Characterization of fruit specific cDNAs from tomato

Summary Differential hybridization was used to screen a cDNA library made from ripe tomato fruit poly(A⁺)RNA. Clones were identified representing genes expressed predominantly at the unripe and/or ripe stage of the fruit development. Northern analysis was used for further characterization of the clones and in this report we describe four cDNA clones expressed at varying stages of fruit development. Three of these cDNAs were found to represent low-copy number genes and one was found to represent a gene family. Dot blot analysis revealed that the expression of these four genes was reduced between 2-fold and 100-fold in three ripening mutants of tomato.     

Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.

Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.     

Simultaneous co-suppression of polygalacturonase and pectinesterase in tomato fruit: inheritance and effect on isoform profiles

The simultaneous down regulation of two, or more, genes can be brought about by the transformation of a plant with a single chimeric transgene containing homologous sequences to both target genes. This has been achieved for the two cell wall hydrolases — polygalacturonase and pectinesterase — in tomato fruit. This paper reports the stable inheritance of this co-ordinated gene silencing over two generations. It has also been shown that only two of the three isoforms of pectinesterase in the tomato fruit are silenced by this chimeric construct thus providing some indication of the relative homologies between the gene sequences for these isoforms.     

Anthocyanin inheritance and instability in purple basil (Ocimum basilicum L.)

The instability of the purple pigments (anthocyanins) in purple basil varieties (Ocimum basilicum L.) limits their use as ornamental plants and as a potential anthocyanin source. Several self-pollinated generations of all purple plants were unsuccessful in stabilizing anthocyanin expression. In this study we investigated the inheritance and stability patterns of leaf traits using the Purple Ruffles variety. The results from the complete diallele crosses indicated anthocyanin expression in vegetative tissue is controlled by two dominant genes and ruffled leaf texture is controlled by a single recessive gene. Genes controlling leaf margin and leaf base structures were tightly linked to leaf texture. Essential oil production and oil constituents in leaves did not change as a result of the reversion in color. Color stability in cuttings was affected by the environment and the location where cuttings were taken. An accumulation of secondary metabolites (apigenin, genistein, and kaempferol) in green-reverted sectors on purple leaves was detected using reverse-phase high-performance liquid chromatography (HPLC) analysis; this suggested a potential block in the anthocyanin pathway. We hypothesize the reversion mutation is occurring in an anthocyanin regulatory gene.     

Anthocyanin accumulation in the hypocotyl of an ABA-over producing male-sterile tomato (Lycopersicon esculentum) mutant

Anthocyanin accumulation is known to be regulated by light and plant hormones but its occurrence varies with plant species and/or organ and tissue, and it has been negatively correlated with male sterility. In this study, we have examined the light responsive changes in anthocyanin in an abscisic acid (ABA) over-producer, male-sterile 7B-1 mutant and wild-type (WT) tomato hypocotyls. The results show that light-induced anthocyanin accumulation in the hypocotyl was more in WT compared with the 7B-1 mutant and more so under white light (W) compared with blue light (B) or red light (R). In contrast, the chlorophyll content was greater in the mutant than in WT. Exogenous ABA caused a transitory increase in anthocyanin content in WT but a reduction in 7B-1 , both in W and B. The high level of anthocyanin in WT under light conditions was not correlated with increased mRNA levels of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR), some of the anthocyanin biosynthetic genes. However, the activity of PAL (EC 4.3.1.5) was higher in the WT than in 7B-1 hypocotyls, and exogenous ABA caused an increase in PAL activity in the WT but a reduction in the mutant. The results presented show that high ABA negatively affects anthocyanin accumulation and that in the 7B-1 mutant it is related, in part, to reduced PAL activity. The results also support the view that the 7B-1 mutant has a defect in light perception and ABA sensitivity.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Three-dimensional structure of (1,4)-beta-D-mannan mannanohydrolase from tomato fruit

The three-dimensional crystal structure of tomato (Lycopersicon esculentum) beta-mannanase 4a (LeMAN4a) has been determined to 1.5 A resolution. The enzyme adopts the (beta/alpha)(8) fold common to the members of glycohydrolase family GH5. The structure is comparable with those of the homologous Trichoderma reesei and Thermomonospora fusca beta-mannanases: There is a conserved three-stranded beta-sheet located near the N terminus that stacks against the central beta-barrel at the end opposite the active site. Three noncanonical beta-helices surround the active site. Similar helices are found in T. reesei but not T. fusca beta-mannanase. By analogy with other beta-mannanases, the catalytic acid/base residue is E204 and the nucleophile residue is E318. The active site cleft of L. esculentum beta-mannanase most closely resembles that of the T. reesei isozyme. A model of substrate binding in LeMAN4a is proposed in which the mannosyl residue occupying the -1 subsite of the enzyme adopts the (1)S(5) skew-boat conformation.     

Weighted correlation network analysis (WGCNA) applied to the tomato fruit metabolome

Background

Advances in “omics” technologies have revolutionized the collection of biological data. A matching revolution in our understanding of biological systems, however, will only be realized when similar advances are made in informatic analysis of the resulting “big data.” Here, we compare the capabilities of three conventional and novel statistical approaches to summarize and decipher the tomato metabolome.

Methodology

Principal component analysis (PCA), batch learning self-organizing maps (BL-SOM) and weighted gene co-expression network analysis (WGCNA) were applied to a multivariate NMR dataset collected from developmentally staged tomato fruits belonging to several genotypes. While PCA and BL-SOM are appropriate and commonly used methods, WGCNA holds several advantages in the analysis of highly multivariate, complex data.

Conclusions

PCA separated the two major genetic backgrounds (AC and NC), but provided little further information. Both BL-SOM and WGCNA clustered metabolites by expression, but WGCNA additionally defined “modules” of co-expressed metabolites explicitly and provided additional network statistics that described the systems properties of the tomato metabolic network. Our first application of WGCNA to tomato metabolomics data identified three major modules of metabolites that were associated with ripening-related traits and genetic background.     

Characterization of abscisic Acid-induced ethylene production in citrus leaf and tomato fruit tissues

Abscisic acid (ABA) significantly stimulated ethylene production in citrus (Citrus sinensis [L.] Osbeck, cv Shamouti orange) leaf discs. The extent of stimulation was dependent upon the concentration of ABA (0.1-1 milimolar) and the duration of treatment (15-300 minutes). Aging the discs before applying ABA increased ABA-induced ethylene production due to enhancement of both ethylene-forming enzyme activity and the responsiveness of ABA. Discs excised from mature leaves were much more responsive to ABA than discs excised from young or senescing leaves. ABA stimulated ethylene production shortly after application, suggesting that ABA does not enhance ethylene production via the acceleration of senescence. The stimulating effect of ABA on ethylene production resulted mainly from the enhancement of 1-aminocylopropane-1-carboxylic acid synthesis. Stimulation of ethylene production by ABA in intact citrus leaves and tomato (Lycopersicon esculentum Mill., cv Castlemart) fruit was small but could be increased by various forms of wounding.     

Recovery of ethylene biosynthesis in diazocyclopentadiene (DACP)-treated tomato fruit

Diazocyclopentadiene (DACP), a competitive ethylene action inhibitor binds irreversibly to the ethylene receptor to reduce tissue responses to ethylene. Tomato fruit (Lycopersicon esculentum Mill cv lsquo;Rondellorsquo;) were treated with DACP at the mature green stage. Ethylene biosynthesis and respiration rate were depressed. Color changes from green to red were delayed. Compared to the control, ACC content increased and ACC oxidase activity in vivo decreased in DACP-treated fruit. Thus, decrease of ethylene production caused by DACP treatment was due to the reduction of ACC oxidase activity. The decline in ripening subsequently recovered after DACP treatment. Results from the Northern analysis for gene expression of ACC synthase and ACC oxidase, showed that expression of both genes declined in DACP-treated fruit, and then recovered. Therefore the recovery of ethylene production was due to the recovery in gene expression and activity of ACC oxidase. We conclude that the effects of DACP on ethylene biosynthesis are on expression of ACC synthase and ACC oxidase genes, and/or regulation of ACC oxidase activity.     

Xyloglucan endotransglucosylase/hydrolases (XTHs) during tomato fruit growth and ripening

Depolymerization of cell wall xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endotransglucosylase/hydrolases (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151) have been proposed to have a dual role integrating newly secreted xyloglucan chains into an existing wall-bound xyloglucan, or restructuring the existing cell wall material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SlXTHs were studied and grouped into three phylogenetic groups to determine which members of each family were expressed during fruit growth and fruit ripening, and the ways in which the expression of different SlXTHs contributed to the total XET and XEH activities. Our results showed that all of the SlXTHs studied were expressed during fruit growth and ripening, and that the expression of all the SlXTHs in Group 1 was clearly related to fruit growth, as were SlXTH12 in Group 2 and SlXTH6 in Group 3-B. Only the expression of SlXTH5 and SlXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SlXTHs were expressed at the red ripe stage. Both total XET and XEH activities were higher during fruit growth, and decreased during fruit ripening. Ethylene production during tomato fruit growth was low and experienced a significant increase during fruit ripening, which was not correlated either with SlXTH expression or with XET and XEH activities. We suggest that the role of XTH during fruit development could be related to the maintenance of the structural integrity of the cell wall, and the decrease in XTHs expression, and the subsequent decrease in activity during ripening may contribute to fruit softening, with this process being regulated through different XTH genes.     

Anthocyanin composition of the fruit of Coriaria myrtifolia L

The anthocyanin composition of the fruit of Coriaria myrtifolia L. and the changes which occur during ripening were studied using HPLC-PAD and LC-MS. Ten anthocyanins were detected and identified by their absorption and mass spectra as the 3-glucoside and 3-galactoside derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin. Fruit ripening was accompanied by substantial changes in the anthocyanin profile, with methoxylated anthocyanins, i.e. malvidin and peonidin, predominating in the final stages of ripening, and the trihydroxylated anthocyanin, delphinidin, during the earlier stages. Furthermore, galactoside derivatives were more abundant than glucosides in the ripe fruit. At full maturity, the fruits of C. myrtifolia were very rich in anthocyanins with a content of 10.7% (on a dry weight basis), a level which is higher than that found in most fruits usually considered to be anthocyanin-rich. The ability to grow C. myrtifolia in damaged and nitrogen poor soils, together with the possibility of using this plant for the extraction of anthocyanin, makes it ideal for consolidating soils and repopulating semi-desert or fire-damaged areas.     

Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin

Peroxidase (EC 1.11.1.7)-mediated stiffening of cell walls within the fruit skin of tomato is hypothesized to regulate fruit growth. However, to date, there is no experimental evidence demonstrating that peroxidase affects the mechanical properties of skin tissue. Here, the mechanical properties of skin strips excised from a range of fruits at different ages were determined using an 'Instron' universal material testing instrument. The stiffness of tomato fruit skin strips increases 3-fold with increasing fruit age. Application of partially-purified peroxidase from the cell walls of mature tomato fruit skin significantly increased the stiffness of fruit skin irrespective of the age of fruit. Furthermore, the application of hydrogen peroxide significantly increased the stiffness of skin strips excised from fruit of an age when endogenous peroxidase isozymes associated with the termination of growth are first detected. The results support the hypothesis that the tomato fruit skin plays an integral role in the regulation of tomato fruit growth, and that changes in its mechanical properties may be mediated by peroxidase. As far as is known, this is the first demonstration that peroxidases alter the mechanical properties of the plant cell wall.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Oviposition responses of Queensland fruit fly (Bactrocera tryoni) to mineral oil deposits on tomato fruit

Behavioural responses of wild and laboratory‐culture females of Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), to mineral oil deposits on tomato fruit dipped in aqueous oil emulsions were assessed in a no‐choice test and three choice tests. The oils were two commercial products used to manage plant pests and diseases, Ampol D‐C‐Tron NR and SK EnSpray 99, one distillation fraction of the base oil of the former, and four distillation fractions of the base oil of the latter. The initial and final boiling points of the fractions were equivalent to those of n‐alkanes with chain lengths of C20–22 (Ampol), and C21–C23, C22–23.5, C22–24, and C22–24.5 (SK). For both fly types in the no‐choice test, numbers of punctures and eggs per fruit declined strongly as concentrations of the nC20–22 Ampol fraction in emulsions rose from 0.25 to 2% (vol/vol). Fly type affected the extent of responses but there was no significant interaction for fly type*oil concentration. Responses of laboratory‐culture females in the choice tests also declined as concentrations of SK and the four fractions of its base oil in emulsions rose from 0 to 0.25%. The SK nC22–24 and nC22–24.5 fractions had least impact. Responses of laboratory‐culture flies to 0.5% emulsions of the nC20–22 Ampol fraction and the nC21–23 SK fraction in choice tests were not significantly different. Likewise, responses of laboratory‐culture flies to 0.5% emulsions of the two commercial products were not significantly different. Emulsifier type did not affect numbers of punctures or eggs per fruit in choice responses of laboratory‐culture flies to 0.5% emulsions of the Ampol nC20–22 fraction or 0.5% emulsions of the SK nC21–23 fraction. If the equivalence of no‐choice and choice responses in the laboratory were to hold in the field, then unsprayed ‘sacrificial’ plants would not be necessary and oil emulsions could be used as cover sprays.     

Characterization of a new xyloglucan endotransglucosylase/hydrolase (XTH) from ripening tomato fruit and implications for the diverse modes of enzymic action

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall-modifying enzymes that align within three or four distinct phylogenetic subgroups. One explanation for this grouping is association with different enzymic modes of action, as XTHs can have xyloglucan endotransglucosylase (XET) or endohydrolase (XEH) activities. While Group 1 and 2 XTHs predominantly exhibit XET activity, to date the activity of only one member of Group 3 has been reported: nasturtium TmXH1, which has a highly specialized function and hydrolyses seed-storage xyloglucan rather than modifying cell wall structure. Tomato fruit ripening was selected as a model to test the hypothesis that preferential XEH activity might be a defining characteristic of Group 3 XTHs, which would be expressed during processes where net xyloglucan depolymerization occurs. Database searches identified 25 tomato XTHs, and one gene (SlXTH5) was of particular interest as it aligned within Group 3 and was expressed abundantly during ripening. Recombinant SlXTH5 protein acted primarily as a transglucosylase in vitro and depolymerized xyloglucan more rapidly in the presence than in the absence of xyloglucan oligosaccharides (XGOs), indicative of XET activity. Thus, there is no correlation between the XTH phylogenetic grouping and the preferential enzymic activities (XET or XEH) of the proteins in those groups. Similar analyses of SlXTH2, a Group 2 tomato XTH, and nasturtium seed TmXTH1 revealed a spectrum of modes of action, suggesting that all XTHs have the capacity to function in both modes. The biomechanical properties of plant walls were unaffected by incubation with SlXTH5, with or without XGOs, suggesting that XTHs do not represent primary cell wall-loosening agents. The possible roles of SlXTH5 in vivo are discussed.