Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier B.V. All rights reserved.

Anatomical changes induced by selection for plant architecture in maize (Zea mays L.)

The original brachytic population 'Dent Single Cross Composite' (DSCC-br2br2) and a selection-derived sub-population with modified plant architecture (DSCC-br2br2-Lg3Lg3, selected for erect leaves), were evaluated for the following characteristics number of vascular bundles of greater and smaller size, total vascular tissue area (phloem and xylem), sustaining tissue area (vascular tissue plus sclerenchyma), phloem and sclerenchyma areas in apical, medial and basal portions from midclub and in apical and basal sheath regions (from second leaf above and first below ear insertion). These variables had different values for the five different sections studied in each leaf and these differences did not have the same pattern in the two DSCC populations (brachytic and with modified architecture). Selection for architectural modification caused some indirect foliar anatomical modifications. With the exception of the phloem and the vascular tissue areas in apical leaf and sheath base regions, the modified plant architecture population showed smaller values of sustaining tissue area, sclerenchyma area, vascular tissue area and number of smaller vascular bundles than the original one. In the ligule region the modified maize leaves had smaller vascular and sustaining tissue areas, reducing transportation area, which could reduce gram yield.

Distribution of Xylella fastidiosa in citrus rootstocks and transmission of citrus variegated chlorosis between sweet orange plants through natural root grafts

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Nicotiana tabacum as an experimental host for the study of plant-Xylella fastidiosa interactions

Xylella fastidiosa causes citrus variegated chlorosis (CVC). Information generated from the X. fastidiosa genome project is being used to study the underlying mechanisms responsible for pathogenicity. However, the lack of an experimental host other than citrus to study plant-X. fastidiosa interaction has been an obstacle to accelerated progress in this area. We present here results of three experiments that demonstrated that tobacco could be an important experimental host for X. fastidiosa. All tobacco plants inoculated with a citrus strain of X. fastidiosa expressed unequivocal symptoms, consisting of orange leaf lesions, approximately 2 months after injection of the pathogen. CVC symptoms were observed in citrus 3 to 6 months after inoculation. The pathogen was readily detected in symptomatic tobacco plants by polymerase chain reaction (PCR) and phase contrast microscopy. In addition, X. fastidiosa was reisolated on agar plates in 4 of 10 plants. Scanning electron microscopy analysis of cross sections of stems and petioles revealed the presence of rod shaped bacteria restricted to the xylem of inoculated plants. The cell size was within the limit typical of X. fastidiosa.

Drought tolerance of two field-grown clones of Coffea canephora

We compared tolerance to soil drought of two field-grown clones of Coffea canephora (clone 46, drought-sensitive; and clone 120, drought-tolerant). Under irrigation, there were no marked differences between the clones in water relation parameters, gas exchange and total leaf area. Under rainfed conditions, clone 46 showed osmotic adjustment and increased tissue rigidity. These adjustments, however, were incapable of preventing substantial decreases in xylem pressure potential. By contrast, clone 120 did not exhibit osmotic adjustment, but was able to increase tissue elasticity and to maintain xylem pressure potentials to a greater extent than clone 46 (despite having twice the total leaf area of this clone). Stomatal conductance was lowered by drought in clone 120 but not in clone 46. Carbon assimilation per unit leaf area in both clones remained unaffected under stress. Long-term water use efficiency (WUE), as estimated through carbon isotope discrimination, was consistently greater in clone 120 than in clone 46. Because of these traits, clone 120 was better able to postpone dehydration and to maintain whole-tree photosynthesis. It is proposed that these features should decisively contribute to buffer its productivity in drought-prone areas. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings

Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Effect of storage of cuttings on the rooting of different cultivars of cut chrysanthemums (Dendranthema grandiflora Tzvelev) for two different seasons

Chrysanthemums are commercially propagated through cuttings. Pre-rooting storage of cuttings in the dark is a common practice among growers and companies that work and trade with chrysanthemum cuttings. Therefore, the maximum storage period for cuttings and differences in tolerance among cultivars has been investigated. Adventitious roots of cuttings can originate in almost any tissue, including the epidermis, stem cortex and pericycle, ray parenchyma, immature xylem and phloem cells, and pith. The aims of this work were to determine the effect of time of cold storage of cuttings (0, 1, 2, 3, 4, 5 and 6 weeks) on the rooting of four cut chrysanthemum cultivars (Super White, Sheena, Dark Orange Reagan and Town Talk) for two seasons of the year (summer and winter), and also to determine the origin of root formation in chrysanthemum cuttings. The study was carried out as a randomized complete block design with 5 replications for each storage treatment. Each plot was comprised of three cuttings that were examined 14 days after the cutting procedure. During the winter, the roots were studied by scanning electron microscopy (SEM) at the Electron Microscope Laboratory. The following conclusions were made: in winter, cold storage affected the rooting of cuttings, mainly after two weeks of storage for all cultivars. The rooting percentage was lower in the winter and the cuttings could be preserved for a shorter period. The source and growth of roots in chrysanthemum cuttings was found to be endogenous. After three days, callus formed in the pericycle and the first emergence of adventitious roots occurred by the fourth day of rooting. During the summer, cold storage could be up to 4 weeks without any problems.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Morfo-anatomia do eixo vegetativo aéreo de Achyrocline alata (Kunth) DC. (Asteraceae)

Achyrocline alata occurs in a dense and low vegetation in Mato Grosso do Sul State, Brazil and it has been studied under physical-chemical and yield aspects, being scarce anatomical data. The present work has as objective the study of the aerial vegetative axis. Stem is cylindrical and hairy, having five wings, which are arisen from their leaves. The blade shows the presence of uniseriate epidermis, which is covered by the cuticle of variable thickness; and non-glandular and glandular trichomes. The non-glandular trichomes are uniseriate and multicellular and have their apex cell in whip form, while the glandular trichomes are multicellular, uniseriate or biseriate. The mesophyll is dorsiventral with uni-stratified palisade parenchyma in most of the cases and lacunal parenchyma formed by three to four layers of irregular cells. Only one collateral vascular bundle occurs in the midvein. Stem in transversal section is covered by epidermis with trichomes similar to leaves; the cortex is constituted by a discontinuous area of angular collenchyma, which is followed by chlorophyll parenchyma. Vascular cylinder that is delimited by pericycle shows vascular bundles of collateral type. In the secondary structure, the periderm is originated from epidermal and subepidermal tissues. In vascular region, the fascicular cambium differs into secondary xylem and phloem, while interfascicular cambium produces sclerenchymatous tissue.

Postharvest characteristics and treatments to overcome latex exuded from cut flowers of lotus

Lotus (Nelumbo nucifera Gaertn.) is a perennial herbaceous aquatic ornamental plant with potential to be used as a new cut flower for the Brazilian ornamental market. It shows exotic and attractive flowers and has a strong market appeal, once it is known as a symbol of purity, holiness and immortality. However, flowers have a short-vase life. Lotus flower stem exudes a large quantity of sticky milky sap from the cut surface, which is produced in laticifers, spatially associated with both xylem and phloem. It has been reported that latex coagulates on the cut surface preventing or reducing water absorption and reducing flowers' vaselife, requiring treatments to stop the flow of latex. The objective of this study was to report observations of lotus postharvest characteristics and evaluate treatments to overcome latex flow. The experiment was conducted as a complete randomized design with three replications of four stems in each vase and eight treatments; a control (distilled water), pretreatment of cut stem-ends with hot water (40° C/1 minute), boiling water (3 seconds), isopropyl alcohol 90% (10 minutes) or citric acid (pH = 2.8/1h) and, maintenance of stems in a holding solution of Tween® 20 (0.01%), citric acid (200 mg L-1) or Tween® 20 (0.01%) plus citric acid (200 mg L-1). Treatments had no significant effect on flowers vaselife which was only about three days, although isopropyl alcohol, hot and boiling water completely stopped latex flow. Cut stem-ends pretreated with citric acid (pH = 2.8/1 h) showed a significantly higher relative water content of petals compared to others treatments. The senescence symptom of lotus cut flowers was mainly characterized by abscission of turgid petals and dehiscence of stamens without any visual change of petal color and brightness.

Nodule growth and nitrogen fixation of Calopogonium mucunoides L. show low sensitivity to nitrate

It is well established that nitrate is a potent inhibitor of nodulation and nitrogen fixation in legumes. The objective of this study was to demonstrate the relative insensitivity of these processes to nitrate with Calopogonium mucunoides, a tropical South American perennial legume, native to the cerrado (savannah) region. It was found that nodule number was reduced by about half in the presence of high levels of nitrate (15 mM) but nodule growth (total nodule mass per plant) and nitrogen fixation (acetylene reduction activity and xylem sap ureide levels) were not affected. Other sources of N (ammonium and urea) were also without effect at these concentrations. At even higher concentrations (30 mM), nitrate did promote significant inhibition (ca. 50%) of acetylene reduction activity, but no significant reduction in xylem sap ureides was found. The extraordinary insensitivity of nodulation and N2 fixation of C. mucunoides to nitrate suggests that this species should be useful in studies aimed at elucidating the mechanisms of nitrate inhibition of these processes. © 2010 Springer Science+Business Media B.V.

Delimitação entre os lenhos juvenil e adulto de pinus elliottii engelm

Trees produce different types of woody tissue during growth. The xylem produced in the first years until a certain cambial age is called juvenile wood and has different physical, mechanical, chemical and anatomical characteristics from that produced later, when the cambial cells have matured. Thus, prior knowledge of the quality of the wood is of fundamental importance for choosing the best use of the material. This work aimed at using data from length of the tracheid and wood density to define the age transition from juvenile to mature wood. For this, three Pinus elliottii var. elliottii trees, aged 35 years, were collected at the Itapetininga Experimental Station, located in the state of São Paulo, Brazil. From each tree, disks of about 5 cm thick were obtained 0.05 m above the ground and used to determine the radial density by the gamma ray attenuation method and to measure the length of the tracheids. The results obtained by linear regression analysis showed that juvenile wood is limited to the first seven years of tree growth and the formation of mature wood occurs after twenty years. For the mature wood, there are significant differences among the averages obtained from the tracheid length and wood density.

Seedling morphology and development in the epiphytic cactus Epiphyllum phyllanthus (L.) Haw. (Cactaceae: Hylocereeae)

Seedling morphology is relevant in classification, taxonomy, and vegetation studies to understand plant life cycles, germination succession and requirements, and developmental progression. However, most morphological studies of seedlings lack analysis of organ anatomy, impeding the comprehension of series of development and establishment in a particular environment. Here, we have taken a traditional anatomical approach to examine the stages of seedling development in Epiphyllum phyllanthus, a holo-epiphytic cactus of tribe Hylocereeae. The goals were 1) to offer a comprehensive description of growth series in E. phyllanthus seedlings based on morphological and anatomical analysis and 2) to examine the initial growth phases in the life cycle of this species to identify organ development and understand their adaptive significance in relation to seedling establishment. Our results include descriptions of seed morphology, embryonic features, and seedling vascularization pattern in the root, hypocotyl, cotyledons, and epicotyl. The morphological and developmental patterns in E. phyllanthus seedlings have potential phylogenetic and ontogenetic implications in the Cactaceae. Characters such as the presence of mucilage on the seed coat, the lack of seed operculum, and large cotyledons in E. phyllanthus are comparable to basal cacti, but the root anatomy is more similar to columnar relatives. At the familial level, there is an apparent trend in decreasing number of phloem and xylem poles in the stele of primary root, correlated with degree of specialization and advanced phylogenetic position: tetrarch to septarch-octarch in basal lineages, tetrarch Cereus-type in columnar species, to the diarch vascular system in Rhipsalideae and some species with cylindric/globose stem. © Torrey Botanical Club.

Resistência de genótipos de eucalipto a ceratocystis spp

Eucalyptus is the most important plantation forest species in Brazil. Wilt and canker caused by Ceratocystis fimbriata on eucalyptus were first reported in 1998 in plantations of an E. grandis × E. urophylla hybrid in southern Bahia, Brazil. This work aimed at studying the reaction of different eucalyptus genotypes after inoculation with C. fimbriata isolates, in order to find a possible source of resistance. The study included four isolates of Ceratocystis collected from eucalyptus in different regions. One disc of fungal mycelium with 1-cm-diameter (from colonies growing for 10 days on malt extract agar medium-MEA) was inoculated on the stem of thus injured eucalyptus plants (six months old). A cotton wool moistened with sterile distilled water was wrapped with plastic film. Control plants were inoculated with discs of MEA without fungal colonies. The inoculated plants were kept in a greenhouse. Wilt symptoms were observed 90 days after inoculation. The seedlings were cut in the longitudinal direction of the stem in order to observe the colonization of fungus in the plant xylem. We tested twenty eucalyptus genotypes, but only five showed resistance to all isolates of Ceratocystis, belonging to different species of Eucalyptus: E. urophylla (C2 and C9), E. grandis (C3), E. saligna (C6 and C13) Most E. gramdis genotypes were more susceptible to all four fungal isolates. These results support future studies related to eucalyptus resistance to Ceratocystis.

Stem anatomy and development of successive cambia in Hebanthe eriantha (Poir.) Pedersen: A neotropical climbing species of the Amaranthaceae

Hebanthe eriantha (Poir.) Pedersen, a climbing species of the Amaranthaceae increases in stem thickness by forming successive cambia. The family is dominated by herbaceous species and is constantly under discussion due to its disputed nature of the meristem. In the young stem small alternate segments of vascular cambium cease to divide and new arc of cambium initiates outside to it. The newly formed arcs connect with pre-existing alternate segments of cambium to complete the ring. On the contrary, in thick stems, instead of small segments, complete ring of cambium is replaced by new one. These new alternate segments/cambia originate from the parenchyma cells located outside to the phloem produced by previous cambium. Cambium is storied and exclusively composed of fusiform initials while ray cells remain absent at least in the early part of the secondary growth. However, large heterocellular rays are observed in 15-mm diameter stems but their frequency is much lower. In some of the rays, ray cells become meristematic and differentiate into radially arranged xylem and phloem elements. In fully grown plants, stems are composed of several successive rings of secondary xylem alternating with secondary phloem. Secondary xylem is diffuse-porous and composed of vessels, fibres, axial parenchyma while exceptionally large rays are observed only in the outermost regions of thick stems. Vessel diameter increases progressively from the centre towards the periphery of stems. Although the origin of successive cambia and composition of secondary xylem of H. eriantha remains similar to other herbaceous members of Amaranthaceae, the occurrence of relatively wider and thick-walled vessels and large rays in fully grown plants is characteristic to climbing habit. © 2013 Springer-Verlag Wien.