove and excretes salt excess by salt gland, and this can attenuate the saline stress. However, when exposed to longer periods of higher salt fluxes the plant growth can be affected.

Morphology, ultrastructure and mineral uptake is affected by copper toxicity in young plants of Inga subnuda subs. luschnathiana (Benth.) T.D. Penn.

Toxic effects of copper (Cu) were analyzed in young plants of Inga subnuda subs. luschnathiana, a species that is highly tolerant to flooding and found in Brazil in wetlands contaminated with Cu. Plants were cultivated in fully nutritive solution, containing different concentrations of Cu (from 0.08 µmol to 0.47 mmol L(-1)). Symptoms of Cu toxicity were observed in both leaves and roots of plants cultivated from 0.16 mmol Cu L(-1). In the leaves, Cu clearly induced alterations in the thickness of the epidermis, mesophyll, palisade parenchyma, and intercellular space of the lacunose parenchyma. Also, this metal induced disorganization in thylakoid membranes, internal and external membrane rupture in chloroplasts, mitochondrial alterations, and electrodense material deposition in vacuoles of the parenchyma and cell walls. The starch grains disappeared; however, an increase of plastoglobule numbers was observed according to Cu toxicity. In the roots, destruction of the epidermis, reduction of the intercellular space, and modifications in the format of initial cells of the external cortex were evident. Cell walls and endoderm had been broken, invaginations of tonoplast and vacuole retractions were found, and, again, electrodense material was observed in these sites. Mineral nutrient analysis revealed higher Cu accumulation in the roots and greater macro- and micronutrients accumulation into shoots. Thus, root morphological and ultrastructural changes induced differential nutrients uptake and their translocations from root toward shoots, and this was related to membrane and endoderm ruptures caused by Cu toxicity.

Nitric oxide increases tolerance responses to moderate water deficit in leaves of Phaseolus vulgaris and Vigna unguiculata bean species.

Drought stress is one of the most intensively studied and widespread constraints, and nitric oxide (NO) is a key signaling molecule involved in the mediation of abiotic stresses in plants. We demonstrated that a sprayed solution of NO from donor sodium nitroprusside increased drought stress tolerance responses in both sensitive (Phaseolus vulgaris) and tolerant (Vigna unguiculata) beans. In intact plants subjected to halting irrigation, NO increased the leaf relative water content and stomatal conductance in both species. After cutting leaf discs and washing them, NO induced increased electrolyte leakage, which was more evident in the tolerant species. These leaf discs were then subjected to different water deficits, simulating moderate and severe drought stress conditions through polyethylene glycol solutions. NO supplied at moderate drought stress revealed a reduced membrane injury index in sensitive species. In hydrated discs and at this level of water deficit, NO increased the electron transport rate in both species, and a reduction of these rates was observed at severe stress levels. Taken together, it can be shown that NO has an effective role in ameliorating drought stress effects, activating tolerance responses at moderate water deficit levels and in both bean species which present differential drought tolerance.

Seeds' physicochemical traits and mucilage protection against aluminum effect during germination and root elongation as important factors in a biofuel seed crop (Ricinus communis).

We determined the length, volume, dry biomass, and density in seeds of five castor bean cultivars and verified notable physicochemical trait differences. Seeds were then subjected to different toxic aluminum (Al) concentrations to evaluate germination, relative root elongation, and the role of root apices' rhizosphere mucilage layer. Seeds' physicochemical traits were associated with Al toxicity responses, and the absence of Al in cotyledons near to the embryo was revealed by Al-hematoxylin staining, indicating that Al did not induce significant germination reduction rates between cultivars. However, in the more sensitive cultivar, Al was found around the embryo, contributing to subsequent growth inhibition. After this, to investigate the role of mucilage in Al tolerance, an assay was conducted using NH4Cl to remove root mucilage before or after exposure to different Al concentrations. Sequentially, the roots were stained with hematoxylin and a quantitative analysis of staining intensity was obtained. These results revealed the significant contribution of the mucilage layer to Al toxicity responses in castor bean seedlings. Root growth elongation under Al toxicity confirmed the role of the mucilage layer, which jointly indicated the differential Al tolerance between cultivars and an efficient Al-exclusion mechanism in the tolerant cultivar.

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a threatened species native to the Brazilian Cerrado.

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N(2)-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N(2) fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N(2)-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.

Morphophysiological differences in leaves of Lavoisiera campos-portoana (Melastomataceae) enhance higher drought tolerance in water shortage events.

Lavoisiera campos-portoana Barreto (Melastomataceae) has two kinds of leaves, pubescent and glabrous, and branches may have one or both types of leaves at the same moment. The plant is endemic to high altitude rocky fields in Brazil where rainfall is very seasonal. We predicted that these two leaf types are adaptations to different regimes of water availability. In experimental conditions of drought stress, we measured relative water content (RWC), photosynthetic pigments, chlorophyll a fluorescence and osmotic potential, and we counted stomates and measured stomatal conductance on both sides of leaves and compared these between the two leaf types. Stomatal conductance and electron transport rate at a given photosynthetic photon flux were greater in pubescent leaves than in glabrous leaves, and both declined during drought stress. Excessive photon flux density in glabrous leaves was greater during stress and after rehydration. Photosynthetic pigment content and RWC did not change between leaves, and values reduced during the stress period. Both types of leaves showed osmotic adjustment capacity, which occurred earlier in glabrous ones. These morphophysiological differences highlight the adaptation strategies of this plant to withstand drought, since the glabrous portion of the plant presents a preventive behavior, while the pubescent portion only shows the same responses in more advanced stages of drought stress.

Cloning and characterization of drought-stimulated phosphatidic acid phosphatase genes from Vigna unguiculata.

Under environmental stresses, several lipolytic enzymes are known to be activated and to contribute to membrane lipid turnover and generation of second messengers. In animal cells, phosphatidic acid phosphatase (PAP, EC 3.1.3.4), which dephosphorylates phosphatidic acid generating diacylglycerol, is long known as an enzyme involved in lipid synthesis and cell signalling. However, knowledge on PAP in plants remains very limited. The aim of this work was to isolate and characterize PAP genes in the tropical legume Vigna unguiculata (cowpea), and to study their expression under different stress conditions. Two cDNAs designated as VuPAPalpha and VuPAPbeta were cloned from the leaves of cowpea. Both proteins share sequence homology to animal type 2 PAP, namely, the six transmembrane regions and the consensus sequences corresponding to the catalytic domain of the phosphatase family, like the recently described Arabidopsis LPP (Lipid Phosphate Phosphatase) proteins. The recombinant protein VuPAPalpha expressed in Escherichia coli cells was able to convert phosphatidic acid into diacylglycerol. Unlike VuPAPbeta, VuPAPalpha has an N-terminal transit peptide and was addressed to chloroplast in vitro. Both genes are expressed in several cowpea organs and their transcripts accumulate in leaves in response to water deficit, including progressive dehydration of whole plants and rapid desiccation of detached leaves. No changes in expression of both genes were observed after wounding or by treatment with jasmonic acid. Furthermore, the in silico analysis of VuPAPalpha promoter allowed the identification of several putative drought-related regulatory elements. The possible physiological role of the two cloned PAPs is discussed.