Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes.

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and 'Dulce' (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.

Rapid method for total carotenoid detection in canary yellow-fleshed watermelon.

Yellow-fleshed watermelons (Citrullus lanatus[Thunb.] Matsum. and Nakai) contain many different carotenoids, all in low to trace amounts. Since there is not 1 predominant carotenoid in yellow-fleshed watermelon, testing the total carotenoid content among watermelon lines is important in determining the antioxidant potential and thus potential health benefits of different varieties. Unfortunately, current methods to assay total carotenoid content are time consuming and require organic solvents. This report describes a rapid and reliable light absorption method to assay total carotenoid content for yellow-fleshed watermelon that does not require organic solvents. Light absorption of 78 watermelon flesh purees was measured with a diode array xenon flash spectrophotometer that can measure actual light absorption from opaque samples; results were compared with a hexane extraction method. The puree absorbance method gave a linear relationship (R(2)= 0.88) to total carotenoid content and was independent of watermelon variety within the total carotenoid concentration range measured (0 to 7 mug/g fresh weight).

Genes expressed during the development and ripening of watermelon fruit.

A normalized cDNA library was constructed using watermelon flesh mRNA from three distinct developmental time-points and was subtracted by hybridization with leaf cDNA. Random cDNA clones of the watermelon flesh subtraction library were sequenced from the 5' end in order to identify potentially informative genes associated with fruit setting, development, and ripening. One-thousand and forty-six 5'-end sequences (expressed sequence tags; ESTs) were assembled into 832 non-redundant sequences, designated as "EST-unigenes". Of these 832 "EST-unigenes", 254 ( approximately 30%) have no significant homology to sequences published so far for other plant species. Additionally, 168 "EST-unigenes" ( approximately 20%) correspond to genes with unknown function, whereas 410 "EST-unigenes" ( approximately 50%) correspond to genes with known function in other plant species. These "EST-unigenes" are mainly associated with metabolism, membrane transport, cytoskeleton synthesis and structure, cell wall formation and cell division, signal transduction, nucleic acid binding and transcription factors, defense and stress response, and secondary metabolism. This study provides the scientific community with novel genetic information for watermelon as well as an expanded pool of genes associated with fruit development in watermelon. These genes will be useful targets in future genetic and functional genomic studies of watermelon and its development.

The development of the refractive state in the newborn Thomson gazelle.

Changes in refractive error during the first 3 months of life were studied retinoscopically in six Thomson gazelles ( Gazella thomsoni). Animals were hand-raised to allow repeat testing over time without chemical restraint. Refraction results were correlated with ultrasound measurements of intraocular dimensions, and with values in adult gazelles. Gazelles are born hyperopic with a mean refractive error of 3.44+/-0.31 D. By day 50, the animals are virtually emmetropic (0.13+/-0.21 D) and remain so in adulthood (0.03+/-0.09 D). The refractive error is highly correlated with the axial length ( r(2)=0.96) and with the vitreous chamber depth ( r(2)=0.83), but not with anterior chamber depth. Significant with-the-rule astigmatism was recorded ( P<0.001).

Enhanced levels of the aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits.

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.

Acetyl-coa: alcohol acetyltransferase activity and aroma formation in ripening melon fruits.

Melon varieties (Cucumis melo L.) differ in a range of physical and chemical attributes. Sweetness and aroma are two of the most important factors in fruit quality and consumer preference. Volatile acetates are major components of the headspace of ripening cv. Arava fruits, a commercially important climacteric melon. In contrast, volatile aldehydes and alcohols are most abundant in cv. Rochet fruits, a nonclimacteric melon. The formation of volatile acetates is catalyzed by alcohol acetyltransferases (AAT), which utilize acetyl-CoA to acetylate several alcohols. Cell-free extract derived from Arava ripe melons exhibited substantial levels of AAT activity with a variety of alcohol substrates, whereas similar extracts derived from Rochet ripe melons had negligible activity. The levels of AAT activity in unripe Arava melons were also low but steadily increased during ripening. In contrast, similar extracts from Rochet fruits displayed low AAT activity during all stages of maturation. In addition, the benzyl- and 2-phenylethyl-dependent AAT activity levels seem well correlated with the total soluble solid content in Arava fruits.

Calculating the contrasts that retinal ganglion cells and LGN neurones encounter in natural scenes.

Visual responses are known to depend on stimulus contrast and not simply on the absolute levels of retinal illumination. Here, we have determined the contrasts that mammalian retinal ganglion cells and lateral geniculate neurones (LGN) are likely to encounter in real world scenes. Local contrasts were calculated in 135 calibrated images of a variety of real world scenes using contrast operators that closely mirror the characteristic receptive-field organisation of mammalian retinal ganglion cells and LGN neurones. We have found that the frequency distribution of the calculated local contrasts has a pronounced peak at zero contrast and that it tails off roughly exponentially with increasing positive and negative contrasts; about 90% of the contrasts in the images were within the equivalent range of +/-0.5 Michelson and Weber contrasts. Further analysis suggests that the characteristic forms of the contrast-response functions of mammalian retinal and LGN neurones are matched to the range of contrasts that they experience when viewing real world images.

The potential of Lagenaria rootstock to confer resistance to the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae) in Cucurbitaceae.

Antibiosis and resistance of six Cucurbita and two Lagenaria accessions to the carmine spider mite, Tetranychus cinnabarinus Boisduval, were evaluated in the laboratory. Significant differences among accessions were observed three days after the inoculation of detached leaf discs. The Lagenaria accessions, Slawi and Sus, proved to be the most resistant to mites, with average populations of mite eggs, 87 and 95%, respectively less than that of the susceptible C. pepo accession, Orangetti. The Cucurbita accessions, Tace, Brava, Tetsukabuto, Phoenix and TZ-148 had mite egg totals 4, 9, 13, 26 and 40%, respectively, less than those of accession Orangetti. The Sus accession of Lagenaria was resistant to T. cinnabarinus from the four-leaf stage until fruit set in laboratory and field tests. Grafting the susceptible Brava onto Sus rootstock increased the resistance of the scion to the same level as that of non-grafted Sus. Grafting the susceptible Cucumis melo Noy Yizre'el on resistant or susceptible rootstocks of Cucurbita and Lagenaria accessions did not affect its susceptibility to T. cinnabarinus. The results indicate that resistance to T. cinnabarinus can be transferred by grafting from Lagenaria stocks to Cucurbita scions but not in the opposite direction.

Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

Discrimination of spectrally blended natural images: optimisation of the human visual system for encoding natural images.

We have developed a protocol for testing experimentally the hypothesis that the human visual system is optimised for making visual discriminations amongst natural scenes. Visual stimuli were made by gradual blending of the Fourier spectra of digitised photographs of natural scenes. The statistics of the stimuli were made unnatural to varying degrees by changing the overall slopes of the amplitude spectra of the stimuli. Thresholds were measured for discriminating small amounts of spectral blending at different spectral slopes. We found that thresholds were lowest when the spectral slope was natural; thresholds were increased when the slopes were either shallower or steeper than natural. A number of spurious cues were considered, such as differences in mean luminance or overall spectral power or contrast between test and reference stimuli. Control experiments were performed to remove such spurious cues, and the discrimination thresholds were still lowest for stimuli that were most natural. Thus, these experiments do provide experimental support for the idea that human vision and the human visual system are optimised for processing natural visual information [corrected].

Discrimination of changes in the slopes of the amplitude spectra of natural images: band-limited contrast and psychometric functions.

Thresholds were measured for discriminating changes in the slopes of the amplitude spectra of stimuli derived from photographs of natural scenes and from random-luminance patterns. The variety and magnitudes of the thresholds could be explained by a model based on the discrimination of the changes in band-limited local contrast. Different spatial scales of local contrast (or different spatial-frequency bands of about 1 octave) were implicated for different reference spectral slopes; the model implicated a lower frequency-band for stimuli with shallower amplitude spectra. The implications of the model were tested experimentally by using stimuli in which the spectra were changed within restricted spatial-frequency bands. When the amplitude spectra of the test and reference stimuli differed only within the implicated frequency bands, thresholds were affected little. However, when the test and reference spectra differed at all frequencies except those in the implicated bands, thresholds were elevated markedly. The forms of the psychometric functions for the discrimination task were entirely compatible with the hypothesis that the task relies upon the ability to discriminate changes of contrast. The Weibull functions fitted to the data had slope parameters (beta) in the range 1 to 3, compatible with discrimination of low (but suprathreshold) contrasts.

Band-limited contrast in natural images explains the detectability of changes in the amplitude spectra.

The psychophysical task of discriminating changes in the slopes of the amplitude spectra of complex images has been used in the past to test whether the human visual system might be optimised for coding the spatial structure in natural images (e.g. Knill et al., 1990; Tadmor & Tolhurst, 1994). We have reported that the dependency of these discrimination thresholds on the reference slope has the same overall general form, regardless of the particular digitised photographs that are used for generating the stimuli. The actual discrimination thresholds, however, differ markedly in magnitude for stimuli that are derived from different digitised photographs. Here, we describe a model that aims at explaining this diversity of threshold magnitudes: we suppose that the observer is detecting small changes in image contrast estimated within limited spatial-frequency bands of about 1 octave bandwidth. This local-contrast analysis reveals that contrast changes in only one frequency band are of comparable magnitudes to the changes that observers need for detecting differences in the Michelson contrast of simple sinusoidal gratings. The success of this band-limited contrast model is further shown in experiments where the slopes of the amplitude spectra of stimuli were changed only within restricted frequency bands. We show that when the slope is changed outside the limited frequency band implicated by the contrast model, the observer's thresholds are greatly elevated. Thresholds remain unchanged when slope changes are made within the implicated band. We also find that the exact bandwidth of the contrast operator is not critical, provided that it is in the range of about 0.6-1.5, which is the characteristic bandwidth range of V1 neurons.

Quantitative trait loci influencing chemical and sensory characteristics of eating quality in sweet corn.

This study was conducted to ascertain the chromosomal location and magnitude of effect of quantitative trait loci (QTL) associated with the chemical and sensory properties of sweet corn (Zea mays L.) eating quality. Eighty-eight RFLPs, 3 cloned genes (sh1, sh2, and dhn1), and 2 morphological markers (a2 and se1) distributed throughout the sweet corn genome were scored in 214 F2:3 families derived from a cross between the inbreds W6786su1Se1 and IL731Asu1se1. Kernel properties associated with eating quality (kernel tenderness and starch, phytoglycogen, sucrose, and dimethyl sulfide concentrations) were quantified on F2:3 sib-pollinated ears harvested at 20 days after pollination. Sensory evaluation was conducted on a subset of 103 F2:3 families to determine intensity of attributes associated with sweet corn eating quality (corn aroma, grassy aroma, sweetness, starchiness, grassy flavor, crispness, tenderness, and juiciness) and overall liking. Single factor analysis of variance revealed significant QTL for all these traits, which accounted for from 3 to 42% of the total phenotypic variation. A proportion of the RFLP markers associated with human sensory response were also found to be associated with kernel characteristics. To our knowledge this is the first report of the identification of QTL associated with human flavor preferences in any food crop. Key words : sweet corn, RFLP, quantitative trait loci, eating quality, sensory evaluation.

RFLP mapping of the sugary enhancer1 gene in maize.

RFLP marker data from an F2∶3 population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F2∶3 families, was derived from each of the 214 F2 plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F2∶3 family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.

Beta subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli.

Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies. Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.

Discrimination of changes in the second-order statistics of natural and synthetic images.

It has been suggested that the second-order statistics of different natural images are all remarkably similar and that neurones and channels in the visual system may exploit this similarity. We have measured the ability of human observers to discriminate changes in these statistics using different natural and synthetic stimulus images and have found that the dependence of their discrimination thresholds upon the reference second-order statistics is similar in form, for both kinds of stimuli. However, there is some variety in the magnitudes of the thresholds for the natural stimulus images; in fact, the second-order statistics of different natural images are more diverse than previously suggested. The discrimination task can be modelled as the discrimination of changes in local contrast within restricted spatial frequency bands and is similar to the discrimination of blur.

Both the phase and the amplitude spectrum may determine the appearance of natural images.

Several studies have demonstrated that the appearance of a natural image is determined mostly by its global phase spectrum; the amplitude spectrum is regarded as providing little specific information. In this paper, we show several cases where the global amplitude spectrum is also essential for specifying the particular content of natural images. Thus, both the amplitude and the phase spectrum may be important for reliable specification of such images. The results suggest that, although the average amplitude spectra of different natural images may be similar in their overall form, a realistic description of the amplitude spectra must also incorporate the particular way in which the energy is distributed across different orientations.

Overproduction of the beta subunit of DNA polymerase III holoenzyme reduces UV mutagenesis in Escherichia coli.

Overproduction of the beta subunit of DNA polymerase III holoenzyme caused a 5- to 10-fold reduction of UV mutagenesis along with a slight increase in sensitivity to UV light in Escherichia coli. The same effects were observed in excision-deficient cells, excluding the possibility that they were mediated via changes in excision repair. In contrast, overproduction of the alpha subunit of the polymerase did not influence either UV mutagenesis or UV sensitivity. The presence of the mutagenesis proteins MucA and MucB expressed from a plasmid alleviated the effect of overproduced beta on UV mutagenesis. We have previously suggested that DNA polymerase III holoenzyme can exist in two forms: beta-rich form unable to bypass UV lesions and a beta-poor form capable of bypassing UV lesions (O. Shavitt and Z. Livneh, J. Biol. Chem. 264:11275-11281, 1989). The beta-poor form may be related to an SOS form of DNA polymerase III designed to perform translesion polymerization under SOS conditions and thereby generate mutations. On the basis of this model, we propose that the overproduced beta subunit affects the relative abundance of the regular replicative beta-rich polymerase and the SOS bypass-proficient polymerase by sequestering the polymerase molecules to the beta-rich form and blocking the SOS form.

Amplitude spectra of natural images.

Several studies have suggested that the amplitude spectra of photographs of natural scenes are remarkably similar and have the form: amplitude varies; is directly proportional to spatial frequency-1.0. This is, of course, a straight line with slope of -1.0 when plotted on double logarithmic coordinates. We have examined the amplitude spectra of 135 digitized photographs of natural scenes and have found that relatively few images conform exactly to the suggestion. About 25% of the images in our sample have spectra which show significant curvature when plotted on log-log coordinates. The best-fitting regression lines have slopes that range from -0.8 to -1.5; the average slope is -1.2, rather steeper than previously suggested.