Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development

procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA(3). The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA(3) or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA(3) application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set.

The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively.

Biochemical and histological characterization of tomato mutants

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H2O2) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.

Solanum americanum as a reservoir of natural enemies of the tomato red spider mite, Tetranychus evansi (Acari: Tetranychidae)

Brazilian populations of the pathogenic fungus Neozygites floridana (Weiser & Muma) Remaudiere & S. Keller (Entomophthoromycotina: Entomophthorales) and the predatory mite Phytoseiulus longipes Evans (Acari: Phytoseiidae) are potential candidates for introduction into Africa for classical biological control of the tomato red spider mite, Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae). The efficiency of these natural enemies against T. evansi has been demonstrated under laboratory conditions, but little is known about their performance on native Solanaceae in the field. The American nightshade, Solanum americanum Mill., is native to the Americas and may serve as an alternative host plant for T. evansi and its natural enemies in the absence of tomato plants. In this work, we studied the population dynamics of T. evansi and its natural enemies on S. americanum in a screen house, semi-field and field plots in Recife, Pernambuco, Brazil, to evaluate the potential of natural enemies for controlling T. evansi. Of the four natural enemies found in association with T. evansi, only N. floridana and P. longipes were clearly associated with the reduction of the populations of T. evansi. Neozygites floridana was observed in a screen house, semi-field and field plots, but P. longipes was only detected in the semi-field plots. Increases in the population of T. evansi were always followed by increases in the density of one of these natural enemies, suggesting that they were important factors regulating T. evansi populations on S. americanum. The presence of this host plant near the release sites in Africa thus might increase the chances for the permanent establishment of these natural enemies.

Bioactivity of neem nanoformulations on tomato pinworm

The use of insecticide plants is an important tool in the management of insect pests. Aiming to control Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), neem nanoformulations were evaluated. After estimating the LC50 for a commercial neem oil formulation, selection bioassays were performed with 22 nanoformulations. In order to do that, newly emerged caterpillars were fed on leaflets treated with nanoformulation solutions for 10 days. The effect on the development and longevity of the insect was evaluated with the two most promising nanoformulations, aqueous NC40 and powdered NC40 (NC 40 = Poly- -hydroxibutirate nanocapsules). The LC50 for neem oil was estimated in 0.20% or 1.31mgL(-1) of azadiractin. The nanoformulations aqueous NC40 and powdered NC40 affected the insect development.

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

Biochemical dissection of diageotropica and Never ripe tomato mutants to Cd-stressful conditions

In order to further address the modulation of signaling pathways of stress responses and their relation to hormones, we used the ethylene-insensitive Never ripe (Nr) and the auxin-insensitive diageotropica (dgt) tomato mutants. The two mutants and the control Micro-Tom (MT) cultivar were grown over a 40-day period in the presence of Cd (0.2 mM CdCl2 and 1 mM CdCl2). Lipid peroxidation, leaf chlorophyll, praline content, Cd content and antioxidant enzyme activities in roots, leaves and fruits were determined. The overall results indicated that the MT genotype had the most pronounced Cd damage effects while Nr and dgt genotypes might withstand or avoid stress imposed by Cd. This fact may be attributed, at least in part, to the fact that the known auxin-stimulated ethylene production is comprised in dgt plants. Conversely, the Nr genotype was more affected by the Cd imposed stress than dgt, which may be explained by the fact that Nr retains a partial sensitivity to ethylene. These results add further information that should help unraveling the relative importance of ethylene in regulating the cell responses to stressful conditions. (C) 2012 Elsevier Masson SAS. All rights reserved.

Relationships of Photosynthetic Photon Flux Density, air Temperature and Humidity with Tomato Leaf Diffusive Conductance and Temperature

The objective was to study the leaf temperature (LT) and leaf diffusive vapor conductance (gs) responses to temperature, humidity and incident flux density of photosynthetically active photons (PPFD) of tomato plants grown without water restriction in a plastic greenhouse in Santa Maria, RS, Brazil. The plants were grown in substrate and irrigated daily. The gs was measured using a steady-state null-balance porometer on the abaxial face of the leaves during the daytime. Both leaf surfaces were measured in one day. The PPFD and LT were measured using the porometer. Leaf temperature was determined using an infrared thermometer, and air temperature and humidity were measured using a thermohygrograph. The leaves on the upper layer of the plants had higher gs than the lower layer. The relationship between the gs and PPFD was different for the two layers in the plants. A consistent relationship between the gs and atmospheric water demand was observed only in the lower layer. The LT tended to be lower than the air temperature. The mean value for the gs was 2.88 times higher on the abaxial than adaxial leaf surface.

A dysflagellar mutant of Leishmania (Viannia) braziliensis isolated from a cutaneous leishmaniasis patient

Background: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features. Methods: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo. Results: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis. Conclusions: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FHR127H) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-gamma were able to synthesize FHR127H, the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-gamma secrete FH without retention in the ER. Moreover, the retention of FHR127H provoked enlargement of ER cisterns after treatment with IFN-gamma. A similar ER retention was observed in Cos-7 cells expressing the mutant FHR127H protein. Despite this deficiency in secretion, we show that the FHR127H mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein. The Journal of Immunology, 2012, 189: 3242-3248.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

Dried Tomato-Flavored Probiotic Cream Cheese with Lactobacillus paracasei

A dried tomato-flavored probiotic cream cheese (P) containing Lactobacillus paracasei Lpc-37 was developed for the purpose of this study. The same product, but without probiotic addition (C) was used as control. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were used as lactic starter cultures. Chemical composition analyses and sensory tests were performed on days 1 and 7, respectively. Titratable acidity, pH value and L. paracasei population were determined every 7 d during the refrigerated storage (21 d) of the cream cheeses. The experiment and analyses were performed in triplicate, using standard methods. Probiotic population remained greater than 10(7) CFU/g throughout the storage period, thereby characterizing the product as potentially probiotic. Cream cheeses C and P did not differ on the sensory tests, both obtaining good overall acceptance by the consumers, of which 82.6% stated that they certainly or probably would buy the product.

Diallel cross among fresh market tomato inbreeding lines

The general combining ability (GCA), specific combining ability (SCA), and heterosis were studied in a complete diallel cross among fresh market tomato breeding lines with reciprocal excluded. Fifteen genotypes (five parents and ten hybrids) were tested using a randomized complete block design, with three replications, and the experiments were conducted in Itatiba, São Paulo state, Brazil, in 2005/06. The yield components evaluated were fruit yield per plant (FP), fruit number per plant (FN), average fruit weight (FW); cluster number per plant (CN); fruit number per cluster (FC), fruit wall thickness (FT) and number of locules per fruit (NL). Fruit quality components evaluated were total soluble solids (SS); total titratable acidity (TA); SS/TA ratio, fruit length (FL); fruit width (WI); length to width ratio (FL/WI). The data for each trait was first subjected to analysis of variance. Griffing's method 2, model 1 was employed to estimate the general (GCA) and specific (SCA) combining abilities. Parental and hybrid data for each trait were used to estimate of mid-parent heterosis. For plant fruit yield, IAC-2 was the best parental line with the highest GCA followed by IAC-4 and IAC-1 lines. The hybrids IAC-1 x IAC-2, IAC-1 x IAC-4 and IAC-2 x IAC-4 showed the highest effects of SCA. High heterotic responses were found for fruit yield and plant fruit number with values up to 49.72% and 47.19%, respectively. The best hybrids for fruit yield and plant fruit number were IAC-1 x IAC-2, IAC-1 x IAC-4 and IAC-2 x IAC-5, for fruit yield and plant fruit number, the main yield components.

Relationships of photosynthetic photon flux density, air temperature and humidity with tomato leaf diffusive conductance and temperature

The objective was to study the leaf temperature (LT) and leaf diffusive vapor conductance (gs) responses to temperature, humidity and incident flux density of photosynthetically active photons (PPFD) of tomato plants grown without water restriction in a plastic greenhouse in Santa Maria, RS, Brazil. The plants were grown in substrate and irrigated daily. The gs was measured using a steady-state null-balance porometer on the abaxial face of the leaves during the daytime. Both leaf surfaces were measured in one day. The PPFD and LT were measured using the porometer. Leaf temperature was determined using an infrared thermometer, and air temperature and humidity were measured using a thermohygrograph. The leaves on the upper layer of the plants had higher gs than the lower layer. The relationship between the gs and PPFD was different for the two layers in the plants. A consistent relationship between the gs and atmospheric water demand was observed only in the lower layer. The LT tended to be lower than the air temperature. The mean value for the gs was 2.88 times higher on the abaxial than adaxial leaf surface.

Her2p molecular modeling, mutant analysis and intramitochondrial localization

Bacterial GatCAB amidotransferases are responsible for the transamidation of mischarged glutamyl-tRNA(Gln) into glutaminyl-tRNA(Gln). Mitochondria matrix also has a multienzymatic complex necessary for the transamidation of glutamyl-tRNA(Gln). Gtf1p, Her2p and Pet112p are the constituents of mitochondrial GatFAB amidotransferase complex. Her2p is subunit A of GatFAB complex, while Gtf1p is subunit F, a connector protein between Pet112p (subunit B) and Her2p. Here we evaluate through molecular modeling and amino acid correlation analysis the HER2 protein family. Localization studies indicated that Her2p is predominantly localized in the mitochondrial outer membrane, but it is also located in the mitochondrial matrix where together with Pet112p and Gtf1p constitutes the GatFAB complex. Finally, HER2 random mutagenesis unveiled important residues that provide thermo stability for the complex and are differently suppressed by overexpression of GTF1 or PET112. For instance, her2/ts11 mutant showed its fermentative growth impaired, and poorly rescued by GTF1 indicating that Her2p unknown function in the mitochondria outer membrane affects cell viability.