Salinity Effect on Horticultural Crops: Morphological, Physiological, and Biomolecular Elements of Salinity Stress Response

Among abiotic stresses, high salinity stress is the most severe environmental stress. High salinity exerts its negative impact mainly by disrupting the ionic and osmotic equilibrium of the cell. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death. Salt tolerance in plants is a multifarious phenomenon involving a variety of changes at molecular, organelle, cellular, tissue as well as whole plant level. In addition, salt tolerant plants show a range of adaptations not only in morphological or structural features but also in metabolic and physiological processes that enable them to survive under extreme saline environments. The main objectives of my dissertation were understanding the main physiological and biomolecular features of plant responses to salinity in different genotypes of horticultural crops that are belonging to different families Solanaceae (tomato) and Cucurbitaceae (melon) and Brassicaceae (cabbage and radish). Several aspects of crop responses to salinity have been addressed with the final aim of combining elements of functional stress response in plants by using several ways for the assessment of plant stress perception that ranging from destructive measurements (eg. leaf area, relative growth rate, leaf area index, and total plant fresh and dry weight), to physiological determinations (eg. stomatal conductance, leaf gas exchanges, water use efficiency, and leaf water relation), to the determination of metabolite accumulation in plant tissue (eg. Proline and protein) as well as evaluation the role of enzymatic antioxidant capacity assay in scavenging reactive oxygen species that have been generated under salinized condition, and finally assessing the gene induction and up-down regulation upon salinization (eg. SOS pathway).

Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue.

CHOLINERGIC MODULATION OF THALAMIC INPUT INTO THE CINGULATE CORTEX

This research demonstrates cholinergic modulation of thalamic input into the limbic cortex. A projection from the mediodorsal thalamus (MD) to the anterior cingulate cortex was defined anatomically and physiologically. Injections of horse-radish peroxidase into the anterior cingulate cortex labels neurons in the lateral, parvocellular, region of MD. Electrical Stimulation of this area produces a complex field potential in the anterior cingulate cortex which was further characterized by current density analysis and single cell recordings.^ The monsynaptic component of the response was identified as a large negative field which is maximal in layer IV of the anterior cingulate cortex. This response shows remarkable tetanic potentiation of frequencies near 7 Hz. During a train of 50 or more stimuli, the response would grow quickly and remain at a fairly stable potentiated level throughout the train.^ Cholinergic modulation of this thalamic response was demonstrated by iontophoretic application of the cholinergic agonist carbachol decreased the effectiveness of the thalamic imput by rapidly attenuation the response during a train of stimuli. The effect was apparently mediated by muscarinic receptors since the effect of carbachol was blocked by atropine but not by hexamethonium.^ To determine the source of the cingulate cortex cholinergic innervation, lesions were made in the anterior and medial thalamus and in the nucleus of the diagonal band of Broca. The effects of these lesions on choline acetyltranferase activity in the cingulate cortex were determined by a micro-radio-enzymatical assay. Only the lesions of the nucleus of the diagonal band significantly decreased the choline acetyltransferase activity in the cingulate cortex regions. Therefore, the diagonal band appears to be a major source of sensory cholinergic innervation and may be involved in gating of sensory information from the thalamus into the limbic cortex. Attempts to modulate the cingulate response to MD stimulation with electrical stimulation of the diagonal band, however were not successful.^

A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the λ integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe–plant interactions.

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls1

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca2+. The complex formed in the presence of Ca2+ was more stable than that without Ca2+. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca2+ and B. Removal of the Ca2+ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca2+ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca2+ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca2+ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca2+, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca2+ but also borate and Ca2+ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1 : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

A influência da luz na germinação das sementes de rabanete (Raphanus sativus, L.)

A germinação das sementes depende de diversos fatores. Estes podem ser dependentes da própria semente: intrínsecos (tais como a constituição da semente ou a sua maturidade e vitalidade), ou extrínsecos (tais como a luz, a temperatura, a humidade, o oxigénio e as características do solo). No presente trabalho pretendemos estudar o efeito de um desses fatores - a luz - na germinação das sementes de rabanete (Raphanus sativus, L.). Sabe-se que certas sementes só germinam à luz (fotoblastismo positivo), outras só o fazem às escuras (fotoblastismo negativo) e que outras são indiferentes ao efeito da luz no que se refere à germinação (fotoblastismo neutro) (Sousa, Braga, Braga & Delachiave, 2008; Ribeiro et al., 2012). Com o nosso estudo pudemos perceber que as sementes do rabanete apresentam fotoblastismo neutro.

Contrary effects of jasmonate treatment of two closely related plant species on attraction of and oviposition by a specialist herbivore

Elevated jasmonic acid (JA) concentrations in response to herbivory can induce wounded plants to produce defences against herbivores. In laboratory and field experiments we compared the effects of exogenous JA treatment to two closely related cabbage species on the host-searching and oviposition preference of the diamondback moth (DBM), Plutella xylostella. JA-treated Chinese cabbage (Brassica campestris) was less attractive than untreated Chinese cabbage to ovipositing DBM, while JA-treatment of common cabbage (B. oleracea) made plants more attractive than untreated controls for oviposition by this insect. Similar effects were observed when plants of the two species were damaged by DBM larvae. In the absence of insect-feeding, or JA application, Chinese cabbage is much more attractive to DBM than common cabbage. Inducible resistance therefore appears to occur in a more susceptible plant and induced susceptibility appears to occur in a more resistant plant, suggesting a possible balance mechanism between constitutive and inducible defences to a specialist herbivore.

Light-activated phytotoxic thiophenes in Flaveria linearis L.

Chromatographic analyses of crude leaf extracts of Flaveria linearis L. revealed the presence of four acetylenic monothiophenes. Three of these metabolites were purified and structurally characterized by UV, NMR, IR, and GC-MS. Germination, growth, and survival/mortality studies with and without UVA, the activating wavelengths of these metabolites, were conducted with the crude leaf extracts and the purified compounds (taken individually and combined) against selected crop species (lettuce, radish, and carrot). Results suggest that acetylenic metabolites are phytotoxic against lettuce, carrot, and radish, but with variability in response among species. These variations in sensitivity and the allelopathic potential of F. linearis is discussed.

High-performance parallel systems for data-intensive computing

Thesis (Ph.D.)--University of Washington, 2016-08

Nitrogênio mineral em um latossolo em função de sistemas de manejo do solo e de culturas de cobertura de inverno

The increasing advancement of agriculture makes providing adequate conditions for the growth and development of plants is the primary purpose of soil management systems. Much of the success of PD is attributed to cultural remains left by cover crops that do not require high nitrogen inputs and can thus be used to reduce nitrogen input in the agro- ecosystem. The nitrogen is one of the elements applied in agriculture, it is absorbed in higher quantities and limiting the yield of grain crops such as corn. Thus, there has been the influence of the no-till and conventional tillage combined with different crops of winter cover and bare soil when in succession to corn, on mineral nitrogen content. The experimental work was made at the experimental station of the Agronomic Institute of Paraná - Iapar. The implemented design was blocks at random split plot with three replications in factorial 6 x 2 x 3 x 5. The main plots were as treatment, beyond the bare soil, 5 winter species (ryegrass, vetch, vetch + oat, oat and radish), while in the subplots were used two tillage systems (No-till and conventional tillage). Three collections made were (before management, the urea before and after the urea), these being held in 5 depths (0-5, 5-10, 10-20, 20-40 and 40-60 cm). So a layer 0-5 cm and a que presents increased amount to NH4 + ion. The use of associated PD system in the presence of winter cover crops decreased as NO3 - losses in soil profile.

Comparison of host range and pathogenicity of isolates of Pythium myriotylum and Pythium zingiberis

Apart from morphology and genetic characteristics, species status of Pythium zingiberis and P. myriotylum may also be confirmed based on their pathogenicity and host range. An Australian putative P. zingiberis isolate and imported type isolates of P. myriotylum and P. zingiberis were subject to both in vitro and in vivo pathogenicity tests. In vitro tests were carried out on excised carrot, ginger, potato, radish, and sweet potato tuber/root sections, and on seeds and seedlings of cucumber, cauliflower, millet, rye, sweet corn, tomato, and wheat. In all assays conducted, the Australian isolate was found to be the most pathogenic, followed by type specimen of P. zingiberis (UOP 275), and then the type specimen P. myriotylum (CBS 254.70). An in vivo experiment on ginger plants at 35°C (with 10 h day light) in quarantine conditions showed that the ginger plants inoculated with the Australian isolate and also the type specimen of P. zingiberis died at 21 days after inoculation, whereas those inoculated with P. myriotylum CBS 254.70 were still green and healthy. Along with cardinal growth rate, the Australian isolate was confirmed to be closely related to P. zingiberis. This is also the first direct comparison in pathogenicity of P. zingiberis and P. myriotylum.

Extraction and characterization of Raphanus Sativus seed oil obtained by different methods

Purpose: To evaluate the impact of three different extraction methods on yield, physicochemical properties and bioactive ingredients of Raphanus sativus seed oil. Methods: Raphanus Sativus seed oil was prepared by traditional solvent extraction (SE), super-critical carbon dioxide extraction (SCE) and sub-critical propane extraction (SPE). The yield, physicochemical properties, fatty acid composition and oxidative stability of the oil extracts were compared. The contents of tocopherol and sulforaphene in the oils were also determined. Results: The oil yield obtained by SPE, SE and SCE were 33.69, 27.17 and 24.10 %, respectively. There were no significant differences in physicochemical properties and fatty acid compositions of oils extracted by the three methods. However, SCE oil had the best oxidative stability, and highest contents of vitamin E and sulforaphene, followed by oils from SPE and SE. Conclusion: SCE is highly selective for tocopherol and sulforaphene, which could explain its high oil oxidative stability. These results suggest that of the three extraction methods, SCE is best suited for preparing medicinal radish seed oil.