Two Pine Endo-β-1,4-Glucanases Are Associated with Rapidly Growing Reproductive Structures

Two cDNA clones encoding endo-β-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (Pinus radiata cellulase 1) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit1

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

Tomato Phosphate Transporter Genes Are Differentially Regulated in Plant Tissues by Phosphorus1

Phosphorus is a major nutrient acquired by roots via high-affinity inorganic phosphate (Pi) transporters. In this paper, we describe the tissue-specific regulation of tomato (Lycopersicon esculentum L.) Pi-transporter genes by Pi. The encoded peptides of the LePT1 and LePT2 genes belong to a family of 12 membrane-spanning domain proteins and show a high degree of sequence identity to known high-affinity Pi transporters. Both genes are highly expressed in roots, although there is some expression of LePT1 in leaves. Their expression is markedly induced by Pi starvation but not by starvation of nitrogen, potassium, or iron. The transcripts are primarily localized in root epidermis under Pi starvation. Accumulation of LePT1 message was also observed in palisade parenchyma cells of Pi-starved leaves. Our data suggest that the epidermally localized Pi transporters may play a significant role in acquiring the nutrient under natural conditions. Divided root-system studies support the hypothesis that signal(s) for the Pi-starvation response may arise internally because of the changes in cellular concentration of phosphorus.

Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: Similarity of functional domains to subtilisin-like endoproteases.

A 69-kDa proteinase (P69), a member of the pathogenesis-related proteins, is induced and accumulates in tomato (Lycopersicon esculentum) plants as a consequence of pathogen attack. We have used the polymerase chain reaction to identify and clone a cDNA from tomato plants that represent the pathogenesis-related P69 proteinase. The nucleotide sequence analysis revealed that P69 is synthesized in a preproenzyme form, a 745-amino acid polypeptide with a 22-amino acid signal peptide, a 92-amino acid propolypeptide, and a 631-amino acid mature polypeptide. Within the mature region the most salient feature was the presence of domains homologous to the subtilisin serine protease family. The amino acid sequences surrounding Asp-146, His-203, and Ser-532 of P69 are closely related to the catalytic sites (catalytic triad) of the subtilisin-like proteases. Northern blot analysis revealed that the 2.4-kb P69 mRNA accumulates abundantly in leaves and stem tissues from viroid-infected plants, whereas the mRNA levels in tissues from healthy plants were undetectable. Our results indicate that P69, a secreted calcium-activated endopeptidase, is a plant pathogenesis-related subtilisin-like proteinase that may collaborate with other defensive proteins in a general mechanism of active defense against attacking pathogens.

Identification and characterization of putative transposable DNA elements in solanaceous plants and Caenorhabditis elegans.

Several families of putative transposable elements (TrEs) in both solanaceous plants and Caenorhabditis elegans have been identified by screening the DNA data base for inverted repeated domains present in multiple copies in the genome. The elements are localized within intron and flanking regions of many genes. These elements consist of two inverted repeats flanking sequences ranging from 5 bp to > 500 bp. Identification of multiple elements in which sequence conservation includes both the flanking and internal regions implies that these TrEs are capable of duplicative transposition. Two of the elements were identified in promoter regions of the tomato (Lycoperiscon esculentum) polygalacturonase and potato (Solanum tuberosum) Win1 genes. The element in the polygalacturonase promoter spans a known regulatory region. In both cases, ancestral DNA sequences, which represent potential recombination target sequences prior to insertion of the elements, have been cloned from related species. The sequences of the inverted repeated domains in plants and C. elegans show a high degree of phylogenetic conservation. While frequency of the different elements is variable, some are present in very high copy number. A member of a single C. elegans TrE family is observed approximately once every 20 kb in the genome. The abundance of the described TrEs suggests utility in the genomic analysis of these and related organisms.

Effect of commercial amino acids on iron nutrition of tomato plants grown under lime-induced iron deficiency

The objective of this work was to study the effect of root and foliar application of two commercial products containing amino acids from plant and animal origin on iron (Fe) nutrition of tomato seedlings cultivated in two nutrient media: lime and normal nutrient solutions. In the foliar-application experiment, each product was sprayed with 0.5 and 0.7 mL L–1 2, 7, 12, and 17 d after transplanting. In the root application experiment, 0.1 and 0.2 mL L–1 of amino acids products were added to the nutrient solutions. In both experiments, untreated control plants were included as well. Foliar and root application of the product containing amino acids from animal origin caused severe plant-growth depression and nonpositive effects on Fe nutrition were found. In contrast, the application of the product from plant origin stimulated plant growth. Furthermore, significantly enhanced root and leaf FeIII-chelate reductase activity, chlorophyll concentration, leaf Fe concentration, and FeII : Fe ratio were found in tomato seedlings treated with the product from plant origin, especially when the amino acids were directly applied to the roots. These effects were more evident in plants developed under lime-induced Fe deficiency. The positive results on Fe uptake may be related to the action of glutamic acid, the most abundant amino acid in the formulation of the product from plant origin.

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.

Fine mapping of the tomato I-3 gene for fusarium wilt resistance and elimination of a co-segregating resistance gene analogue as a candidate for I-3

The I-3 gene from the wild tomato species Lycopersicon pennellii confers resistance to race 3 of the devastating vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici. As an initial step in a positional cloning strategy for the isolation of I-3, we converted restriction fragment length polymorphism and conserved orthologue set markers, known genes and a resistance gene analogue (RGA) mapping to the I-3 region into PCR-based sequence characterised amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers. Additional PCR-based markers in the I-3 region were generated using the randomly amplified DNA fingerprinting (RAF) technique. SCAR, CAPS and RAF markers were used for high-resolution mapping around the I-3 locus. The I-3 gene was localised to a 0.3-cM region containing a RAF marker, eO6, and an RGA, RGA332. RGA332 was cloned and found to correspond to a putative pseudogene with at least two loss-of-function mutations. The predicted pseudogene belongs to the Toll interleukin-1 receptor-nucleotide-binding site-leucine-rich-repeat sub-class of plant disease resistance genes. Despite the presence of two RGA332 homologues in L. esculentum, DNA gel blot and PCR analysis suggests that no other homologues are present in lines carrying I-3 that could be alternative candidates for the gene.

Assessment of P availability in heavily fertilized soils using the diffusive gradient in thin films (DGT) technique

Phosphorus-availability tests typically provide an indication of quantity of P available (Colwell bicarbonate-extractable P), or of the intensity of supply (0.01 M CaCl2-extractable P). The soil's capacity to buffer P is more difficult to assess, and is generally estimated using a P-adsorption curve. The diffusive gradient in thin films (DGT) approach may provide a simpler means of assessing a soil's ability to maintain soil solution P. Optimal extraction conditions were found to be 24 h exposure of DGT samplers to saturated soil. The DGT approach was evaluated on a range of 24 soils, some of which had high Colwell- (>100 mu g g(-1)) and Bray 1- (>30 mu g g(-1)) extractable P content, but showed a tomato (Lycopersicon esculentum Mill.) yield response to the addition of P fertilizer. The DGT approach provided an excellent separation of soils on which tomato showed a yield response, from those where fertilizer P did not increase dry-matter yield. Phosphorus accumulation was strongly correlated with soil solution P concentration and anion exchange resin-extractable P, but showed poor correlation with Colwell- or Bray 1-extractable P. The DGT P accumulation rate of 3.62 x 10(-7) to 4.79 x 10(-5) mol s(-1) m(-3) for the soils tested was comparable to the uptake rate of roots of tomato plants that were adequately supplied with P (2.25 x 10(-5) mol s(-1) m(-3)).

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Crocin and β-Carotene bleaching assays as analytical tools in the optimization of the extraction of hydrophilic and lipophilic antioxidants from tomato

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop worldwide and a rich source of hydrophilic (H) and lipophilic (L) antioxidants. The H fraction is constituted mainly by ascorbic acid and soluble phenolic compounds, while the L fraction contains carotenoids (mostly lycopene), tocopherols, sterols and lipophilic phenolics [1,2]. To obtain these antioxidants it is necessary to follow appropriate extraction methods and processing conditions. In this regard, this study aimed at determining the optimal extraction conditions for H and L antioxidants from a tomato surplus. A 5-level full factorial design with 4 factors (extraction time (I, 0-20 min), temperature (T, 60-180 •c), ethanol percentage (Et, 0-100%) and solid/liquid ratio (S/L, 5-45 g!L)) was implemented and the response surface methodology used for analysis. Extractions were carried out in a Biotage Initiator Microwave apparatus. The concentration-time response methods of crocin and P-carotene bleaching were applied (using 96-well microplates), since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively [3]. Measurements were carried out at intervals of 3, 5 and 10 min (initiation, propagation and asymptotic phases), during a time frame of 200 min. The parameters Pm (maximum protected substrate) and V m (amount of protected substrate per g of extract) and the so called IC50 were used to quantify the response. The optimum extraction conditions were as follows: r~2.25 min, 7'=149.2 •c, Et=99.1 %and SIL=l5.0 giL for H antioxidants; and t=l5.4 min, 7'=60.0 •c, Et=33.0% and S/L~l5.0 g/L for L antioxidants. The proposed model was validated based on the high values of the adjusted coefficient of determination (R2.wi>0.91) and on the non-siguificant differences between predicted and experimental values. It was also found that the antioxidant capacity of the H fraction was much higher than the L one.

Optimization of microwave-assisted extration of phenolic compounds from tomao by full factorial design coupled with response surface methodology

Tomato (Lycopersicon esculentum Mill.), apart from being a functional food rich in carotenoids, vitamins and minerals, is also an important source of phenolic compounds [1 ,2]. As antioxidants, these functional molecules play an important role in the prevention of human pathologies and have many applications in nutraceutical, pharmaceutical and cosmeceutical industries. Therefore, the recovery of added-value phenolic compounds from natural sources, such as tomato surplus or industrial by-products, is highly desirable. Herein, the microwave-assisted extraction of the main phenolic acids and flavonoids from tomato was optimized. A S-Ieve! full factorial Box-Behnken design was implemented and response surface methodology used for analysis. The extraction time (0-20 min), temperature (60-180 "C), ethanol percentage (0-100%), solidlliquid ratio (5-45 g/L) and microwave power (0-400 W) were studied as independent variables. The phenolic profile of the studied tomato variety was initially characterized by HPLC-DAD-ESIIMS [2]. Then, the effect of the different extraction conditions, as defined by the used experimental design, on the target compounds was monitored by HPLC-DAD, using their UV spectra and retention time for identification and a series of calibrations based on external standards for quantification. The proposed model was successfully implemented and statistically validated. The microwave power had no effect on the extraction process. Comparing with the optimal extraction conditions for flavonoids, which demanded a short processing time (2 min), a low temperature (60 "C) and solidlliquid ratio (5 g/L), and pure ethanol, phenolic acids required a longer processing time ( 4.38 min), a higher temperature (145.6 •c) and solidlliquid ratio (45 g/L), and water as extraction solvent. Additionally, the studied tomato variety was highlighted as a source of added-value phenolic acids and flavonoids.