2005 Publications

Despite the hundreds of existing stone fruit (Prunus spp.) cultivars used for fresh market, there is a continuing need to develop new stone fruit cultivars as the requirements of the industry change. Over the last 20 years, there has been a shift toward private breeding as the public sector decreases its support of these long-range programs. As a result, there are fewer public breeding programs and many of those still operating protect their releases and partially fund their programs with royalty payments. Other trends that are shaping the development of new stone fruit cultivars are a need for small or more easily managed tree architecture, a trend toward the use of fewer agricultural chemicals, the expansion of production zones into the milder winter zones to allow year-round availability of stone fruit, a general diversifiction of fruit tyes being marketed, the increased awareness of the health benefits of fruit consumption, the need for better and more consistent quality, and given the global marketing of these fruit the increased need for enhanced postharvest qualities. The breeding programs of the world are responding to these trends and working toward developing the cultivars for the world markets of the future.

Cation levels within the cytosol are coordinated by a network of transporters. Here, we examine the functional roles of calcium exchanger 1 (CAX1), a vacuolarH1/Ca21 transporter, and the closely related transporter CAX3.We demonstrate that like CAX1, CAX3 is also localized to the tonoplast. We show that CAX1 is predominately expressed in leaves, while CAX3 is highly expressed in roots. Previously, using a yeast assay, we demonstrated that an N-terminal truncation of CAX1 functions as an H1/Ca21 transporter. Here, we use the same yeast assay to show that full-length CAX1 and full-length CAX3 can partially, but not fully, suppress the Ca21 hypersensitive yeast phenotype and coexpression of full-length CAX1 and CAX3 conferred phenotypes not produced when either transporter was expressed individually. In planta, CAX3 null alleles were modestly sensitive to exogenous Ca21 and also displayed a 22% reduction in vacuolar H1-ATPase activity. cax1/cax3 double mutants displayed a severe reduction in growth, including leaf tip and flower necrosis and pronounced sensitivity to exogenous Ca21 and other ions. These growth defects were partially suppressed by addition of exogenous Mg21. The double mutant displayed a 42% decrease in vacuolar H1/Ca21 transport, and a 47% decrease inH1-ATPase activity. While the ionome of cax1 and cax3 lines were modestly perturbed, the cax1/cax3 lines displayed increased PO32 4 , Mn21, and Zn21 and decreased Ca21 and Mg21 in shoot tissue. These findings suggest synergistic function of CAX1 and CAX3 in plant growth and nutrient acquisition.

Tomatoes are a principal dietary source of carotenoids and flavonoids, both of which are highly beneficial for human health1, 2. Overexpression of genes encoding biosynthetic enzymes or transcription factors have resulted in tomatoes with improved carotenoid or flavonoid content, but never with both3, 4, 5, 6, 7. We attempted to increase tomato fruit nutritional value by suppressing an endogenous photomorphogenesis regulatory gene, DET1, using fruit-specific promoters combined with RNA interference (RNAi) technology. Molecular analysis indicated that DET1 transcripts were indeed specifically degraded in transgenic fruits. Both carotenoid and flavonoid contents were increased significantly, whereas other parameters of fruit quality were largely unchanged. These results demonstrate that manipulation of a plant regulatory gene can simultaneously influence the production of several phytonutrients generated from independent biosynthetic pathways, and provide a novel example of the use of organ-specific gene silencing to improve the nutritional value of plant-derived products.

The phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II, consisting of (1YSPTSPS7)n heptad repeats, encodes information about the state of the transcriptional apparatus that can be conveyed to factors that regulate mRNA synthesis and processing. Here we describe how the CTD code is read by two classes of protein phosphatases, plant CPLs and yeast Ssu72, that specifically dephosphorylate Ser5 in vitro. The CPLs and Ssu72 recognize entirely different positional cues in the CTD primary structure. Whereas the CPLs rely on Tyr1 and Pro3 located on the upstream side of the Ser5-PO4 target site, Ssu72 recognizes Thr4 and Pro6 flanking the target Ser5-PO4 plus the downstream Tyr1 residue of the adjacent heptad. We surmise that the reading of the CTD code does not obey uniform rules with respect to the location and phasing of specificity determinants. Thus, CTD code, like the CTD structure, is plastic.

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four ArabidopsisDicer-like proteins (DCL1-4), ArabidopsisHYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein–protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.

The gene of the Arabidopsis thaliana H(+)/Ca(2+) transporter, CAX1 (cation exchanger 1) was introduced into Japonica cultivars of rice (Ilpumbyeo) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice callus. The introduction of the CAX1 gene was also proven by PCR using CAX1-specific oligonucleotide primers in regenerated plants. Stable integration and expression of the CAX1 gene in T(0) plants and T(1) progeny were confirmed by DNA hybridization, Northern blot analysis, and luminescent analysis.

Bulb color in onions (Allium cepa) is an important trait, but its complex, unclear mechanism of inheritance has been a limiting factor in onion cultivar improvement. The identity of the L locus, which is involved in the color difference between Brazilian yellow and red onions, is revealed in this study. A cross was made between a US-type yellow breeding line and Brazilian yellow cultivar. The segration ratio of nine red to seven yellow onions in the F2 population supports the involvement of two complementary genes in anthocyanin production in the F1 hybrids. The high-performance liquid chromatography (HPLC) and reverse-transcriptase (RT)-PCR analysis of the Brazilian yellow onions indicated that the genes are involved late in the anthocyanin synthesis pathway. The genomic sequence of the anthocyanidin pathway. The genomic sequence of the anthocyandin synthase (ANS) gene in Brazilian yellow onions showed a point mutation, which results in an amino acid change of a glycine to an arginine at residue 229. Because this residue is located adjacent to a highly conserved iron-binding active site, this muttion is likely responsible for the inactivation of the ANS gene in Brazilian yellow onions. Following the isolation of the promoter sequence of the mutant allele, a PCR-based marker for allelic selection of the ANS gene was designed. This assay is based on an insertion (larger than 3 kb) mutation. The marker perfectly co-segregated with the color phenotypes in the F2 populations, thereby indicating that the L locus encodes ANS.

Bulb color in onions (Allium cepa) is an important trait and is inherited in a complex manner. However, the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that pink bulb color in onions is inherited as a significantly reduced transcription of the anthocyanidin synthase (ANS) gene. In this study, we developed a PCR-based marker for an allelic selection of the ANS gene to avoid the laborious progency tests traditionally employed. To identify polymorphisms between pink and red alleles of the ANS gene, promoter sequences of both alleles were isolated. There was 97% nucleotide sequence identify between the promoter sequences of the two alleles. A 390-bp insertion was identified 632 bp upstream from the putative transcription start site in the pink allele. A pair of primers was designed on the flanking sequences of the inserted region and utilized as a PCR-based marker for allelic selection of the ANS gene. The reliability of the marker was tested using parents, F1 hybrids, and F3 lines whose genotypes had been identified by progeny tests. The marker was also used to evaluate the distribution of the pink allele in white and yellow breeding lines. The results indicated that a majority of the breeding lines tested were homozygous recessive.

In onions (Allium cepa), a variety of bulb colors exist ranging from white, yellow, to red, with different intermediate shades. In order to identify the function of the basic color factor, the C locus, which is required for any color production, a candidate gene approach was attempted utilizing anthocyanin synthesis path genes. RT-PCR was carried out to examine differential expression of the genes involved in the anthocyanin synthesis pathway among four different bulb colors: recessive white, dominant white, yellow, and red. The transcription of two homologous chalcone synthase (CHS) genes (CHS-A and CHS-B) was significantly reduced in both dominant and recessive white onions. The reduced transcription of CHS genes was also observed in white, but not yellow, F2 plants originating from the cross between white and yellow onions. Single nucleotide polymorphism (SNP) markers tagging parental alleles of CHS genes were utilized to determine whether the reduced transcription of CHS genes was caused by mutation in CHS genes or other regulatory genes. In the F2 populations originating from the cross between recessive white and yellow or red parents, the SNP markers tagging two parental alleles of the CHS-A and CHS-B genes did not co-segregate with the genotype of the C locus. These results suggest that the basic color factor is likely to be a regulatory gene controlling CHS gene transcription.

Bulb color in onions (Allium cepa) is an important trait, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that inactivation of the dihydroflavonol 4 reductase (DFR) gene at the transcriptional level resulted in a lack of anthocyanin production in yellow onions. The objectives of the present study were the identification of the critical mutations in the DR gene (DFR-a) and the development of a PCR-based marker for allelic selection. We report the isolation of two additional DFR homologs (DFR-B and DFR-C). No unique sequences were identified in either DFR homolog, even in the untranslated region (UTR). Both genes shared more than 95% nucleotide sequence identity with the DFRA gene. To obtain a unique sequence from each gene, we isolated the promoter regions. Sequences of the DFR-A and DFR-B promoters differed completely from one another, except for an approximately 100-bp sequence adjacent to the 5¢UTR. It was possible to specifically amplify only the DFR-A gene using primers designed to anneal to the unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800 bp deletion within the 3¢ region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a eletion mutation in the yellow DRF-A gene results in the lack of anthocyanin production in yellow onions.

Peppers (Capsicum spp.) were grown for phytochemical analyses at three different locations including a greenhouse at Colleg Station and field plots at Uvalde and Weslaco, Texas. Cultivar effets were significant at each location for all compounds. The best sources of b-carotene were mature greenhouse-grown fruit of Fidel (23.7mg/g0 and PI 357509 (9.2 mg/g). Mature greenhouse fruit of Tropic Bell (10.1 mg/g) and PI 357509 (9.2 mg/g) had high lutein, but Uvalde field-grown mature fruit of these lines were low in this compound, (1.4 and 0.5 mg/g, respectively). MJ 201 fruit had the highest zeaxanthin levels (10 mg/g) at both College Station and Uvalde. The best sources of quercetin over all locations were the yellow wax types, Banana Supreme j(186 mg/g), PI 357509 (86 mg/g) and Rio Grande Gold (26 mg/g). Fidel (37 mg/g) and Banana Supreme (21.5 mg/g) were the best sources of luteolin. Immature fruit generally contained lower levels of lutein and xeaxanthin than mature, colored fruit. These differences were not always statistically statistically significant. Greenhouse-grown peppers at College Station contained more carotenoids than the field-grown peppers in Uvalde and Weslaco, but there were no significant differences among locations for flavonoid concentrations. Several good candidate parents were identifited for the breeding program to develop novel pepper varieties with increased health benefits. Families of these varieties are currently being examined to assess the impact of specific environmental factors and identify genes involved in regulating synthesis of these beneficial phytochemicals.

Restrictions placed on water usage for farmers have prompted the development of irrigation management projects aiming at water savings of economically important crops. The objective of this work was to determine yield, water use efficiency, and leaf quality responses to deficit irrigation rates of processing spinach (Spinacea oleracea L.) cultivars. Three irrigation treatments were imposed with a center pivot system, 100%, 75%, and 50% crop evapotranspiration rates (ETc). Commercial cultivars used were ‘DMC 09’, ‘ASR 157’, and ‘ACX 3665’. Leaf quality was significantly affected by deficit irrigation rate and cultivar. Leaf yellowness was highest at 50% ETc, and was more evident for ‘ACX 3665’. The percent excess stem (>10 cm) was higher at 100% ETc. This response was greater in ‘ACX 3665’ than in ‘ASR 157’ and ‘DMC 09’. Marketable yields were significantly higher for ‘ASR 157’ at either 100% or 75% ETc rates, compared to ‘DMC 09’ and ‘ACX 3665’. High water use efficiency was also measured at 75% ETc for ‘ASR 157’. Minimum canopy temperature differences were detected among the irrigation treatments. This work demonstrated that it is possible to reach a 25% water savings in one season, without reducing yields when using vigorous cultivars.

Working with the largest cantaloupe and honeydew melon growers in Texas and with other collaborators, we have used a balanced nutrition approach to address the issue of improving and preserving fruit quality. Calcium (Ca) and potassium (K) are key minerals involved not only in processes determining fruit quality and storability, but also in human nutrition and health. Not only does Ca contribute to building strong bones in humans, it also helps melons develop strong rind tissue. Due to many environmental factors, plants cannot absorb enough of these minerals from the soil during fruit formation to ensure optimum fruit quality standards. We have, therefore, looked at the possibility of using foliar sprays as a supplement to soil-derived nutrients to improve and preserve fruit quality.

Muskmelon [Cucumis melo L. (Reticulatus Group)] fruit sugar content is directly related to potassium (K) - mediated phloem transport of sucrose into the fruit. However, during fruit growth and maturation, soil fertilization alone is often inadequate (due to poor root uptake and competitive uptake inhibition from calcium and magnesium) to satisfy the numerous K-dependent process, such as photosynthesis, phloem transport, and fruit growth. Experiments were conducted during Spring 2003 and 2004 to determine if supplemental foliar K applications during the fruit growth and maturation period would alleviate this apparent inadequate K availability in orange-flesh muskmelon ‘Cruiser’. Plants were grown in a greenhouse and fertilized throughout the study with a soil-applied N-P-K fertilizer. Flowers were hand pollinated and only one fruit per plant was allowed to develop. Starting at 3 to 5 days after fruit set, and acid-complexed potassium (potassium metalosate, 24% K) solution, diluted to 4.0 mL-L1. Three sets of plants were sprayed either weekly (once per week), biweekly (once every 2 weeks) or not sprayed (control). Fruit from plants receiving supplemental foliar K matured on average 2 days earlier than those from control plants. In general, there were no difference in fruit maturity or quality aspects between the weekly and biweekly treatments except for fruit sugar and beta-carotene concentration, which were significantly higher in the weekly compared to the biweekly or control treatments. Supplemental foliar K applications also resulted in significantly firmer fruit with higher K, soluble solids, total sugars, ascorbic acid (vitamin C) and beta-carotene concentrations than fruit from control plants. These results demonstrate that carefully timed foliar K nutrition can alleviate the developmentally induced K deficiency effects on fruit quality and marketability.

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the protein's secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.

Trials were conducted during summer months of 2002 and 2003 to evaluate the effects of a kaolin-based particle film (Surround WP, Engelhard Corp.) on gas exchange, nut quality, casebearer density and population of natural enemies (insects and arachnids) on pecan (Carya illinoinensis ‘Pawnee’) trees. Film application was repeated for seven (2002) or nine (2003) times during the growing season. In both years, treated trees showed lower leaf temperature (up to 4 °C) than untreated trees. During the warmest hours of the day, kaolin-treated leaves were generally 0 to 2 °C cooler than air temperatures, compared to 4 to 6 °C for control leaves. Leaf net assimilation rate, stomatal conductance and stem water potential were not affected by film application. Nut size and quality did not differ between the two treatments. Shellout (percentage of nut consisting of kernel) was not affected by treatment and averaged about 55%. Crop grade distribution (fancy, choice, standard, and damaged) was also similar among treatments in both years. In both years, numbers of green lacewing eggs was less on kaolin-treated compared to control leaves. The density of common natural enemies (lady beetles, green lacewings, spiders) of pecan pests did not differ between treatments. The average number of developing nuts damaged by pecan nut casebearer (Acrobasis nuxvorella Neunzig) was significantly higher in kaolin-sprayed trees (24.2%) compared to control trees sprayed with conventional insecticides (9.3%). The results suggest that kaolin-based particle film may not be a viable alternative to conventional methods of controlling pecan pests. Also, under adequate irrigation conditions, carbon assimilation, water relations and productivity may not benefit from kaolin particle film application.

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H1/ cation exchangers (CAX) have more calcium (Ca21) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells,CAXtransporters are activated whenthe N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca21 hypersensitive yeast phenotypes and also appear to be more specific Ca21 transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4- expressing tomato lines. The sCAX1-expressing tomato linesdemonstrate increased vacuolarH1/Ca21 transport,whenmeasured in root tissue, elevated fruit Ca21 level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca21 levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca21 and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.

The Arabidopsis Ca2+/H+ antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca2+ into the vacuole to help mediate Ca2+ homeostasis. Previous whole plant studies indicate that activity of Ca2+/H+ antiporters is regulated by pH. However, the pH regulation of individual Ca2+/H+ antiporters has not been examined. To determine whether CAX1 and CAX2 activity is affected by pH, Ca2+/H+ antiport activity was measured in vacuolar membrane vesicles isolated from yeast heterologously expressing either transporter. Ca2+ transport by CAX1 and CAX2 was regulated by cytosolic pH and each transporter had a distinct cytosolic pH profile. Screening of CAX1/CAX2 chimeras identified an amino acid domain within CAX2 that altered the pH-dependent Ca2+ transport profile so that it was almost identical to the pH profile of CAX1. Results from mutagenesis of a specific His residue within this domain suggests a role for this residue in pH regulation.

Several citrus limonoid glycosides have proved to be particularly difficult to purify using conventional techniques. A reversed-phase flash chromatographic technique has been developed for the separation and isolation of the closely related limonoid glucosides, nomilin 17--d-glucopyranoside and nomilinic acid 17--d-glucopyranoside, with confirmation of their identities by electrospray ionisation mass spectrometry. Furthermore, the semipurification of the mixture of glucosides enriched with flavanone glucosides such as naringin, narirutin and other limonoid glucosides was obtained. The closely eluting glucosides were successfully separated to achieve a good yield and purity of 93%.

Flavanoids exist as secondary plant metabolites, which displays a wide variety of biological effects. The polymethoxylated flavones, such as nobiletin and tangeretin, present in Citrus reticulata peels are of great interest due to their pharmacological effects. Separation and isolation of these structurally very similar flavones has been achieved by using commercially available ion exchange resins. The strong cation exchange resin [H+] selectively adsorbs the tangeretin and other phenolic compounds present in the matrix in comparison to the hexamethoxylated flavone, i.e. nobiletin. The cation exchange resin can be used successfully to isolate nobiletin and tangeretin from a plant extract. Purity of the isolated compounds was monitored by HPLC using a C-18 column with photometric detection at 280 nm. The structures of the isolated compounds have been confirmed by NMR and tandem mass spectrometry.

In plants, yeast, and bacteria, cation/H_ exchangers (CAXs) have been shown to translocate Ca2_ and other metal ions utilizing the H_ gradient. The best characterized of these related transporters is the plant vacuolar localized CAX1. We have used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within Arabidopsis CAX1. The mutants were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2_ and other metals. By utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca2_ transport activity. The CAX1 His338 residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His338 to every other amino acid residue and utilized yeast growth assays to estimate the transport properties of the 19 CAX mutants. Expression of 16 of these His338 mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca2_. Most interestingly, H338N exhibited increased tolerance to Cd2_ and Zn2_. Endomembrane fractions from yeast cells were used to measure directly the transport of H338N. Although the H338N mutant demonstrated 25% of the wild type Ca2_/H_ transport, it showed an increase in transport for both Cd2_ and Zn2_ reflected in a decrease in the Km for these substrates. This study provides insights into the CAX cation filter and novel mechanisms by which metals may be partitioned across membranes.
In plants, yeast, and bacteria, cation/H_ exchangers (CAXs) have been shown to translocate Ca2_ and other metal ions utilizing the H_ gradient. The best characterized of these related transporters is the plant vacuolar localized CAX1. We have used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within Arabidopsis CAX1. The mutants were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2_ and other metals. By utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca2_ transport activity. The CAX1 His338 residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His338 to every other amino acid residue and utilized yeast growth assays to estimate the transport properties of the 19 CAX mutants. Expression of 16 of these His338 mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca2_. Most interestingly, H338N exhibited increased tolerance to Cd2_ and Zn2_. Endomembrane fractions from yeast cells were used to measure directly the transport of H338N. Although the H338N mutant demonstrated 25% of the wild type Ca2_/H_ transport, it showed an increase in transport for both Cd2_ and Zn2_ reflected in a decrease in the Km for these substrates. This study provides insights into the CAX cation filter and novel mechanisms by which metals may be partitioned across membranes.

In order to make use of the large collection of Riken Arabidopsis full-length cDNA (RAFL) clones, it has previously been necessary to find or generate an expression vector with Sfil sites into which the insert can be cloned or to PCR amplify the inserts in order to add more suitable restriction sites for subcloning. However, the former can be time-consuming, and the latter can introduce unwanted errors. To circumvent these problems, Shigaki et al. (p. 301) instead construct a small shuttle univector compatible with Sfil digestion and containing a single loxP site, enabling them to make use of the Cre-loxP recombination system for their cloning reactions. A second host vector was also developed containing selection genes for both bacteria and plants, as well as the requisite loxP site to accept the univector-transported RAFL clone cDNA fragment. The univector carries a conditional replication origin and can therefore be propagated only in select Escherichia coli strains, while the host vector uses the more permissive ColE1 replication origin. The efficacy of the system was demonstrated, not with a RAFL clone, but using a modified GFP cDNA engineered with flanking Sfil sites. Following recombination by addition of Cre protein, the resulting fused vector was transformed into Arabidopsis tumefaciens for transformation of Arabidopsis using the floral dip method, yielding a GFP-expressing plant. This simplified and straightforward adaptation promises to make the generation of transgenic plants both easier and more economical.

To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary vectors containing different reporter genes precisely at near-perfect efficiency. We have constructed univector-adapted vectors with three reporters, b-glucuronidase, luciferase, and green fluorescent protein, and a BASTA-resistance gene for selection of plant transformants. Expression in plants using the new system was validated by comparison to conventional reporter vectors. These new vectors are efficient and economical alternatives to the other plant reporter vectors currently available. The royalty-free Cre-loxP system serves as a platform for the future expansion of recombination-based cloning vectors for plant research.

The characteristic red pigmentation of watermelon and tomato fruits is determined by accumulation of the carotenoid pigment lycopene and this phenotype is polyphyletic. Since several carotenoids are known to have health promoting activity, and watermelon can be a significant source of lycopene and other carotenoids, it is important to understand the genetic basis of watermelon fruit-specific carotenoid biosynthesis. Unlike tomato, very little is known about the regulation of carotenoid biosynthesis during fruit development in watermelon, a non-climacteric fruit. We have HPLC analyzed the carotenoids of red, yellow and orange watermelons and compared their carotenoid patterns with those of known fruit colour mutants of tomato. Interestingly, we could detect tomato mutant equivalents to most watermelon fruit colour phenotypes, including r, og, B and t.

The objectives of this study were to evaluate the hydrophilic antioxidant activity (AOAH) and the lipophilic antioxidant activity (AOAL); and their correlations with vitamin C, and total phenolic and â-carotene contents in fresh guava fruits of one white flesh clone (‘Allahabad Safeda’) and three pink flesh clones (‘Fan Retief’, ‘Ruby Supreme,’ and an advanced selection). A ferric reducing antioxidant power assay was used to estimate both AOAH and AOAL from methanol and dichloromethane extracts, respectively. The white flesh clone, ‘Allahabad Safeda,’ showed higher levels of both AOAH [33.3 ìM Trolox equivalents (TE)/g fresh weight (FW)] and AOAL (0.25 ìM TE/g FW) than the pink flesh clones that ranged from 15.5 to 30.4 and from 0.12 to 0.13 ìM TE/g FW for AOAH and AOAL, respectively. The AOAH was positively correlated with vitamin C (r = 0.92, p < 0.01) and total phenolic (r = 0.97, p < 0.01) but was negatively correlated with â-carotene (r = -0.73, p = 0.03). The AOAL was not correlated with these antioxidants.

‘Waco’ is a new pecan [Carya illinoinesis (Wangenh.) K. Koch] cultivar released 11 Feb. 2005 by the U. S. Department of agriculture (USDA), Agricultural Research Service (ARS), and the Agricultural Experiment Station of Texas. When compared to other protandrous cultivars. ‘Waco’ has excellent nut quality, and is suitable for planting in the western U.S. pecan production area. ‘Waco’ pecans are large enough to be sold in shell, or they can be shelled to produce a high proportion of intact halves and large pieces of very high quality.

Dysregulation of cell proliferation and apoptosis are important aspects of carcinogenesis. We have shown that grapefruit pulp and its functional components protect against colon carcinogenesis, in part by suppressing cell proliferation. The present study evaluated the hypothesis that grapefruit and its compounds are also protective by enhancing apoptosis through a reduction in iNOS and COX-2 levels. To test this, 100 male SD rats were provided one of five diets: control diet, natural or irradiated (300 Gy, 137Cs) grapefruit pulp power (200 mg/kg). Rats were injected with saline or azoxymethane (AOM; 15 mg/kg) during the 3rd and 4th wk after initiation of the diets, and colons were resected (6 wk post 2nd injection) for evaluation of apoptosis by TUNEL assay and COX-2 and iNOS by immunoblotting. All diets increased (P < 0.05) the apoptotic index, compared to the control die; however, the greatest enhancement was seen with natural GFPP and limonin in AOM-injected rats. Furthermore, the apoptotic indices in AOM injected rats provided with natural GFPP or limonin diets were (P < 0.05) greater than that of saline-injected rats consuming the same diets. AOM-injected rats fed natural GFPP or limonin also had reduced (P < 0.05) COX-2 and iNOS levels compared to the control diet. Since reduced iNOS and COX-2 levels and enhanced apoptosis inhibits tumor development, these results suggest that consumption of natural grapefruit or limonin may contribute toward colon cancer prevention. Funded by USDA 2001-52102-11257, THECB 003658-0359c-2001, and NIEHS P30-#S09106.

A variety of in vitro models such as 3-carotene-linoleic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH) superoxide, and hamster low density lipoprotein (LDL) were used to measure the antioxidant activity of 11 citrus bioactive compounds. The compounds tested included two limonoids, limonin (Lim) and Limonin 17-β-D-glucopyranoside (LG); eight flavonoids, apigenin (Api), scutellarein (Scu), kaempferol (Kae), rutin trihydrate (Rut), neohesperidin (Neh), neoeriocitrin (Nee), naringerin (Ngri), and naringin (Ng), and a coumarin (bergapten). The above compounds were tested at concentration of 10 mM in all four methods. It was found that Lim, LG, and Ber inhibited < 7%, whereas Scu, Kae, and Rut inhibited 51.3%, 47.0%, and 44.4%, respectively, using the 3-carotene-linoleate model system. Lim, LG, Rut, Scu, Nee, and Kae showed 0.5%, 0.25%, 32.2%, 18.3%, 17.2%, and 12.2%, respectively, free radical scavenging activity using the DPPH method. In the superoxide model, Lim, LG, and Ber inhibited the production of superoxide radicals by 2.5-10%, while the flavonoids such as Rut, Scu, Nee, and Neh inhibited superoxide formation by 64.1%, 52.1%, 48.3%, and 37.7%, respectively. However, LG did not inhibit LDL oxidation in the hamster LDL model. But, Lim and Ber offered some protection against LDL oxidation, increasing lag time to 345 min (3-fold) and 160 min (33% increase), respectively, while both Rut and Nee increased lag time to 2800 min (23-fold). Scu and Kae increased lag time to 2140 min (18-fold) and 1879 min (15.7-fold), respectively. In general, it seems that flavonoids, which contain a chromanol ring system, had stronger antioxidant activity as compared to limonoids and hergapten, which lack the hydroxyl groups. The present study confirmed that several structural features were linked to the strong antioxidant activity of flavonoids. This is the first report on the antioxidant activity of limonin, limonin glucoside, and neoerioctrin.

The prediction of human population growth worldwide indicates there will be a need to substantially increase food production in order to meet the demand on food supply. This can be achieved in part by the effective management of insect pests. Since plants have co-evolved with herbivorous insects for millions of years, they have developed an array of defense genes to protect themselves against a wide variety of chewing and sucking insects. Using these naturally-occurring genes via genetic engineering represents an environmentally friendly insect pest-control measure. Insects, however, have been actively evolving adaptive mechanisms to evade natural plant defenses. Such evolved adaptability undoubtedly has helped insects during the last century to rapidly overcome a great many human-imposed management practices and agents, including chemical insecticides and genetically engineered plants. Thus, better understanding of the molecular and genetic basis of plant defense and insect counter-defense mechanisms is imperative, not only from a basic science perspective, but also for biotechnology-based pest control practice. In this review, we emphasize the recent advance and understanding of molecular strategies of attack-counterattack and defense-counter-defense between plants and their herbivores.